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US20140200261A1 - Signal-sensor polynucleotides for the alteration of cellular phenotypes - Google Patents

Signal-sensor polynucleotides for the alteration of cellular phenotypes Download PDF

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Publication number
US20140200261A1
US20140200261A1 US14/041,011 US201314041011A US2014200261A1 US 20140200261 A1 US20140200261 A1 US 20140200261A1 US 201314041011 A US201314041011 A US 201314041011A US 2014200261 A1 US2014200261 A1 US 2014200261A1
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cancer
sensor
signal
sensor polynucleotide
synthetic signal
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Stephen G. Hoge
Tirtha Chakraborty
Joshua P. Frederick
Matthias John
Antonin de Fougerolles
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ModernaTx Inc
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Moderna Therapeutics Inc
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Priority to US14/135,876 priority patent/US20140206852A1/en
Publication of US20140200261A1 publication Critical patent/US20140200261A1/en
Assigned to MODERNATX, INC. reassignment MODERNATX, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: MODERNA THERAPEUTICS, INC.
Assigned to MODERNA THERAPEUTICS, INC. reassignment MODERNA THERAPEUTICS, INC. CORRECTIVE ASSIGNMENT TO CORRECT THE EXECUTION DATE OF ASSIGNOR JOSHUA P. FREDERICK PREVIOUSLY RECORDED ON REEL 031638 FRAME 0919. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Assignors: JOHN, MATTHIAS, DE FOUGEROLLES, ANTONIN, HOGE, Stephen G., CHAKRABORTY, TIRTHA, FREDERICK, JOSHUA P.
Priority to US16/036,170 priority patent/US20190185529A1/en
Priority to US17/162,061 priority patent/US11708396B2/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the invention relates to compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of signal-sensor polynucleotides, primary constructs and mRNA molecules for the alteration of cellular phenotypes and micro environments.
  • Cancer is a disease characterized by uncontrolled cell division and growth within the body. In the United States, roughly a third of all women and half of all men will experience cancer in their lifetime. Polypeptides are involved in every aspect of the disease including cancer cell biology (carcinogenesis, cell cycle suppression, DNA repair and angiogenesis), treatment (immunotherapy, hormone manipulation, enzymatic inhibition), diagnosis and determination of cancer type (molecular markers for breast, prostate, colon and cervical cancer for example). With the host of undesired consequences brought about by standard treatments such as chemotherapy and radiotherapy used today, genetic therapy for the manipulation of disease-related peptides and their functions provides a more targeted approach to disease diagnosis, treatment and management.
  • PCT/US2013/030068 filed Mar. 9, 2013, entitled Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides
  • International Application No. PCT/US2013/030070 filed Mar. 9, 2013, entitled Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides
  • International Patent Application No. PCT/US2013/031821 filed Mar. 15, 2013, entitled In Vivo Production of Proteins; the contents of each of which are herein incorporated by reference in their entireties.
  • Formulations and delivery of modified polynucleotides are described in, for example, co-pending and co-owned International Publication No WO2013090648, filed Dec. 14, 2012, entitled Modified Nucleoside, Nucleotide, Nucleic Acid Compositions and US Publication No US20130156849, filed Dec. 14, 2012, entitled Modified Nucleoside, Nucleotide, Nucleic Acid Compositions; the contents of each of which are herein incorporated by reference in their entireties.
  • next generation of therapeutics must also address the complex cellular microenvironment of the cancer and have the capacity for cell, tissue, organ or patient stratification, whether structurally or functionally.
  • nucleic acid based compounds or polynucleotide-encoding nucleic acid-based compounds e.g., signal-sensor polynucleotides
  • nucleic acid-based compounds e.g., signal-sensor polynucleotides
  • compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of signal-sensor polynucleotide molecules encoding at least one oncology-related polypeptide of interest may be chemically modified mRNA (mmRNA) molecules.
  • mmRNA mRNA
  • the present invention provides an isolated signal-sensor polynucleotide comprising a region encoding an oncology-related polypeptide of interest that functions, when translated, to send a death or survival signal.
  • death or survival signals include those which (i) alter (increase or decrease) the expression of one or more proteins, nucleic acids, or non-coding nucleic acids, (ii) alter the binding properties of biomolecules within the cell, and/or (iii) perturb the cellular microenvironment in a therapeutically beneficial way.
  • the signal-sensor polynucleotide may also encode in a flanking region, one or more sensor sequences.
  • sensor sequences function to “sense” the cell, tissue or organ microenvironment and confer upon the signal-sensor polynucleotide an altered expression or half life profile (increased or decreased) depending on the interactions of the sensor sequence with the cell, tissue or organ microenvironment.
  • signal-sensor polynucleotide comprising, a first region of linked nucleosides, a first flanking region located 5′ relative to said first region and a second flanking region located 3′ relative to said first region.
  • the first region may encode an oncology-related polypeptide of interest such as, but not limited to, SEQ ID NOs: 1321-2487, 6611-6616 and 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516 and 7517
  • the first flanking region may include a sequence of linked nucleosides such as, but not limited to, the native 5′ untranslated region (UTR) of any of the nucleic acids that encode any of SEQ ID NOs: 1321-2487, 6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516, 7517, SEQ ID NO: 1-4 and functional variants thereof.
  • UTR native 5′ untranslated region
  • the first region may comprise at least an open reading frame of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2488-2496, 6617-6621, 7348-7354, 7362-7489, 7491, 7494, 7506, 7511 and 7513.
  • the second flanking region may include a sequence of linked nucleosides such as, but not limited to, the native 3′ UTR of any of the nucleic acids that encode any of SEQ ID NOs: 1321-2487, 6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516, 7517, SEQ ID NO: 5-21 and functional variants thereof, and one or more sensor sequences located such as, but not limited to, SEQ ID NOs: 3529-4549, SEQ ID NOs: 5571-6591 and functional variants thereof.
  • the signal-sensor polynucleotide may also include a 3′ tailing sequence of linked nucleosides.
  • a signal-sensor polynucleotide which comprises an mRNA encoding an oncology-related polypeptide of interest and one or more sensor sequences such as, but not limited to, SEQ ID NOs: 3529-4549, SEQ ID NOs: 5571-6591 and functional variants thereof.
  • the oncology-related polypeptide of interest may be, but is not limited to, SEQ ID NOs: 1321-2487, 6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516 and 7517.
  • the mRNA may include at least one open reading frame of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2488-2496, 6617-6621, 7348-7354, 7362-7489, 7491, 7494, 7506, 7511 and 7513.
  • the signal-sensor polynucleotides may comprise one, two, three or more than three stop codons.
  • the signal-sensor polynucleotides comprise two stop codons.
  • the first stop codon is “TGA” and the second stop codon is selected from the group consisting of “TAA,” “TGA” and “TAG.”
  • signal-sensor polynucleotides comprise three stop codons.
  • the signal-sensor polynucleotides may have a 3′ tailing sequence of linked nucleosides such as, but not limited to, a poly-A tail of at least 140 nucleotides, a triple helix, and a poly A-G quartet.
  • the signal-sensor polynucleotides may have a 5′cap such as, but not limited to, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • a 5′cap such as, but not limited to, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • the signal-sensor polynucleotides may include at least one chemical modification such as, but not limited to, modifications located on one or more of a nucleoside and/or the backbone of the nucleotides.
  • the signal-sensor polynucleotides comprise a pseudouridine analog such as, but not limited to, 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine, 1-methyl-pseudouridine (m 1 ⁇ ), 1-methyl-4-thio-pseudouridine (m 1 s 4 ⁇ ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ⁇ ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio
  • the signal-sensor polynucleotides comprise the pseudouridine analog 1-methylpseudouridine. In yet another embodiment, the signal-sensor polynucleotides comprise the pseudouridine analog 1-methylpseudouridine and the modified nucleoside 5-methylcytidine.
  • the signal-sensorpolynucleotides may include at least two chemical modifications such as, but not limited to, modifications located on one or more of a nucleoside and/or the backbone of the nucleotides.
  • the signal-sensor polynucleotide comprises the chemical modifications 1-methylpseudouridine and 5-methylcytidine.
  • the signal-sensor polynucleotides may comprise at least one translation enhancer element (TEE) such as, but not limited to, TEE-001-TEE-705.
  • TEE translation enhancer element
  • the signal-sensor polynucleotide encodes a factor modulating the affinity between HIF subunits and/or HIF-dependent gene expression such as, but not limited to, SEQ ID NO: 6611-6616.
  • the signal-sensor polynucleotides may be purified and/or formulated.
  • the present invention provides a method of treating a disease, disorder and/or condition in a subject in need thereof by increasing the level of an oncology-related polypeptide of interest comprising administering to said subject an isolated signal-sensor polynucleotide encoding said oncology-related polypeptide.
  • the disease, disorder and/or condition may include, but is not limited to, adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin
  • the present invention provides a method of reducing, eliminating, or preventing tumor growth in a subject in need thereof by increasing the level of an oncology-related polypeptide of interest comprising administering to said subject an isolated signal-sensor polynucleotide encoding said oncology-related polypeptide.
  • the tumor growth may be associated with or results from a disease, disorder and/or condition such as, but not limited to, adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoi
  • the present invention provides a method of reducing and/or ameriorating at least one symptom of cancer in a subject in need thereof by increasing the level of a polypeptide of interest comprising administering to said subject an isolated signal-sensor polynucleotide encoding said oncology-related polypeptide.
  • Non-limiting examples of symptoms include weakness, aches and pains, fever, fatigue, weight loss, blood clots, increased blood calcium levels, low white blood cell count, short of breath, dizziness, headaches, hyperpigmentation, jaundice, erthema, pruritis, excessive hair growth, change in bowel habits, change in bladder function, long-lasting sores, white patches inside the mouth, white spots on the tongue, unusual bleeding or discharge, thickening or lump on parts of the body, indigestion, trouble swallowing, changes in warts or moles, change in new skin and nagging cough and hoarseness.
  • the present invention provides a method of preferentially inducing cell death in cancer cells in a tissue or organ comprising contacting the tissue or organ with a signal-sensor polynucleotide encoding an oncology-related polypeptide whose expression triggers apoptosis or cell death and at least one microRNA binding site of a microRNA where the expression of the microRNA in the cancer cell is lower than the expression of the mircroRNA in normal non-cancerous cells.
  • the signal-sensor polynucleotide may be administered at a total daily dose of between 0.001 ug and 150 ug.
  • Administration of a signal-sensor polynucleotide may be by injection, topical administration, ophthalmic administration or intranasal administration.
  • administration may be by injection such as, but not limited to, intradermal, subcutaneous and intramuscular.
  • administration may be topical such as, but not limited to, using creams, lotions, ointments, gels, sprays, solutions and the like.
  • FIG. 1 is a schematic of a primary construct of the present invention.
  • FIG. 2 is an expanded schematic of the second flanking region of a primary construct of the present invention illustrating the signal-sensor elements of the polynucleotide.
  • FIG. 3 is a gel profile of Apoptosis-Inducing Factor short (AIFsh) protein from AIFsh modified mRNA in mammals.
  • FIG. 3A shows the expected size of AIFsh.
  • FIG. 3B shows the expected size of AIFsh.
  • FIG. 4 is a gel profile of Siah E3 ubiquitin protein ligase 1 (SIAH1) protein from SIAH1 modified mRNA in mammals.
  • FIG. 4A shows the expected size of SIAH1.
  • FIG. 4B shows the expected size of SIAH1.
  • FIG. 5 is a gel profile of constitutively active (C.A.) caspase 3 (also known as reverse caspase 3 (Rev-Caspase 3)) protein from C.A. caspase 3 modified mRNA in mammals.
  • FIG. 5A shows the expected size of C.A. caspase 3.
  • FIG. 5B shows the expected size of C.A. caspase 3.
  • FIG. 6 is a gel profile of Granulysin protein from granulysin modified mRNA in mammals.
  • FIG. 6A shows the expected size of granulysin.
  • FIG. 6B shows the expected size of granulysin.
  • FIG. 7 is a western blot of C.A. caspase 3 and C.A. caspase 6.
  • FIG. 7A shows protein from C.A. caspase 3 modified mRNA fully modified with 5-methylcytidine and 1-methylpseudouridine or fully modified with 1-methylpseudouridine.
  • FIG. 7B shows protein from C.A. caspase 6 modified mRNA fully modified with 5-methylcytidine and 1-methylpseudouridine or fully modified with 1-methylpseudouridine.
  • RNA ribonucleic acid
  • RNA ribonucleic acid
  • compositions including pharmaceutical compositions
  • methods for the design, preparation, manufacture and/or formulation of polynucleotides encoding one or more polypeptides of interest are also provided.
  • polypeptides of the present invention are encoded by a new class of polynucleotide therapeutics, termed “signal-sensor polynucleotides” which are particularly useful in the stratification, profiling and/or personalization of the polynucleotide therapeutice (e.g., mRNA) and which are tailored to a particular cell type, disease or cell microenvironment or biological profile.
  • signal-sensor polynucleotides e.g., mRNA
  • cancers exhibit diverse gene expression patterns, physicochemical environments and metastatic or motility behaviors and according to Hanahan and Weinberg (Cell, 2011, 144:646-674) there are six hallmarks of cancer. These include sustaining a proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. These hallmarks or functions of cancer allow the cancer to survive, proliferate and disseminate and each arises at different times and in different patterns depending on the cancer type.
  • the polynucleotides of the present invention represent such therapeutics; having the ability to selectively stabilize or destabilize cell systems, signal proliferation (survival) or death, trigger the cell cycle or senescence and/or activate or avoid the immune response depending on the cell type, e.g., cancer or normal cell.
  • signal-sensor polynucleotide therapeutics may be used to destabilize the survival advantages or hallmarks of a cancer cell (hence they would be cytotoxic).
  • diagnostic efforts would include the profiling of the cancer (although this would not be required a priori) including metabolic state (hypoxic, acidotic), apoptotic vs. survival gene profiles, cell cycle vs. senescent stage, immune status, and stromal factors present.
  • the signal-sensor polynucleotide disrupts the transcriptome of the cancer cell.
  • the disruption may affect one or more signaling or expression events.
  • the encoded oncology-related polypeptide may act upstream of a transcription factor known to induce or enhance the expression of genes associated with a cancer. Delivery of the signal-sensor polynucleotide encoding the oncology-related polypeptide which inhibits such a transcription factor (either by binding or sequestration or degradation) would thereby alter the transcriptome of the cancer cell and have a therapeutic benefit.
  • a transcription factor is HIF-1alpha.
  • a signal-sensor polynucleotide encoding a protein which is capable of binding HIF-1alpha or whose expression results in lower HIF-1alpha, would effectively turn down HIF-1alpha regulated genes, e.g., VEGFA or SLC2A1, and destabilize the cancer.
  • the profile of the cancer may be evaluated before the signal-sensor polynucleotide is selected.
  • profiling data would inform the selection of which oncology-related polypeptide to be delivered.
  • the profile of gene expression categorized by hallmark class such as apoptosis, replicative capacity or metabolic signature would allow dynamic instability scoring for a polypeptide and an optimization of therapeutic window for the signal-sensor polynucleotide.
  • a “dynamic instability index” refers to a dose of signal-sensor polynucleotide sufficient to induce 50% increase of the oncology-related target protein in vitro in a cancer cell as compared to a normal matched cell.
  • Profiling may also be done within hallmark classes such as the distinction between caspase-dependent and caspase independent gene expression for the apoptosis class.
  • profiling could be conducted across classes such as gene profiling of apoptosis, senescence (replicative capacity), and metabolic classes.
  • the signal-sensor polynucleotides described herein may be used to reduce the expression and/or amount of a polypeptide in a cell.
  • MYC inhibitor A, MYC inhibitor B, MYC inhibitor C or MYC inhibitor D may be used on Hep3B cells in order to determine the potency of MYC inhibitor A, MYC inhibitor B, MYC inhibitor C or MYC inhibitor D at various concentrations (see e.g., Example 55).
  • the signal-sensor polynucleotides described herein may direct either cytotoxic or cytoprotective therapeutic benefit to specific cells, e.g., normal vs. cancerous.
  • signal-sensor polynucleotides would not only encode an oncology-related polypeptide but also a sensor sequence.
  • Sensor sequences include, for example, microRNA binding sites, transcription factor binding sites, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules.
  • a “sensor region” is a region of linked nucleosides of the signal-sensor polynucleotide comprising at least one sensor sequence.
  • the signal-sensor polynucleotides of the present invention may have one or more sensor regions.
  • one or more sensor regions may be located in the first flanking region.
  • the sensor region in the first flanking region may comprise at least one sensor sequence.
  • the sensor sequence may be, but is not limited to, mir-122, mir-142-3p, mir-142-5p, mir-146, fragments or variants thereof.
  • the sensor region in the first flanking region may comprise at least one sensor sequence such as a mir-122 sequence.
  • the mir-122 sequence may be, but is not limited to, a mir-122 binding site, mir-122 seed sequence, mir-122 binding site without the seed sequence or a combination thereof.
  • one or more sensor regions may be located in the second flanking region.
  • the sensor region in the second flanking region may include a sensor sequence such as mir-122, mir-142-3p, mir-142-5p, mir-146, fragments or variants thereof.
  • the sensor region in the second flanking region may include three sensor sequences.
  • the sensor sequences may be, but are not limited to, mir-122 sequences such as mir-122 binding sites, mir-122 seed sequences, mir-122 binding sites without the seed sequence or a combination thereof.
  • the sensor region in the second flanking region is located in the 3′UTR and the sensor region may include a sensor sequence which is a mir-122 sequence.
  • the mir-122 sequence may be, but is not limited to, a mir-122 binding site, mir-122 seed sequence, mir-122 binding site without the seed sequence or a combination thereof.
  • two or more sensor regions may be located in the same region of the signal-sensor polynucleotide such as, but not limited to, a first region first region of linked nucleotides, the first flanking region and/or the second flanking region.
  • the two or more sensor regions are located in the second flanking region.
  • three sensor regions are located in the 3′ UTR in the second flanking region.
  • the three sensor regions may include, mir-122 binding sites, mir-122 seed sequences, mir-122 binding sites without the seed sequence or a combination thereof.
  • two or more sensor regions may be located in different regions of the signal-sensor polynucleotide such as, but not limited to, the first region of linked nucleotides, the first flanking region and/or the second flanking region.
  • a first sensor region is located in the first flanking region and a second sensor region is located in the second flanking region.
  • the sensor regions may comprise the same sensor sequence or different sensor sequences.
  • a start codon is located within a sensor region.
  • a sensor region may comprise two or more sensor sequences.
  • the sensor sequences may be the same or different.
  • the sensor region may comprise two or more sensor sequence which are different from each other but they may be based on the same mir binding site.
  • the sensor region may include at least one miR binding site sequence and at least one mir binding site sequence with the seed removed.
  • the sensor region may include at least one miR binding site sequence and at least one miR seed sequence.
  • the sensor region may include at least one miR binding site sequence with the seed removed and at least one miR seed sequence.
  • the sensor region may comprise two or more sensor sequences which are in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times.
  • a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times.
  • each letter, A, B, or C represent a different miR sequence.
  • the signal-sensor polynucleotide may include two or more sensor regions with each sensor region having one or more sensor sequences.
  • the sensor sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times in each of the sensor regions.
  • the sensor sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times across the entire signal-sensor polynucleotide. In these patterns, each letter, A, B, or C represent a different miR sequence.
  • the first sensor region may have sensor sequences in the pattern ABA and the second sensor region may have sensor sequences in the pattern BAB so the overall pattern of the sensor sequences in the signal-sensor polynucleotide is ABABAB.
  • the first sensor region may have sensor sequences AA
  • the second sensor region may have sensor sequences BB
  • the third sensor region may have sensor sequences AA
  • the fourth sensor region may have sensor sequences BB so the overall pattern of the sensor sequences in the signal-sensor polynucleotide is AABBAABB.
  • the sensor sequences in the signal-sensor polynucleotides of the present invention may include one or more regulatory sequences in the 3-UTR and/or 5′UTR of natural mRNAs, which regulate mRNA stability and translation in different tissues and cells.
  • cis-regulatory elements may include, but are not limited to, Cis-RNP (Ribonucleoprotein)/RBP (RNA binding protein) regulatory elements, AU-rich element AUE, structured stem-loop, constitutive decay elements (CDEs), GC-richness and other structured mRNA motifs (Parker B J et al., Genome Research, 2011, 21, 1929-1943, which is herein incorporated by reference in its entirety.).
  • CDEs are a class of regulatory motifs that mediate mRNA degradation through their interaction with Roquin proteins.
  • CDEs are found in many mRNAs that encode regulators of development and inflammation to limit cytokine production in macrophage (Leppek K et al., Cell, 2013, 153, 869-881, which is herein incorporated by reference in its entirety.).
  • a particular CDE can be introduced to the signal-sensor polynucleotide when the degradation of polypeptides in a cell or tissue is desired.
  • a particular CDE can also be removed from the signal-sensor polynucleotide in order to maintain a more stable mRNA in a cell or tissue for sustaining protein expression.
  • microRNA profiling of the cancer cells or tissues may be conducted to determine the presence or absence of miRNA in the cells or tissues to determine the appropriate microRNA to use as sensor sequences in the signal sensor polynucleotides.
  • MicroRNA gene regulation may be influenced by the sequence surrounding the microRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous and artificial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence.
  • the microRNA may be influenced by the 5′UTR and/or the 3′UTR.
  • a non-human 3′UTR may increase the regulatory effect of the microRNA sequence on the expression of a polypeptide of interest compared to a human 3′UTR of the same sequence type.
  • microRNA mediated gene regulation can influence microRNA mediated gene regulation.
  • a structured IRES Internal Ribosome Entry Site
  • EIF4A2 binding to this secondarily structured element in the 5′UTR is necessary for microRNA mediated gene expression (Meijer H A et al., Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
  • the sensor-signal polynucleotide can further be modified to include this structured 5′-UTR in order to enhance microRNA mediated gene regulation.
  • At least one microRNA site can be engineered into the 3′ UTR of the signal-sensor polynucleotides of the present invention.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more microRNA sites may be engineered into the 3′ UTR of the signal-sensor polynucleotides of the present invention.
  • the microRNA sites incorporated into the signal-sensor polynucleotides may be the same or may be different microRNA sites.
  • the microRNA sites incorporated into the signal-sensor polynucleotides may target the same or different tissues in the body.
  • tissue-, cell-type-, or disease-specific microRNA binding sites in the 3′ UTR of a signal-sensor polynucleotide can be reduced.
  • tissue-, cell-type-, or disease-specific microRNA binding sites in the 3′ UTR of a signal-sensor polynucleotide e.g. hepatocytes, myeloid cells, endothelial cells, cancer cells, etc.
  • a microRNA site can be engineered near the 5′ terminus of the 3′UTR, about halfway between the 5′ terminus and 3′terminus of the 3′UTR and/or near the 3′terminus of the 3′UTR.
  • a microRNA site may be engineered near the 5′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′terminus of the 3′UTR.
  • a microRNA site may be engineered near the 3′terminus of the 3′UTR and about halfway between the 5′ terminus and 3′terminus of the 3′UTR.
  • a microRNA site may be engineered near the 5′ terminus of the 3′UTR and near the 3′ terminus of the 3′UTR.
  • a 3′UTR can comprise 4 microRNA sites.
  • the microRNA sites may be complete microRNA binding sites, microRNA seed sequences and/or microRNA binding site sequences without the seed sequence.
  • a signal-sensor polynucleotide may be engineered to include microRNA sites which are expressed in different tissues of a subject.
  • a signal-sensor polynucleotide of the present invention may be engineered to include miR-192 and miR-122 to regulate expression of the signal-sensor polynucleotide in the liver and kidneys of a subject.
  • a signal-sensor polynucleotide may be engineered to include more than one microRNA sites for the same tissue.
  • a signal-sensor polynucleotide of the present invention may be engineered to include miR-17-92 and miR-126 to regulate expression of the signal-sensor polynucleotide in endothelial cells of a subject.
  • the therapeutic window and or differential expression associated with the oncology-related polypeptide encoded by the signal-sensor polynucleotide of the invention may be altered.
  • signal-sensor polynucleotides may be designed whereby a death signal is more highly expressed in cancer cells (or a survival signal in a normal cell) by virtue of the miRNA signature of those cells.
  • the signal-sensor polynucleotide encoding the binding site for that miRNA (or miRNAs) would be more highly expressed.
  • the oncology-related polypeptide encoded by the signal-sensor polynucleotide is selected as a protein which triggers or induces cell death.
  • Neighboring noncancer cells, harboring a higher expression of the same miRNA would be less affected by the encoded death signal as the signal-sensor polynucleotide would be expressed at a lower level due to the affects of the miRNA binding to the binding site or “sensor” encoded in the 3′UTR.
  • cell survival or cytoprotective signals may be delivered to tissues containing cancer and non cancerous cells where a miRNA has a higher expression in the cancer cells—the result being a lower survival signal to the cancer cell and a larger survival signature to the normal cell.
  • Multiple signal-sensor polynucleotides may be designed and administered having different signals according to the previous paradigm.
  • the expression of a signal-sensor polynucleotide may be controlled by incorporating at least one sensor sequence in the signal-sensor polynucleotide and formulating the signal-sensor polynucleotide.
  • a polynucleotide may be targeted to an orthotopic tumor by having a polynucleotide incorporating a miR-122 binding site and formulated in a lipid nanoparticle comprising the cationic lipid DLin-KC2-DMA (see e.g., the experiments described in Example 56A and 56B).
  • signal-sensor polynucleotides can be engineered for more targeted expression in specific cell types or only under specific biological conditions.
  • signal-sensor polynucleotides could be designed that would be optimal for protein expression in a tissue or in the context of a biological condition such as cancer.
  • Transfection experiments can be conducted in relevant cell lines, using engineered signal-sensor polynucleotides and protein production can be assayed at various time points post-transfection.
  • cells can be transfected with different microRNA binding site-engineering nucleic acids or signal-sensor polynucleotides and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, 72 hr and 7 days post-transfection.
  • In vivo experiments can also be conducted using microRNA-binding site-engineered molecules to examine changes in tissue-specific expression of formulated signal-sensor polynucleotides.
  • the signal-sensor polynucleotides of the invention may include at least one microRNA in order to dampen the antigen presentation by antigen presenting cells.
  • the microRNA may be the complete microRNA sequence, the microRNA seed sequence, the microRNA sequence without the seed or a combination thereof.
  • the microRNA incorporated into the signal-sensor polynucleotide may be specific to the hematopoietic system.
  • the microRNA incorporated into the signal-sensor polynucleotides of the invention to dampen antigen presentation is miR-142-3p.
  • the signal-sensor polynucleotides of the invention may include at least one microRNA in order to dampen expression of the encoded polypeptide in a cell of interest.
  • the signal-sensor polynucleotides of the invention may include at least one miR-122 binding site in order to dampen expression of an encoded polypeptide of interest in the liver.
  • the signal-sensor polynucleotides of the invention may include at least one miR-142-3p binding site, miR-142-3p seed sequence, miR-142-3p binding site without the seed, miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5p binding site without the seed, miR-146 binding site, miR-146 seed sequence and/or miR-146 binding site without the seed sequence (see e.g., the experiment outlined in Example 47 and Example 60).
  • the signal-sensor polynucleotides described herein may be modified as to avoid the deficiencies of other polypeptide-encoding molecules of the art.
  • the signal-sensor polynucleotides are referred to as modified signal-sensor polynucleotides or primary constructs, modified mRNA or mmRNA.
  • signal-sensor polynucleotide polynucleotides, primary constructs and/or mmRNA encoding oncology-related polypeptides of interest which have been designed to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency (when applicable), accessibility to circulation, protein half-life and/or modulation of a cell's status, function and/or activity.
  • the present invention provides nucleic acid molecules, specifically signal-sensor polynucleotides, primary constructs and/or mmRNA which encode one or more oncology-related polypeptides of interest.
  • the invention contemplates signal-sensor polynucleotides which are useful in cancer or cancer related diseases, disorders.
  • signal-sensor polynucleotides are nucleic acid transcripts which encode one or more oncology-related polypeptides of interest that, when translated, delivers a “signal” to the cell (cancer or noncancerous) which results in the therapeutic benefit to the organism of either being detrimental to the cancer cell or beneficial to normal cells or both detrimental to cancer cells and advantageous to normal cells.
  • the signal-sensor polynucleotides may optionally further comprise a sequence (translatable or not) which “senses” the microenvironment of the polynucleotide and alters (a) the function or phenotypic outcome associated with the peptide or protein which is translated, (b) the expression level of the signal-sensor polynucleotide, and/or both.
  • nucleic acid in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides.
  • Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′-amino functionalization) or hybrids thereof.
  • RNAs ribonucleic acids
  • DNAs deoxy
  • the signal-sensor polynucleotide or nucleic acid molecule is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.
  • Signal-sensor polynucleotides of the invention may be mRNA or any nucleic acid molecule and may or may not be chemically modified.
  • the basic components of an mRNA molecule include at least a coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail.
  • the present invention expands the scope of functionality of traditional mRNA molecules by providing signal-sensor polynucleotides or primary RNA constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the signal-sensor polynucleotide is introduced.
  • modified mRNA molecules of the present invention which may be synthetic, are termed “mmRNA.”
  • mmRNA modified mRNA molecules of the present invention, which may be synthetic, are termed “mmRNA.”
  • a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a signal-sensor polynucleotide polynucleotide, primary construct or mmRNA without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification
  • the signal-sensor polynucleotides of the present invention are distinguished from wild type mRNA in their functional and/or structural design features which serve to, as evidenced herein, overcome existing problems of effective polypeptide production using nucleic acid-based therapeutics.
  • FIG. 1 shows a representative signal-sensor primary construct 100 of the present invention.
  • the term “primary construct” or “primary mRNA construct” refers to a signal-sensor polynucleotide transcript which encodes one or more polypeptides of interest and which retains sufficient structural and/or chemical features to allow the polypeptide of interest encoded therein to be translated.
  • Signal-sensor primary constructs may be polynucleotides of the invention. When structurally or chemically modified, the signal-sensor primary construct may be referred to as a mmRNA.
  • the primary construct 100 here contains a first region of linked nucleotides 102 that is flanked by a first flanking region 104 and a second flaking region 106 .
  • the “first region” may be referred to as a “coding region” or “region encoding” or simply the “first region.”
  • This first region may include, but is not limited to, the encoded oncology-related polypeptide of interest.
  • the oncology-related polypeptide of interest may comprise at its 5′ terminus one or more signal peptide sequences encoded by a signal peptide sequence region 103 .
  • the flanking region 104 may comprise a region of linked nucleotides comprising one or more complete or incomplete 5′ UTRs sequences.
  • the flanking region 104 may also comprise a 5′ terminal cap 108 .
  • the second flanking region 106 may comprise a region of linked nucleotides comprising one or more complete or incomplete 3′ UTRs.
  • the flanking region 106 may also comprise a 3′ tailing sequence 110 and a 3′UTR 120 .
  • first operational region 105 Bridging the 5′ terminus of the first region 102 and the first flanking region 104 is a first operational region 105 .
  • this operational region comprises a start codon.
  • the operational region may alternatively comprise any translation initiation sequence or signal including a start codon.
  • this operational region comprises a stop codon.
  • the operational region may alternatively comprise any translation initiation sequence or signal including a stop codon. According to the present invention, multiple serial stop codons may also be used.
  • the operation region of the present invention may comprise two stop codons.
  • the first stop codon may be “TGA” and the second stop codon may be selected from the group consisting of “TAA,” “TGA” and “TAG.”
  • the operation region may further comprise three stop codons.
  • the third stop codon may be selected from the group consisting of “TAA,” “TGA” and “TAG.”
  • the 3′UTR 120 of the second flanking region 106 may comprise one or more sensor sequences 130 .
  • a region comprising at least one sensor sequence is referred to as a “sensor region.”
  • These sensor sequences as discussed herein operate as pseudo-receptors (or binding sites) for ligands of the local microenvironment of the primary construct or signal-sensor polynucleotide.
  • microRNA binding sites or miRNA seeds may be used as sensors such that they function as pseudoreceptors for any microRNAs present in the environment of the polynucleotide.
  • the shortest length of the first region of the signal-sensor primary construct of the present invention can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.
  • the length may be sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids.
  • the length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.
  • the length of the first region encoding the oncology-related polypeptide of interest of the present invention is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).
  • the “first region” may be referred to as a “coding region” or “region encoding” or simply the “first region.”
  • the signal-sensor polynucleotide, primary construct, or mmRNA includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000, from 500 to 2,000,
  • the first and second flanking regions may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
  • 15-1,000 nucleotides in length e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides.
  • the tailing sequence may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides).
  • the length may be determined in units of or as a function of polyA binding protein binding.
  • the polyA tail is long enough to bind at least 4 monomers of polyA binding protein.
  • PolyA binding protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.
  • the capping region may comprise a single cap or a series of nucleotides forming the cap.
  • the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length.
  • the cap is absent.
  • the first and second operational regions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a start and/or stop codon, one or more signal and/or restriction sequences.
  • a signal-sensor primary construct or mmRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5′-end binding proteins.
  • the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
  • the newly formed 5′-/3′-linkage may be intramolecular or intermolecular.
  • the 5′-end and the 3′-end of the nucleic acid may contain chemically reactive groups that, when close together, form a new covalent linkage between the 5′-end and the 3′-end of the molecule.
  • the 5′-end may contain an NHS-ester reactive group and the 3′-end may contain a 3′-amino-terminated nucleotide such that in an organic solvent the 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNA molecule will undergo a nucleophilic attack on the 5′-NHS-ester moiety forming a new 5′-/3′-amide bond.
  • T4 RNA ligase may be used to enzymatically link a 5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage.
  • 1 ⁇ g of a nucleic acid molecule is incubated at 37° C. for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol.
  • the ligation reaction may occur in the presence of a split oligonucleotide capable of base-pairing with both the 5′- and 3′-region in juxtaposition to assist the enzymatic ligation reaction.
  • either the 5′- or 3′-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5′-end of a nucleic acid molecule to the 3′-end of a nucleic acid molecule.
  • the ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • the ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37° C.
  • multiple distinct signal-sensor polynucleotides, primary constructs or mmRNA may be linked together through the 3′-end using nucleotides which are modified at the 3′-terminus.
  • Chemical conjugation may be used to control the stoichiometry of delivery into cells.
  • the glyoxylate cycle enzymes, isocitrate lyase and malate synthase may be supplied into HepG2 cells at a 1:1 ratio to alter cellular fatty acid metabolism.
  • This ratio may be controlled by chemically linking signal-sensor polynucleotides, primary constructs or mmRNA using a 3′-azido terminated nucleotide on one signal-sensor polynucleotide, primary construct or mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide on the opposite signal-sensor polynucleotide, primary construct or mmRNA species.
  • the modified nucleotide is added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol.
  • the two signal-sensor polynucleotide, primary construct or mmRNA species may be combined in an aqueous solution, in the presence or absence of copper, to form a new covalent linkage via a click chemistry mechanism as described in the literature.
  • a functionalized saccharide molecule may be chemically modified to contain multiple chemical reactive groups (SH—, NH 2 —, N 3 , etc. . . . ) to react with the cognate moiety on a 3′-functionalized signal-sensorpolynucleotide molecule (i.e., a 3′-maleimide ester, 3′-NHS-ester, alkynyl).
  • the number of reactive groups on the modified saccharide can be controlled in a stoichiometric fashion to directly control the stoichiometric ratio of conjugated signal-sensor polynucleotide, primary construct or mmRNA.
  • signal-sensor polynucleotide primary constructs or mmRNA of the present invention can be designed to be conjugated to other polynucleotides, oncology-related polypeptides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
  • intercalating agents e.g. acridines
  • cross-linkers e.g. psoralene, mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g.
  • alkylating agents phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.
  • biotin e.g., aspirin, vitamin E, folic acid
  • transport/absorption facilitators e.g., aspirin, vitamin E, folic acid
  • synthetic ribonucleases proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
  • a specified cell type such as a cancer cell, endothelial cell, or bone cell
  • hormones and hormone receptors non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
  • Conjugation may result in increased stability and/or half life and may be particularly useful in targeting the signal-sensor polynucleotides, primary constructs or mmRNA to specific sites in the cell, tissue or organism.
  • the signal-sensor polynucleotide mmRNA or primary constructs may be administered with, or further encode one or more of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.
  • RNAi agents siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.
  • the signal-sensor polynucleotides described herein may be conjugated with a moiety to target various cancer cells such as, but not limited to, the moieties described in US Patent Application No. US20130216561, the contents of which are herein incorporated by reference in its entirety.
  • the linkage between the signal-sensor polynucleotides and the cancer targeting moiety may be an acid cleavable linkage that can increase the efficacy of the conjugate such as, but not limited to, the linkages described in US Patent Application No. US20130216561, the contents of which are herein incorporated by reference in its entirety.
  • bifunctional signal-sensor polynucleotides e.g., bifunctional primary constructs or bifunctional mmRNA.
  • bifunctional signal-sensor polynucleotides are those having or capable of at least two functions. These molecules may also by convention be referred to as multi-functional.
  • bifunctional signal-sensor polynucleotides may be encoded by the RNA (the function may not manifest until the encoded product is translated) or may be a property of the polynucleotide itself. It may be structural or chemical.
  • Bifunctional modified signal-sensor polynucleotides may comprise a function that is covalently or electrostatically associated with the polynucleotides. Further, the two functions may be provided in the context of a complex of a signal-sensor polynucleotide and another molecule.
  • Bifunctional signal-sensor polynucleotides may encode oncology-related peptides which are anti-proliferative. These peptides may be linear, cyclic, constrained or random coil. They may function as aptamers, signaling molecules, ligands or mimics or mimetics thereof. Anti-proliferative peptides may, as translated, be from 3 to 50 amino acids in length. They may be 5-40, 10-30, or approximately 15 amino acids long. They may be single chain, multichain or branched and may form complexes, aggregates or any multi-unit structure once translated.
  • signal-sensor polynucleotides and primary constructs having sequences that are partially or substantially not translatable e.g., having a noncoding region.
  • Such noncoding region may be the “first region” of the signal-sensor primary construct.
  • the noncoding region may be a region other than the first region.
  • Such molecules are generally not translated, but can exert an effect on protein production by one or more of binding to and sequestering one or more translational machinery components such as a ribosomal protein or a transfer RNA (tRNA), thereby effectively reducing protein expression in the cell or modulating one or more pathways or cascades in a cell which in turn alters protein levels.
  • tRNA transfer RNA
  • the signal-sensor polynucleotide and/or primary construct may contain or encode one or more long noncoding RNA (lncRNA, or lincRNA) or portion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
  • lncRNA long noncoding RNA
  • miRNA micro RNA
  • siRNA small interfering RNA
  • piRNA Piwi-interacting RNA
  • the signal-sensor polynucleotides of the present invention may be auxotrophic.
  • auxotrophic refers to signal-sensor polynucleotides that comprise at least one feature that triggers, facilitates or induces the degradation or inactivation of the itself in response to spatial or temporal cues such that oncology-related protein expression is substantially prevented or reduced.
  • spatial or temporal cues include the location of the signal-sensor polynucleotide to be translated such as a particular tissue or organ or cellular environment. Also contemplated are cues involving temperature, pH, ionic strength, moisture content, and the like.
  • the feature is located in a terminal region of the signal-sensor polynucleotides of the present invention.
  • the auxotrophic mRNA may contain a miR binding site in the terminal region which binds to a miR expressed in a selected tissue so that the expression of the auxotrophic mRNA is substantially prevented or reduced in the selected tissue.
  • an auxotrophic mRNA containing a miR-122 binding site will not produce protein if localized to the liver since miR-122 is expressed in the liver and binding of the miR would effectuate destruction of the auxotrophic mRNA.
  • HEK293 cells do not express miR-122 so there would be little to no downregulation of a signal-sensor polynucleotide having a miR-122 sequence in HEK293 but for hepatocytes which do expression miR-122 there would be a downregulation of a signal-sensor polynucleotide having a miR-122 sequence in hepatocytes (see e.g., the study outlined Example 19).
  • the miR-122 level can be measured in HeLa cells, primary human hepatocytes and primary rat hepatocytes prior to administration with a signal-sensor polynucleotide encoding having at least one miR-122 binding site, miR-122 binding site without the seed sequence or a miR-122 binding site After administration the expression of the signal-sensor polynucleotide can be measured to determine the dampening effect of the miR-122 in the signal-sensor polynucleotide (see e.g., the studies outlined in Examples 41, 42, 43 57, 58 and 59).
  • the effectiveness of the miR-122 binding site, miR-122 seed or the miR-122 binding site without the seed in different 3′UTRs may be evaluated in order to determine the proper UTR for the desired outcome such as, but not limited to, the highest dampening effect (see e.g., the study outlined in Example 46).
  • the degradation or inactivation of auxotrophic mRNA may comprise a feature responsive to a change in pH.
  • the auxotrophic mRNA may be triggered in an environment having a pH of between pH 4.5 to 8.0 such as at a pH of 5.0 to 6.0 or a pH of 6.0 to 6.5.
  • the change in pH may be a change of 0.1 unit, 0.2 units, 0.3 units, 0.4 units, 0.5 units, 0.6 units, 0.7 units, 0.8 units, 0.9 units, 1.0 units, 1.1 units, 1.2 units, 1.3 units, 1.4 units, 1.5 units, 1.6 units, 1.7 units, 1.8 units, 1.9 units, 2.0 units, 2.1 units, 2.2 units, 2.3 units, 2.4 units, 2.5 units, 2.6 units, 2.7 units, 2.8 units, 2.9 units, 3.0 units, 3.1 units, 3.2 units, 3.3 units, 3.4 units, 3.5 units, 3.6 units, 3.7 units, 3.8 units, 3.9 units, 4.0 units or more.
  • the degradation or inactivation of auxotrophic mRNA may be triggered or induced by changes in temperature.
  • a change of temperature from room temperature to body temperature may be less than 1° C., less than 5° C., less than 10° C., less than 15° C., less than 20° C., less than 25° C. or more than 25° C.
  • the degradation or inactivation of auxotrophic mRNA may be triggered or induced by a change in the levels of ions in the subject.
  • the ions may be cations or anions such as, but not limited to, sodium ions, potassium ions, chloride ions, calcium ions, magnesium ions and/or phosphate ions.
  • the signal-sensor primary construct is designed to encode one or more oncology-related polypeptides of interest or fragments thereof.
  • An oncology-related polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned.
  • the term “oncology-related polypeptides of interest” refers to any polypeptide which is selected to be encoded in the signal-sensor primary construct of the present invention.
  • polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
  • polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides.
  • the term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
  • variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.
  • variant mimics are provided.
  • the term “variant mimic” is one which contains one or more amino acids which would mimic an activated sequence.
  • glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine.
  • variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.
  • “Homology” as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • homologs as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.
  • Analogs is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.
  • compositions which are polypeptide based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives.
  • derivative is used synonymously with the term “variant” but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.
  • sequence tags or amino acids such as one or more lysines
  • Sequence tags can be used for peptide purification or localization.
  • Lysines can be used to increase peptide solubility or to allow for biotinylation.
  • amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C-terminal or N-terminal residues
  • substitutional variants when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • conservative amino acid substitution refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine.
  • substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions.
  • non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • “Insertional variants” when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. “Immediately adjacent” to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.
  • “Deletional variants” when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
  • Covalent derivatives when referring to polypeptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
  • Certain post-translational modifications are the result of the action of recombinant host cells on the expressed oncology-related polypeptide.
  • Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the oncology-related polypeptides produced in accordance with the present invention.
  • post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).
  • Features when referring to polypeptides are defined as distinct amino acid sequence-based components of a molecule.
  • Features of the polypeptides encoded by the mmRNA of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
  • surface manifestation refers to a polypeptide based component of a protein appearing on an outermost surface.
  • local conformational shape means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
  • fold refers to the resultant conformation of an amino acid sequence upon energy minimization.
  • a fold may occur at the secondary or tertiary level of the folding process.
  • secondary level folds include beta sheets and alpha helices.
  • tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
  • turn as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
  • loop refers to a structural feature of a polypeptide which may serve to reverse the direction of the backbone of a peptide or polypeptide. Where the loop is found in a polypeptide and only alters the direction of the backbone, it may comprise four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997). Loops may be open or closed. Closed loops or “cyclic” loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties.
  • Such bridging moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in polypeptides having disulfide bridges or alternatively bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
  • Cys-Cys cysteine-cysteine bridge
  • bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
  • domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
  • sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
  • site As used herein when referring to polypeptides the terms “site” as it pertains to amino acid based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.”
  • a site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.
  • terminal refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions.
  • the polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)).
  • Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini.
  • the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
  • any of the features have been identified or defined as a desired component of a polypeptide to be encoded by the signal-sensor primary construct or mmRNA of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.
  • Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis.
  • the resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
  • the oncology-related polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation.
  • a “consensus” sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites.
  • protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of oncology-related polypeptides of interest of this invention.
  • any protein fragment meaning an oncology-related polypeptide sequence at least one amino acid residue shorter than a reference oncology-related polypeptide sequence but otherwise identical
  • a reference oncology-related protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length.
  • any oncology-related protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the invention.
  • a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
  • the signal-sensor primary constructs or mmRNA of the present invention may be designed to encode oncology-related polypeptides of interest such as oncology-related peptides and proteins.
  • signal-sensor primary constructs or mmRNA of the present invention may encode variant polypeptides which have a certain identity with a reference oncology-related polypeptide sequence.
  • a “reference oncology-related polypeptide sequence” refers to a starting oncology-related polypeptide sequence. Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence.
  • a “reference polypeptide sequence” may, e.g., be any one of the protein sequence listed in Table 6.
  • identity refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.
  • the polypeptide variant may have the same or a similar activity as the reference oncology-related polypeptide.
  • the variant may have an altered activity (e.g., increased or decreased) relative to a reference oncology-related polypeptide.
  • variants of a particular signal-sensor polynucleotide or oncology-related polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference signal-sensor polynucleotide or oncology-related polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.)
  • Other tools are described herein, specifically in the definition of “identity.”
  • BLAST algorithm Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match/Mismatch Scores 1, ⁇ 2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be used to treat a disease, disorder and/or condition in a subject.
  • the polynucleotides, primary constructs and/or mmRNA may be used to reduce, eliminate or prevent tumor growth in a subject.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be used to reduce and/or ameliorate at least one symptom of cancer in a subject.
  • a symptom of cancer may include, but is not limited to, weakness, aches and pains, fever, fatigue, weight loss, blood clots, increased blood calcium levels, low white blood cell count, short of breath, dizziness, headaches, hyperpigmentation, jaundice, erthema, pruritis, excessive hair growth, change in bowel habits, change in bladder function, long-lasting sores, white patches inside the mouth, white spots on the tongue, unusual bleeding or discharge, thickening or lump on parts of the body, indigestion, trouble swallowing, changes in warts or moles, change in new skin and nagging cough or hoarseness.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may reduce a side-effect associated with cancer such as, but not limited to, chemo brain, peripheral neuropathy, fatigue, depression, nausea, vomiting, pain, anemia, lymphedema, infections, sexual side effects, reduced fertility or infertility, ostomics, insomnia and hair loss.
  • a side-effect associated with cancer such as, but not limited to, chemo brain, peripheral neuropathy, fatigue, depression, nausea, vomiting, pain, anemia, lymphedema, infections, sexual side effects, reduced fertility or infertility, ostomics, insomnia and hair loss.
  • the signal-sensor primary constructs or mmRNA disclosed herein may encode one or more validated or “in testing” oncology-related proteins or oncology-related peptides.
  • one or more oncology-related proteins or oncology-related peptides currently being marketed or in development may be encoded by the oncology-related signal-sensor polynucleotide, primary constructs or mmRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation into the signal-sensor primary constructs or mmRNA of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may alter a biological and/or physiological process and/or compound such as, but not limited to, the cell cycle, the DNA damage response (e.g., DNA damage repair), apoptosis, angiogenesis, cell motility, the epithelial to mesenchymal transition in epithelial cells, the phosphatidyl inositol 3 (PI3) kinase/Akt cellular signaling pathway, telomerase activity and/or expression, tumor metastasis, tumorigenesis, cathepsins, cell senescence, receptor tyrosine kinase signaling, metabolism and drug metabolism, G protein signaling, growth factors and receptors, heat shock proteins, histone deacetylases, hormone receptors, hypoxia, poly ADP-ribose polymerases, protein kinases, RAS signaling, topisomerases, transcription factors and tumor suppressor activity in cancerous, precancerous and
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be used to express a polypeptide in cells or tissues for the purpose of replacing the protein produced from a deleted or mutated gene.
  • polynucleotides, primary constructs or mmRNA of the invention may be used to treat cancer which has been caused by carcinogens of natural and/or synthetic origin.
  • use of the polynucleotides, primary constructs and/or mmRNA may be used to treat cancer caused by other organisms and/or cancers caused by viral infection.
  • Untranslated Regions UTRs
  • Untranslated regions (UTRs) of a gene are transcribed but not translated.
  • the 5′UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3′UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory features of a UTR can be incorporated into the signal-sensor polynucleotides, primary constructs and/or mmRNA of the present invention to enhance the stability of the molecule.
  • the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • the untranslated regions may be incorporated into a vector system which can produce mRNA and/or be delivered to a cell, tissue and/or organism to produce a polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA of the present may comprise at least one terminal modification.
  • terminal modifications are described in U.S. Provisional Patent Application No. 61/729,933, filed Nov. 26, 2012, entitled Terminally Optimized Modified RNAs, U.S. Provisional Patent application No. 61/737,224, filed Dec. 14, 2012, entitled Terminally Optimized RNAs, U.S. Provisional Patent Application No. 61/758,921, filed Jan. 31, 2013, entitled Differential Targeting Using RNA Constructs, U.S. Provisional Patent Application No. 61/781,139, filed Mar. 14, 2013, entitled Differential Targeting Using RNA Constructs, U.S.
  • Provisional Patent Application No. 61/829,359 filed May 31, 2013, entitled Differential Targeting Using RNA Constructs
  • U.S. Provisional Patent Application No. 61/842,709 filed Jul. 3, 2013, entitled Differential Targeting Using RNA Constructs
  • These terminal modifications include, but are not limited to, 5′caps, microRNA binding sites in the terminal region, chain terminating nucleosides, translation enhancer elements in the terminal region and tailing sequences including a polyA-G quartet and stem loop sequences.
  • Natural 5′UTRs bear features which play roles in for translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5′UTR also have been known to form secondary structures which are involved in elongation factor binding. For example, one of the secondary 5′-UTR structures is the structured IRES for eIF4A2 elongation factor binding, which is necessary for the microRNA mediated gene repression at 3′-UTR.
  • 5′UTR secondary structures involved in elongation factor binding can interact with other RNA binding molecules in the 5′UTR or 3′UTR to regulate gene expression.
  • the elongation factor EIF4A2 binding to a secondarily structured element in the 5′UTR is necessary for microRNA mediated repression (Meijer H A et al., Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
  • the different secondary structures in the 5′UTR can be incorporated into the flanking region to either stabilize or selectively destalized mRNAs in specific tissues or cells.
  • mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII
  • tissue-specific mRNA for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D).
  • non-UTR sequences may be incorporated into the 5′ (or 3′ UTR) UTRs.
  • introns or portions of introns sequences may be incorporated into the flanking regions of the signal-sensor polynucleotides, primary constructs or mmRNA of the invention. Incorporation of intronic sequences may increase protein production as well as mRNA levels.
  • TAEs Translation Enhancer Elements
  • the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA may include at least one translational enhancer polynucleotide, translation enhancer element, translational enhancer elements (collectively referred to as “TEE”s).
  • TEE translational enhancer polynucleotide, translation enhancer element, translational enhancer elements
  • the TEE may be located between the transcription promoter and the start codon.
  • the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA with at least one TEE in the 5′UTR may include a cap at the 5′UTR.
  • at least one TEE may be located in the 5′UTR of signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA undergoing cap-dependent or cap-independent translation.
  • translational enhancer element or “translation enhancer element” (herein collectively referred to as “TEE”) refers to sequences that increase the amount of polypeptide or protein produced from an mRNA.
  • TEEs are conserved elements in the UTR which can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.
  • a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.
  • the TEE may be any of the TEEs listed in Table 35 in Example 45, including portion and/or fragments thereof.
  • the TEE sequence may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more than 99% of the TEE sequences disclosed in Table 35 and/or the TEE sequence may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in Table 35.
  • the TEEs known may be in the 5′-leader of the Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004, herein incorporated by reference in their entirety).
  • TEEs are disclosed as SEQ ID NOs: 1-35 in US Patent Publication No. US20090226470, SEQ ID NOs: 1-35 in US Patent Publication US20130177581, SEQ ID NOs: 1-35 in International Patent Publication No. WO2009075886, SEQ ID NOs: 1-5, and 7-645 in International Patent Publication No. WO2012009644, SEQ ID NO: 1 in International Patent Publication No. WO1999024595, SEQ ID NO: 1 in U.S. Pat. No. 6,310,197, and SEQ ID NO: 1 in U.S. Pat. No. 6,849,405, each of which is herein incorporated by reference in its entirety.
  • the TEE may be an internal ribosome entry site (IRES), HCV-IRES or an IRES element such as, but not limited to, those described in U.S. Pat. No. 7,468,275, US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055369, each of which is herein incorporated by reference in its entirety.
  • the IRES elements may include, but are not limited to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci.
  • Translational enhancer polynucleotides or “translation enhancer polynucleotide sequences” are polynucleotides which include one or more of the specific TEE exemplified herein and/or disclosed in the art (see e.g., U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No.
  • TEE transglutaminase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinasulfene, WO2001055371 WO1999024595, and EP2610341A1 and EP2610340A1; each of which is herein incorporated by reference in its entirety) or their variants, homologs or functional derivatives.
  • One or multiple copies of a specific TEE can be present in the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA.
  • the TEEs in the translational enhancer polynucleotides can be organized in one or more sequence segments.
  • a sequence segment can harbor one or more of the specific TEEs exemplified herein, with each TEE being present in one or more copies.
  • multiple sequence segments can be homogenous or heterogeneous.
  • the multiple sequence segments in a translational enhancer polynucleotide can harbor identical or different types of the specific TEEs exemplified herein, identical or different number of copies of each of the specific TEEs, and/or identical or different organization of the TEEs within each sequence segment.
  • the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA may include at least one TEE that is described in International Patent Publication No. WO1999024595, WO2012009644, WO2009075886, WO2007025008, WO1999024595, European Patent Publication No. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395, US Patent Publication No.
  • the TEE may be located in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA.
  • the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA may include at least one TEE that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity with the TEEs described in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication No. WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European Patent Publication No. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395, each of which is herein incorporated by reference in its entirety.
  • the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences.
  • the TEE sequences in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may be the same or different TEE sequences.
  • the TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level.
  • the 5′UTR may include a spacer to separate two TEE sequences.
  • the spacer may be a 15 nucleotide spacer and/or other spacers known in the art.
  • the 5′UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times or more than 9 times in the 5′UTR.
  • the spacer separating two TEE sequences may include other sequences known in the art which may regulate the translation of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention such as, but not limited to, miR sequences described herein (e.g., miR binding sites and miR seeds).
  • miR sequences described herein e.g., miR binding sites and miR seeds.
  • each spacer used to separate two TEE sequences may include a different miR sequence or component of a miR sequence (e.g., miR seed sequence).
  • the TEE in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more than 99% of the TEE sequences disclosed in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication No.
  • the TEE in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication No. WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European Patent Publication No.
  • EP2610341A1 and EP2610340A1 U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395; each of which are herein incorporated by reference in their entirety.
  • the TEE in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more than 99% of the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al.
  • the TEE in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al.
  • the TEE used in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention is an IRES sequence such as, but not limited to, those described in U.S. Pat. No. 7,468,275 and International Patent Publication No. WO2001055369, each of which is herein incorporated by reference in its entirety.
  • the TEEs used in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may be identified by the methods described in US Patent Publication No. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2012009644, each of which is herein incorporated by reference in its entirety.
  • the TEEs used in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may be a transcription regulatory element described in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is herein incorporated by reference in their entirety.
  • the transcription regulatory elements may be identified by methods known in the art, such as, but not limited to, the methods described in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is herein incorporated by reference in their entirety.
  • the TEE used in the 5′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention is an oligonucleotide or portion thereof as described in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is herein incorporated by reference in their entirety.
  • the 5′ UTR comprising at least one TEE described herein may be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid vector.
  • a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid vector.
  • the vector systems and nucleic acid vectors may include those described in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, US Patent Publication No. US20070048776, US20090093049 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055371, each of which is herein incorporated by reference in its entirety.
  • the TEEs described herein may be located in the 5′UTR and/or the 3′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA.
  • the TEEs located in the 3′UTR may be the same and/or different than the TEEs located in and/or described for incorporation in the 5′UTR.
  • the 3′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences.
  • the TEE sequences in the 3′UTR of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention may be the same or different TEE sequences.
  • the TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level.
  • the 3′UTR may include a spacer to separate two TEE sequences.
  • the spacer may be a 15 nucleotide spacer and/or other spacers known in the art.
  • the 3′UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times or more than 9 times in the 3′UTR.
  • the spacer separating two TEE sequences may include other sequences known in the art which may regulate the translation of the signal-sensor polynucleotides, primary constructs, modified nucleic acids and/or mmRNA of the present invention such as, but not limited to, miR sequences described herein (e.g., miR binding sites and miR seeds).
  • miR sequences described herein e.g., miR binding sites and miR seeds.
  • each spacer used to separate two TEE sequences may include a different miR sequence or component of a miR sequence (e.g., miR seed sequence).
  • the incorporation of a miR sequence and/or a TEE sequence changes the shape of the stem loop region which may increase and/or decrease translation.
  • a miR sequence and/or a TEE sequence changes the shape of the stem loop region which may increase and/or decrease translation.
  • the 5′UTR may comprise at least one microRNA sequence.
  • the microRNA sequence may be, but is not limited to, a 19 or 22 nucleotide sequence and/or a microRNA sequence without the seed.
  • microRNA sequence in the 5′UTR may be used to stabilize the nucleic acid and/or mRNA described herein.
  • a microRNA sequence in the 5′UTR may be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon.
  • Matsuda et al (PLoS One. 2010 11(5):e15057; herein incorporated by reference in its entirety) used antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) around a start codon ( ⁇ 4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the first start codon (AUG).
  • LNA antisense locked nucleic acid
  • EJCs exon-junction complexes
  • the signal-sensor polynucleotides of the present invention may comprise a microRNA sequence, instead of the LNA or EJC sequence described by Matsuda et al, near the site of translation initiation in order to decrease the accessibility to the site of translation initiation.
  • the site of translation initiation may be prior to, after or within the microRNA sequence.
  • the site of translation initiation may be located within a microRNA sequence such as a seed sequence or binding site.
  • the site of translation initiation may be located within a miR-122 sequence such as the seed sequence or the mir-122 binding site.
  • the nucleic acids or mRNA of the present invention comprises at least one microRNA sequence in a region of the nucleic acid or mRNA which may interact with a RNA binding protein.
  • RNA Motifs for RNA Binding Proteins (RBPs)
  • RNA binding proteins can regulate numerous aspects of co- and post-transcription gene expression such as, but not limited to, RNA splicing, localization, translation, turnover, polyadenylation, capping, modification, export and localization.
  • RNA-binding domains such as, but not limited to, RNA recognition motif (RR) and hnRNP K-homology (KH) domains, typically regulate the sequence association between RBPs and their RNA targets (Ray et al. Nature 2013. 499:172-177; herein incorporated by reference in its entirety).
  • the canonical RBDs can bind short RNA sequences.
  • the canonical RBDs can recognize structure RNAs.
  • the nucleic acids and/or mRNA may comprise at least one RNA-binding motif such as, but not limited to a RNA-binding domain (RBD).
  • RBD RNA-binding domain
  • the RBD may be any of the RBDs, fragments or variants thereof descried by Ray et al. (Nature 2013. 499:172-177; herein incorporated by reference in its entirety).
  • the nucleic acids or mRNA of the present invention may comprise a sequence for at least one RNA-binding domain (RBDs).
  • RBDs RNA-binding domains
  • At least one flanking region may comprise at least one RBD.
  • the first flanking region and the second flanking region may both comprise at least one RBD.
  • the RBD may be the same or each of the RBDs may have at least 60% sequence identity to the other RBD.
  • at least on RBD may be located before, after and/or within the 3′UTR of the nucleic acid or mRNA of the present invention.
  • at least one RBD may be located before or within the first 300 nucleosides of the 3′UTR.
  • the nucleic acids and/or mRNA of the present invention may comprise at least one RBD in the first region of linked nucleosides.
  • the RBD may be located before, after or within a coding region (e.g., the ORF).
  • the first region of linked nucleosides and/or at least one flanking region may comprise at least on RBD.
  • the first region of linked nucleosides may comprise a RBD related to splicing factors and at least one flanking region may comprise a RBD for stability and/or translation factors.
  • the nucleic acids and/or mRNA of the present invention may comprise at least one RBD located in a coding and/or non-coding region of the nucleic acids and/or mRNA.
  • At least one RBD may be incorporated into at least one flanking region to increase the stability of the nucleic acid and/or mRNA of the present invention.
  • a microRNA sequence in a RNA binding protein motif may be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon.
  • the signal-sensor polynucleotides of the present invention may comprise a microRNA sequence, instead of the LNA or EJC sequence described by Matsuda et al, near the site of translation initiation in order to decrease the accessibility to the site of translation initiation.
  • the site of translation initiation may be prior to, after or within the microRNA sequence.
  • the site of translation initiation may be located within a microRNA sequence such as a seed sequence or binding site.
  • the site of translation initiation may be located within a miR-122 sequence such as the seed sequence or the mir-122 binding site.
  • an antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) may be used in the RNA binding protein motif.
  • the LNA and EJCs may be used around a start codon ( ⁇ 4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the first start codon (AUG).
  • 3′UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined.
  • AREs 3′ UTR AU rich elements
  • Introduction, removal or modification of 3′ UTR AU rich elements can be used to modulate the stability of signal-sensor polynucleotides, primary constructs or mmRNA of the invention.
  • AREs 3′ UTR AU rich elements
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
  • Transfection experiments can be conducted in relevant cell lines, using signal-sensor polynucleotides, primary constructs or mmRNA of the invention and protein production can be assayed at various time points post-transfection.
  • cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and 7 days post-transfection.
  • signal-sequence polynucleotides of the present invention may include a triple helix on the 3′ end of the signal-sequence polynucleotides.
  • the 3′ end of the nucleic acids of the present invention may include a triple helix alone or in combination with a Poly-A tail.
  • the signal-sequence polynucleotides of the present invention may comprise at least a first and a second U-rich region, a conserved stem loop region between the first and second region and an A-rich region.
  • the first and second U-rich region and the A-rich region may associate to form a triple helix on the 3′ end of the nucleic acid. This triple helix may stabilize the nucleic acid, enhance the translational efficiency of the nucleic acid and/or protect the 3′ end from degradation.
  • triple helices include, but are not limited to, the triple helix sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), MEN- ⁇ and polyadenylated nuclear (PAN) RNA (See Wilusz et al., Genes & Development 2012 26:2392-2407; herein incorporated by reference in its entirety).
  • MALAT1 metastasis-associated lung adenocarcinoma transcript 1
  • MEN- ⁇ and polyadenylated nuclear (PAN) RNA
  • the 3′ end of the modified nucleic acids, enhanced modified RNA or ribonucleic acids of the present invention comprises a first U-rich region comprising TTTTTCTTTT (SEQ ID NO: 1), a second U-rich region comprising TTTTGCTTTTT (SEQ ID NO: 2) or TTTTGCTTTT (SEQ ID NO: 3), an A-rich region comprising AAAAAGCAAAA (SEQ ID NO: 4).
  • the 3′ end of the nucleic acids of the present invention comprises a triple helix formation structure comprising a first U-rich region, a conserved region, a second U-rich region and an A-rich region.
  • the triple helix may be formed from the cleavage of a MALAT1 sequence prior to the cloverleaf structure.
  • MALAT1 is a long non-coding RNA which, when cleaved, forms a triple helix and a tRNA-like cloverleaf structure.
  • the MALAT1 transcript then localizes to nuclear speckles and the tRNA-like cloverleaf localizes to the cytoplasm (Wilusz et al. Cell 2008 135(5): 919-932; herein incorporated by reference in its entirety).
  • the terminal end of the nucleic acid of the present invention comprising the MALAT1 sequence can then form a triple helix structure, after RNaseP cleavage from the cloverleaf structure, which stabilizes the nucleic acid (Peart et al. Non - mRNA 3 ′ end formation: how the other half lives ; WIREs RNA 2013; herein incorporated by reference in its entirety).
  • the signal-sequence polynucleotides described herein comprise a MALAT1 sequence.
  • the signal-sequence polynucleotides may be polyadenylated.
  • the signal-sequence polynucleotides is not polyadenylated but has an increased resistance to degradation compared to unmodified nucleic acids or mRNA.
  • the signal-sequence polynucleotides of the present invention may comprise a MALAT1 sequence in the second flanking region (e.g., the 3′UTR).
  • the MALAT1 sequence may be human or mouse.
  • the cloverleaf structure of the MALAT1 sequence may also undergo processing by RNaseZ and CCA adding enzyme to form a tRNA-like structure called mascRNA (MALAT1-associated small cytoplasmic RNA).
  • mascRNA MALAT1-associated small cytoplasmic RNA
  • the mascRNA may encode a protein or a fragment thereof and/or may comprise a microRNA sequence.
  • the mascRNA may comprise at least one chemical modification described herein.
  • the nucleic acids of the present invention may include a stem loop such as, but not limited to, a histone stem loop.
  • the stem loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length such as, but not limited to, SEQ ID NOs: 7-17 as described in International Patent Publication No. WO2013103659, herein incorporated by reference in its entirety.
  • the histone stem loop may be located 3′ relative to the coding region (e.g., at the 3′ terminus of the coding region). As a non-limiting example, the stem loop may be located at the 3′ end of a nucleic acid described herein.
  • the stem loop may be located in the second terminal region.
  • the stem loop may be located within an untranslated region (e.g., 3′UTR) in the second terminal region.
  • the nucleic acid such as, but not limited to mRNA, which comprises the histone stem loop may be stabilized by the addition of at least one chain terminating nucleoside.
  • the addition of at least one chain terminating nucleoside may slow the degradation of a nucleic acid and thus can increase the half-life of the nucleic acid.
  • the chain terminating nucleoside may be, but is not limited to, those described in International Patent Publication No. WO2013103659, herein incorporated by reference in its entirety.
  • the chain terminating nucleosides which may be used with the present invention includes, but is not limited to, 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine, 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, or a —O-methylnucleoside.
  • the nucleic acid such as, but not limited to mRNA, which comprises the histone stem loop may be stabilized by a modification to the 3′region of the nucleic acid that can prevent and/or inhibit the addition of oligio(U) (see e.g., International Patent Publication No. WO2013103659, herein incorporated by reference in its entirety).
  • the nucleic acid such as, but not limited to mRNA, which comprises the histone stem loop may be stabilized by the addition of an oligonucleotide that terminates in a 3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′′-0-methylnucleosides, 3′-0-ethylnucleosides, 3′-arabinosides, and other modified nucleosides known in the art and/or described herein.
  • an oligonucleotide that terminates in a 3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′′-0-methylnucleosides, 3′-0-ethylnucleosides, 3′-arabinosides, and other modified nucleosides known in the art and/or described herein.
  • the nucleic acids of the present invention may include a histone stem loop, a polyA tail sequence and/or a 5′cap structure.
  • the histone stem loop may be before and/or after the polyA tail sequence.
  • the nucleic acids comprising the histone stem loop and a polyA tail sequence may include a chain terminating nucleoside described herein.
  • the nucleic acids of the present invention may include a histone stem loop and a 5′cap structure.
  • the 5′cap structure may include, but is not limited to, those described herein and/or known in the art.
  • the conserved stem loop region may comprise a miR sequence described herein.
  • the stem loop region may comprise the seed sequence of a miR sequence described herein.
  • the stem loop region may comprise a miR-122 seed sequence.
  • the conserved stem loop region may comprise a miR sequence described herein and may also include a TEE sequence.
  • the incorporation of a miR sequence and/or a TEE sequence changes the shape of the stem loop region which may increase and/or decrease translation.
  • a miR sequence and/or a TEE sequence changes the shape of the stem loop region which may increase and/or decrease translation.
  • the 5′ cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5′ proximal introns removal during mRNA splicing.
  • Endogenous mRNA molecules may be 5′-end capped generating a 5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA molecule.
  • This 5′-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5′ end of the mRNA may optionally also be 2′-O-methylated.
  • 5′-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • Modifications to the signal-sensor polynucleotides, primary constructs, and mmRNA of the present invention may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with ⁇ -thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap. Additional modified guanosine nucleotides may be used such as ⁇ -methyl-phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the mRNA (as mentioned above) on the 2′-hydroxyl group of the sugar ring.
  • Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as an mRNA molecule.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/linked to a nucleic acid molecule.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5′-5′-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m 7 G-3′mppp-G; which may equivalently be designated 3′ 0-Me-m7G(5′)ppp(5′)G).
  • the 3′-O atom of the other, unmodified, guanine becomes linked to the 5′-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).
  • the N7- and 3′-O-methylated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA or mmRNA).
  • mCAP is similar to ARCA but has a 2′-O-methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m 7 Gm-ppp-G).
  • cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5′-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
  • Signal-sensor polynucleotides, primary constructs and mmRNA of the invention may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5′-cap structures.
  • the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
  • Non-limiting examples of more authentic 5′cap structures of the present invention are those which, among other things, have enhanced binding of cap binding proteins, increased half life, reduced susceptibility to 5′ endonucleases and/or reduced 5′decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural or physiological 5′cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl.
  • Cap1 structure is termed the Cap1 structure.
  • Cap structures include 7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and 7mG(5′)-ppp(5′)NlmpN2mp (cap 2).
  • the signal-sensor polynucleotides, primary constructs or mmRNA may be capped post-transcriptionally, and because this process is more efficient, nearly 100% of the signal-sensor polynucleotides, primary constructs or mmRNA may be capped. This is in contrast to ⁇ 80% when a cap analog is linked to an mRNA in the course of an in vitro transcription reaction.
  • 5′ terminal caps may include endogenous caps or cap analogs.
  • a 5′ terminal cap may comprise a guanine analog.
  • Useful guanine analogs include inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • Additional viral sequences such as, but not limited to, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV) can be engineered and inserted in the 3′ UTR of the signal-sensor polynucleotides, primary constructs or mmRNA of the invention and can stimulate the translation of the construct in vitro and in vivo.
  • Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection.
  • signal-sensor polynucleotides, primary constructs or mmRNA which may contain an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
  • signal-sensor polynucleotides, primary constructs or mmRNA containing more than one functional ribosome binding site may encode several oncology-related peptides or oncology-related polypeptides that are translated independently by the ribosomes (“multicistronic nucleic acid molecules”).
  • IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
  • picornaviruses e.g. FMDV
  • CFFV pest viruses
  • PV polio viruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot-and-mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV murine leukemia virus
  • SIV simian immune deficiency viruses
  • CrPV cricket paralysis viruses
  • a long chain of adenine nucleotides may be added to a polynucleotide such as an mRNA molecule in order to increase stability.
  • a polynucleotide such as an mRNA molecule
  • the 3′ end of the transcript may be cleaved to free a 3′ hydroxyl.
  • poly-A polymerase adds a chain of adenine nucleotides to the RNA.
  • the process called polyadenylation, adds a poly-A tail that can be between 100 and 250 residues long.
  • the length of a poly-A tail of the present invention is greater than 30 nucleotides in length.
  • the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).
  • the signal-sensor polynucleotides, primary construct, or mmRNA includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to
  • the poly-A tail is designed relative to the length of the overall signal-sensor polynucleotides, primary constructs or mmRNA. This design may be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions), or based on the length of the ultimate product expressed from the polynucleotides, primary constructs or mmRNA.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the signal-sensor polynucleotides, primary constructs or mmRNA or feature thereof.
  • the poly-A tail may also be designed as a fraction of polynucleotides, primary constructs or mmRNA to which it belongs.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail.
  • engineered binding sites and/or conjugation of signal-sensor polynucleotides, primary constructs or mmRNA for Poly-A binding protein may be used to enhance expression.
  • the engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the nucleic acids and/or mRNA.
  • the nucleic acids and/or mRNA may comprise at least one engineered binding site to alter the binding affinity of Poly-A binding protein (PABP) and analogs thereof.
  • PABP Poly-A binding protein
  • the incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof.
  • multiple distinct signal-sensor polynucleotides, primary constructs or mmRNA may be linked together to the PABP (Poly-A binding protein) through the 3′-end using modified nucleotides at the 3′-terminus of the poly-A tail.
  • Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection.
  • the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site.
  • the signal-sensor polynucleotides and primary constructs of the present invention are designed to include a polyA-G quartet.
  • the G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail.
  • the resultant mmRNA construct is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.
  • the nucleic acids or mRNA of the present invention may comprise a polyA tail and may be stabilized by the addition of a chain terminating nucleoside.
  • the nucleic acids and/or mRNA with a polyA tail may further comprise a 5′ cap structure.
  • the nucleic acids or mRNA of the present invention may comprise a polyA-G quartet.
  • the nucleic acids and/or mRNA with a polyA-G quartet may further comprise a 5′ cap structure.
  • the chain terminating nucleoside which may be used to stabilize the nucleic acid or mRNA comprising a polyA tail or polyA-G quartet may be, but is not limited to, those described in International Patent Publication No. WO2013103659, herein incorporated by reference in its entirety.
  • the chain terminating nucleosides which may be used with the present invention includes, but is not limited to, 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine, 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, or a —O-methylnucleoside.
  • 3′-deoxyadenosine cordycepin
  • 3′-deoxyuridine 3′-deoxycytosine
  • 3′-deoxyguanosine 3′-deoxythymine
  • the nucleic acid such as, but not limited to mRNA, which comprise a polyA tail or a polyA-G quartet may be stabilized by a modification to the 3′region of the nucleic acid that can prevent and/or inhibit the addition of oligio(U) (see e.g., International Patent Publication No. WO2013103659, herein incorporated by reference in its entirety).
  • the nucleic acid such as, but not limited to mRNA, which comprise a polyA tail or a polyA-G quartet may be stabilized by the addition of an oligonucleotide that terminates in a 3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′-0-methylnucleosides, 3′-0-ethylnucleosides, 3′-arabinosides, and other modified nucleosides known in the art and/or described herein.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be quantified in exosomes derived from one or more bodily fluid.
  • bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid
  • exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
  • the level or concentration of signal-sensor polynucleotides, primary construct or mmRNA may be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker.
  • the assay may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
  • Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • Signal-sensor polynucleotides, primary constructs or mmRNA for use in accordance with the invention may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT) or enzymatic or chemical cleavage of a longer precursor, etc.
  • IVT in vitro transcription
  • Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach , Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P.
  • the process of design and synthesis of the signal-sensor primary constructs of the invention generally includes the steps of gene construction, mRNA production (either with or without modifications) and purification.
  • a target signal-sensor polynucleotide sequence encoding the oncology-related polypeptide of interest is first selected for incorporation into a vector which will be amplified to produce a cDNA template.
  • the target signal-sensor polynucleotide sequence and/or any flanking sequences may be codon optimized.
  • the cDNA template is then used to produce mRNA through in vitro transcription (IVT). After production, the mRNA may undergo purification and clean-up processes. The steps of which are provided in more detail below.
  • the step of gene construction may include, but is not limited to gene synthesis, vector amplification, plasmid purification, plasmid linearization and clean-up, and cDNA template synthesis and clean-up.
  • a signal-sensor primary construct is designed.
  • a first region of linked nucleosides encoding the polypeptide of interest may be constructed using an open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript.
  • the ORF may comprise the wild type ORF, an isoform, variant or a fragment thereof.
  • an “open reading frame” or “ORF” is meant to refer to a nucleic acid sequence (DNA or RNA) which is capable of encoding an oncology-related polypeptide of interest. ORFs often begin with the start codon, ATG and end with a nonsense or termination codon or signal.
  • nucleotide sequence of the first region may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g.
  • Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies) and/or DNA2.0 (Menlo Park Calif.).
  • the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 1.
  • nucleotide sequence after a nucleotide sequence has been codon optimized it may be further evaluated for regions containing restriction sites. At least one nucleotide within the restriction site regions may be replaced with another nucleotide in order to remove the restriction site from the sequence but the replacement of nucleotides does alter the amino acid sequence which is encoded by the codon optimized nucleotide sequence.
  • flanking regions may be incorporated into the signal-sensor primary construct before and/or after optimization of the ORF. It is not required that a signal-sensor primary construct contain both a 5′ and 3′ flanking region. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags and may include multiple cloning sites which may have XbaI recognition.
  • a 5′ UTR and/or a 3′ UTR may be provided as flanking regions. Multiple 5′ or 3′ UTRs may be included in the flanking regions and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical modifications, before and/or after codon optimization. Combinations of features may be included in the first and second flanking regions and may be contained within other features.
  • the ORF may be flanked by a 5′ UTR which may contain a strong Kozak translational initiation signal and/or a 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • Tables 2 and 3 provide a listing of exemplary UTRs which may be utilized in the signal-sensor primary construct of the present invention as flanking regions. Shown in Table 2 is a representative listing of a 5′-untranslated region of the invention. Variants of 5′ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.
  • Table 3 Shown in Table 3 is a representative listing of 3′-untranslated regions of the invention. Variants of 3′ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.
  • any UTR from any gene may be incorporated into the respective first or second flanking region of the primary construct.
  • multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5′ UTRs or 3′ UTRs.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3′ or 5′ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.
  • a double, triple or quadruple UTR such as a 5′ or 3′ UTR may be used.
  • a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.
  • a double beta-globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.
  • patterned UTRs are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.
  • flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property.
  • oncology-related polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new chimeric primary transcript.
  • a “family of proteins” is used in the broadest sense to refer to a group of two or more oncology-related polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
  • the signal-sensor primary construct components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • the optimized construct may be reconstituted and transformed into chemically competent E. coli , yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein.
  • the signal-sensor primary constructs of the present invention may include at least two stop codons before the 3′ untranslated region (UTR).
  • the stop codon may be selected from TGA, TAA and TAG.
  • the signal-sensor primary constructs of the present invention include the stop codon TGA and one additional stop codon.
  • the addition stop codon may be TAA.
  • the vector containing the signal-sensor primary construct is then amplified and the plasmid isolated and purified using methods known in the art such as, but not limited to, a maxi prep using the Invitrogen PURELINKTM HiPure Maxiprep Kit (Carlsbad, Calif.).
  • the plasmid may then be linearized using methods known in the art such as, but not limited to, the use of restriction enzymes and buffers.
  • the linearization reaction may be purified using methods including, for example Invitrogen's PURELINKTM PCR Micro Kit (Carlsbad, Calif.), and HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen's standard PURELINKTM PCR Kit (Carlsbad, Calif.).
  • the purification method may be modified depending on the size of the linearization reaction which was conducted.
  • the linearized plasmid is then used to generate cDNA for in vitro transcription (IVT) reactions.
  • a cDNA template may be synthesized by having a linearized plasmid undergo polymerase chain reaction (PCR).
  • Table 4 is a listing of primers and probes that may be useful in the PCR reactions of the present invention. It should be understood that the listing is not exhaustive and that primer-probe design for any amplification is within the skill of those in the art.
  • Probes may also contain chemically modified bases to increase base-pairing fidelity to the target molecule and base-pairing strength. Such modifications may include 5-methyl-Cytidine, 2,6-di-amino-purine, 2′-fluoro, phosphoro-thioate, or locked nucleic acids.
  • the cDNA may be submitted for sequencing analysis before undergoing transcription.
  • the process of signal-sensor polynucleotide production may include, but is not limited to, in vitro transcription, cDNA template removal and RNA clean-up, and capping and/or tailing reactions.
  • the cDNA produced in the previous step may be transcribed using an in vitro transcription (IVT) system.
  • the system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
  • NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein.
  • the NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.
  • the polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to be incorporated into modified nucleic acids.
  • RNA polymerases or variants may be used in the design of the signal-sensor primary constructs of the present invention.
  • RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence.
  • the RNA polymerase may be modified to exhibit an increased ability to incorporate a 2′-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication WO2008078180 and U.S. Pat. No. 8,101,385; herein incorporated by reference in their entireties).
  • Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art.
  • T7 RNA polymerase variants may be evolved using the continuous directed evolution system set out by Esvelt et al.
  • T7 RNA polymerase may encode at least one mutation such as, but not limited to, lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H5
  • T7 RNA polymerase variants may encode at least mutation as described in U.S. Pub. Nos. 20100120024 and 20070117112; herein incorporated by reference in their entireties.
  • Variants of RNA polymerase may also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives.
  • the signal-sensor primary construct may be designed to be recognized by the wild type or variant RNA polymerases. In doing so, the signal-sensor primary construct may be modified to contain sites or regions of sequence changes from the wild type or parent primary construct.
  • the signal-sensor primary construct may be designed to include at least one substitution and/or insertion upstream of an RNA polymerase binding or recognition site, downstream of the RNA polymerase binding or recognition site, upstream of the TATA box sequence, downstream of the TATA box sequence of the signal-sensor primary construct but upstream of the coding region of the primary construct, within the 5′UTR, before the 5′UTR and/or after the 5′UTR.
  • the 5′UTR of the signal-sensor primary construct may be replaced by the insertion of at least one region and/or string of nucleotides of the same base.
  • the region and/or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 nucleotides and the nucleotides may be natural and/or unnatural.
  • the group of nucleotides may include 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein and/or combinations thereof.
  • the 5′UTR of the signal-sensor primary construct may be replaced by the insertion of at least two regions and/or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein and/or combinations thereof.
  • the 5′UTR may be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases.
  • the 5′UTR may be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.
  • the signal-sensor primary construct may include at least one substitution and/or insertion downstream of the transcription start site which may be recognized by an RNA polymerase.
  • at least one substitution and/or insertion may occur downstream the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6). Changes to region of nucleotides just downstream of the transcription start site may affect initiation rates, increase apparent nucleotide triphosphate (NTP) reaction constant values, and increase the dissociation of short transcripts from the transcription complex curing initial transcription (Brieba et al, Biochemistry (2002) 41: 5144-5149; herein incorporated by reference in its entirety).
  • the modification, substitution and/or insertion of at least one nucleic acid may cause a silent mutation of the nucleic acid sequence or may cause a mutation in the amino acid sequence.
  • the signal-sensor primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 guanine bases downstream of the transcription start site.
  • the signal-sensor primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine bases in the region just downstream of the transcription start site.
  • the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides.
  • the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 cytosine bases.
  • the guanine bases in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the nucleotides described herein.
  • the signal-sensor primary construct may include at least one substitution and/or insertion upstream of the start codon.
  • the start codon is the first codon of the protein coding region whereas the transcription start site is the site where transcription begins.
  • the signal-sensor primary construct may include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or insertions of nucleotide bases.
  • the nucleotide bases may be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4 or at least 5 locations upstream of the start codon.
  • the nucleotides inserted and/or substituted may be the same base (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T or A, C and T) or at least four different bases.
  • the guanine base upstream of the coding region in the signal-sensor primary construct may be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein.
  • the substitution of guanine bases in the signal-sensor primary construct may be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472(7344):499-503; herein incorporated by reference in its entirety).
  • at least 5 nucleotides may be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 nucleotides may be the same base type.
  • RNA clean-up may also include a purification method such as, but not limited to, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • the signal-sensor primary construct or mmRNA may also undergo capping and/or tailing reactions.
  • a capping reaction may be performed by methods known in the art to add a 5′ cap to the 5′ end of the signal-sensor primary construct. Methods for capping include, but are not limited to, using a Vaccinia Capping enzyme (New England Biolabs, Ipswich, Mass.).
  • a poly-A tailing reaction may be performed by methods known in the art, such as, but not limited to, 2′ O-methyltransferase and by methods as described herein. If the signal-sensor primary construct generated from cDNA does not include a poly-T, it may be beneficial to perform the poly-A-tailing reaction before the signal-sensor primary construct is cleaned.
  • Signal-sensor primary construct or mmRNA purification may include, but is not limited to, mRNA or mmRNA clean-up, quality assurance and quality control.
  • mRNA or mmRNA clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • AGENCOURT® beads Beckman Coulter Genomics, Danvers, Mass.
  • poly-T beads poly-T beads
  • LNATM oligo-T capture probes EXIQON® Inc, Vedbaek, Denmark
  • HPLC based purification methods such as, but not limited to, strong
  • purified when used in relation to a polynucleotide such as a “purified mRNA or signal-sensor mmRNA” refers to one that is separated from at least one contaminant.
  • a “contaminant” is any substance which makes another unfit, impure or inferior.
  • a purified signal-sensor polynucleotide e.g., DNA and RNA
  • a quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
  • the signal-sensor mRNA or mmRNA may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.
  • the signal-sensor mRNA or mmRNA may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
  • UV/Vis ultraviolet visible spectroscopy
  • a non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.).
  • the quantified signal-sensor mRNA or mmRNA may be analyzed in order to determine if the signal-sensor mRNA or mmRNA may be of proper size, check that no degradation of the signal-sensor mRNA or mmRNA has occurred.
  • Degradation of the signal-sensor mRNA and/or mmRNA may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • the signal-sensor primary constructs or mmRNA may also encode additional features which facilitate trafficking of the polypeptides to therapeutically relevant sites.
  • One such feature which aids in protein trafficking is the signal peptide sequence.
  • a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5′ (or N-terminus) of the coding region or polypeptide encoded, respectively. Addition of these sequences result in trafficking of the encoded oncology-related polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.
  • Table 5 is a representative listing of signal proteins or peptides which may be incorporated for encoding by the signal-sensor polynucleotides, primary constructs or mmRNA of the invention.
  • SS secretion signal
  • MLS mitochondrial leader signal.
  • the signal-sensor primary constructs or mmRNA of the present invention may be designed to encode any of the signal peptide sequences of SEQ ID NOs 94-155, or fragments or variants thereof. These sequences may be included at the beginning of the oncology-related polypeptide coding region, in the middle or at the terminus or alternatively into a flanking region. Further, any of the signal-sensor polynucleotide primary constructs of the present invention may also comprise one or more of the sequences defined by SEQ ID NOs 32-93. These may be in the first region or either flanking region.
  • Additional signal peptide sequences which may be utilized in the present invention include those taught in, for example, databases such as those found at http://www.signalpeptide.de/ or http://proline.bic.nus.edu.sg/spdb/. Those described in U.S. Pat. Nos. 8,124,379; 7,413,875 and 7,385,034 are also within the scope of the invention and the contents of each are incorporated herein by reference in their entirety.
  • the signal-sensor polynucleotide, primary constructs or mmRNA may include a nucleic acid sequence encoding a nuclear localization signal (NLS) and/or a nuclear export signal (NES).
  • a signal-sensor polynucleotide, primary constructs or mmRNA may include a nucleic acid sequence encoding a nuclear localization signal (NLS).
  • the signal-sensor polynucleotide, primary construct or mmRNA encoding a NLS would be able to traffic an oncology related polypeptide into the nucleus and deliver a survival or death signal to the nuclear microenvironment.
  • the signal-sensor polynucleotide, primary constructs or mmRNA may include a nucleic acid sequence encoding a nuclear export signal such as NES 1 and/or NES2.
  • the signal-sensor polynucleotide, primary constructs or mmRNA may encode a NES1, NES2 and a NLS signal and an oncology related polypeptide or a scambled sequence which is not translatable in order to interact with HIF1-alpha to alter the transcritome of the cancer cells.
  • the signal-sensor primary constructs comprise at least a first region of linked nucleosides encoding at least one oncology-related polypeptide of interest.
  • the oncology-related polypeptides of interest or “targets” or oncology-related proteins and oncology-related peptides of the present invention are listed in Table 6, Table 7 and Table 41.
  • Oncology-related polypeptides may be divided into classes based on their function and area of cancer intervention. For example, the classes may include targets associated with (1) apoptosis or Survival signal imbalance (AS targets).
  • AS targets apoptosis or Survival signal imbalance
  • caspase dependent or caspase independent targets may be caspase dependent or caspase independent targets; (2) replicative potential or anti-senescence (CC/S targets); (3) metabolic stress including the involvement of acidosis or hypoxia (O 2 >1%) (M targets); (4) immune response (I targets); and (5) DNA damage/protection (DDR targets).
  • ENSEMBL Transcript ID (ENST)
  • ENSEMBL Protein ID (ENSP)
  • OPT. SEQ ID the optimized sequence ID
  • the targets are also categorized by group where “AS” refers to targets involved in apoptotic signaling; “M” refers to targets involved in metabolic processes and “CC/S” refers to targets involved in cell cycle and senescense.
  • CC/S CDK2 cyclin-dependent kinase 2 266970 972 266970 2133 CC/S CDK2 cyclin-dependent kinase 2 354056 973 243067 2134 CC/S CDK4 cyclin-dependent kinase 4 257904 974 257904 2135 CC/S CDK4 cyclin-dependent kinase 4 312990 975 316889 2136 CC/S CDK4 cyclin-dependent kinase 4 540325 976 439076 2137 CC/S CDK4 cyclin-dependent kinase 4 552254 977 449179 2138 CC/S CDK4 cyclin-dependent kinase 4 552388 978 448963 2139 CC/S CDK4 cyclin-dependent kinase 4 552862 979 446763 2140 CC/S CDK6 cyclin-dependent kinase 6 265734 980 265734
  • pombe 307980 1179 311360 2340 CC/S Rb retinoblastoma 1 267163 1180 267163 2341 protein CC/S Rb retinoblastoma 1 467505 1181 434702 2342 protein CC/S Rb retinoblastoma 1 542917 1182 437642 2343 protein CC/S SMC1 structural maintenance of 322213 1183 323421 2344 chromosomes 1A CC/S SMC1 structural maintenance of 340213 1184 344906 2345 chromosomes 1A CC/S SMC1 structural maintenance of 375340 1185 364489 2346 chromosomes 1A CC/S SMC1 structural maintenance of 428014 1186 413509 2347 chromosomes 1A CC/S USP1 ubiquitin specific peptidase 1 339950 1187 343526 2348 CC/S USP1 ubiquitin specific peptidase 1 371146 1188 360188 23
  • Signal-sensor polynucleotides of the present invention can be designed as a therapeutic for any of those listed in the table.
  • Familial Cancer Syndrome Targets Familial Tumor Cancer Suppressor Chromosomal Tumor Types Syndrome Gene Function Location Observed Li-Fraumeni P53 cell cycle 17p13.1 brain tumors, Syndrome regulation, sarcomas, leukemia, apoptosis breast cancer Familial RB1 cell cycle 13q14.1-q14.2 retinoblastoma, Retinoblastoma regulation osteogenic sarcoma Wilms Tumor WT1 transcriptional 11p13 pediatric kidney regulation cancer, most common form of childhood solid tumor Neurofibromatosis NF1 catalysis of RAS 17q11.2 neurofibromas, Type 1 inactivation sarcomas, gliomas Neurofibromatosis NF2 linkage of cell 22q12.2 Schwann cell Type 2 membrane to actin tumors, cytoskeleton astrocytomas, meningiomas, ependymonas Familial APC signaling through 5q21-q22 colon cancer Adenomato
  • the oncology-related polypeptides may include any “death signal” protein that can be recognized by active T cells of immune system.
  • Such suicide signal proteins encoded by the sensor-signal polynucleotides can be selectively expressed in particular tissues or cells (e.g. cancer cells) through engineered microRNA binding sites and/or other regulatory elements as described herein.
  • the group of proteins when they are expressed on the surface of a cancer cell, can prime T cell to induce T cell mediated immune response, thus killing the cancer cell.
  • a group of proteins that are known to present a “death signal” include, CD80, CD86, B7 and MHC II, etc.
  • the oncology-related polypeptides of the present invention may include at least one protein cleavage signal containing at least one protein cleavage site.
  • the protein cleavage site may be located at the N-terminus, the C-terminus, at any space between the N- and the C-termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half way point, between the half way point and the C-terminus, and combinations thereof.
  • the oncology-related polypeptides of the present invention may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin or Factor Xa protein cleavage signal.
  • Proprotein convertases are a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as prohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basic amino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilases that cleave at non-basic residues, called subtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9).
  • Non-limiting examples of protein cleavage signal amino acid sequences are listing in Table 8.
  • “X” refers to any amino acid
  • “n” may be 0, 2, 4 or 6 amino acids
  • “*” refers to the protein cleavage site.
  • the signal-sensor primary constructs and the mmRNA of the present invention may be engineered such that the primary construct or mmRNA contains at least one encoded protein cleavage signal.
  • the encoded protein cleavage signal may be located before the start codon, after the start codon, before the coding region, within the coding region such as, but not limited to, half way in the coding region, between the start codon and the half way point, between the half way point and the stop codon, after the coding region, before the stop codon, between two stop codons, after the stop codon and combinations thereof.
  • the signal-sensor primary constructs or mmRNA of the present invention may include at least one encoded protein cleavage signal containing at least one protein cleavage site.
  • the encoded protein cleavage signal may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin and/or Factor Xa protein cleavage signal.
  • a proprotein convertase or prohormone convertase
  • thrombin or Factor Xa protein cleavage signal.
  • Factor Xa protein cleavage signal may be used as Table 1 above or other known methods to determine the appropriate encoded protein cleavage signal to include in the signal-sensor primary constructs or mmRNA of the present invention. For example, starting with the signal of Table 8 and considering the codons of Table 1 one can design a signal for the signal-sensor primary construct which can produce a protein signal in the resulting oncology-related polypeptide.
  • the oncology-related polypeptides of the present invention include at least one protein cleavage signal and/or site.
  • the polypeptides of the present invention include at least one protein cleavage signal and/or site with the proviso that the polypeptide is not GLP-1.
  • the signal-sensor primary constructs or mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site.
  • the signal-sensor primary constructs or mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site with the proviso that the signal-sensor primary construct or mmRNA does not encode GLP-1.
  • the signal-sensor primary constructs or mmRNA of the present invention may include more than one coding region. Where multiple coding regions are present in the signal-sensor primary construct or mmRNA of the present invention, the multiple coding regions may be separated by encoded protein cleavage sites.
  • the signal-sensor primary construct or mmRNA may be signed in an ordered pattern. On such pattern follows AXBY form where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different oncology-related polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
  • a second such pattern follows the form AXYBZ where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different oncology-related polypeptides, and X, Y and Z are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
  • a third pattern follows the form ABXCY where A, B and C are coding regions which may be the same or different coding regions and/or may encode the same or different oncology-related polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
  • the oncology-related polypeptides, signal-sensor primary constructs and mmRNA can also contain sequences that encode protein cleavage sites so that the polypeptides, signal-sensor primary constructs and mmRNA can be released from a carrier region or a fusion partner by treatment with a specific protease for said protein cleavage site.
  • microRNAs are 19-25 nucleotide long noncoding RNAs that bind to the 3′UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
  • the modified nucleic acids (mRNA), enhanced modified RNA or ribonucleic acids of the invention may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA such as those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of which are incorporated herein by reference in their entirety.
  • known microRNAs, their sequences and their binding site sequences in the human genome are listed below in Table 9.
  • a microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence.
  • a microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.
  • a microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1.
  • a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1.
  • A adenine
  • the bases of the microRNA seed have complete complementarity with the target sequence.
  • miR-122 a microRNA abundant in liver, can inhibit the expression of the gene of interest if one or multiple target sites of miR-122 are engineered into the 3′UTR of the signal-sensor polynucleotide.
  • Introduction of one or multiple binding sites for different microRNA can be engineered to further decrease the longevity, stability, and protein translation of a signal-sensor polynucleotide.
  • microRNA site refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that “binding” may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.
  • microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues.
  • miR-122 binding sites may be removed to improve protein expression in the liver.
  • signal-sensor polynucleotides may include at least one miRNA-binding site in the 3′UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells (e.g., HEP3B or SNU449).
  • a strong apoptotic signal and at least one miR-122a binding site is encoded by the signal-sensor polynucleotide where the at least one miR-122a binding site is located in the 3′UTR.
  • apoptosis inducing factor short isoform (AIFsh) and at least one miR-122a binding site is encoded by the signal-sensor polynucleotide where the at least one miR-122a binding site is located in the 3′UTR.
  • HSV1-tk and at least one miR-122a binding site is encoded by the signal-sensor polynucleotide where the at least one miR-122a binding site is located in the 3′UTR.
  • signal-sensor polynucleotides may include three miRNA-binding sites in the 3′UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells (e.g., HEP3B or SNU449).
  • cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells (e.g., HEP3B or SNU449).
  • a strong apoptotic signal and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • apoptosis inducing factor short isoform (AIFsh) and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • constitutively active (C.A.) caspase 6 and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • HSV1-tk and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • microRNAs which are differentially expressed in different tissues and cells, and often associated with different types of diseases (e.g. cancer cells).
  • the decision of removal or insertion of microRNA binding sites, or any combination, is dependent on microRNA expression patterns and their profilings in cancer cells.
  • tissues where microRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), nervous system (mir-124a, miR-9), pluripotent cells (miR-302, miR-367, miR-290, miR-371, miR-373), pancreatic islet cells (miR-375), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
  • liver miR-122
  • muscle miR-133, miR-206,
  • microRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g. dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granuocytes, natural killer cells, etc.
  • APCs antigen presenting cells
  • Immune cell specific microRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells specific microRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells).
  • miR-142 and miR-146 are exclusively expressed in the immune cells, particularly abundant in myeloid dendritic cells.
  • Introducing the miR-142 binding site into the 3′-UTR of a signal-sensor polypeptide of the present invention can selectively suppress the gene expression in the antigen presenting cells through miR-142 mediated mRNA degradation, limiting antigen presentation in professional APCs (e.g. dendritic cells) and thereby preventing antigen-mediated immune response after gene delivery (see, Annoni A et al., blood, 2009, 114, 5152-5161, the content of which is herein incorporated by reference in its entirety.)
  • microRNAs binding sites that are known to be expressed in immune cells can be engineered into the signal-sensor polynucleotides to suppress the expression of the sensor-signal polynucleotide in APCs through microRNA mediated RNA degradation, subduing the antigen-mediated immune response, while the expression of the sensor-signal polynucleotide is maintained in non-immune cells where the immune cell specific microRNAs are not expressed.
  • the miR-122 binding site can be removed and the miR-142 (and/or mirR-146) binding sites can be engineered into the 3-UTR of the signal-sensor polynucleotide (e.g., see the constructs described in Example 38 and the experiment outlined in Examples 39 and 40).
  • the signal-sensor polynucleotide may include another negative regulatory element in the 3-UTR, either alone or in combination with mir-142 and/or mir-146 binding sites.
  • one regulatory element is the Constitutive Decay Elements (CDEs).
  • Immune cells specific microRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-
  • microRNAs that are enriched in specific types of immune cells. Furthermore, novel microRNAs are discovered in the immune cells in the art through micro-array hybridization and microtome analysis (Jima D D et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, the content of each of which is incorporated herein by reference in its entirety).
  • MicroRNAs that are known to be expressed in the liver include, but are not limited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, miR-939-5p.
  • microRNA binding sites from any liver specific microRNA can be introduced to or removed from the signal-sensor polynucleotides to regulate the expression of the signal-sensor polynucleotides in the liver.
  • Liver specific microRNAs binding sites can be engineered alone or further in combination with immune cells (e.g. APCs) microRNA binding sites in order to prevent an immune reaction against protein expression in the liver.
  • immune cells e.g. APCs
  • MicroRNAs that are known to be expressed in the lung include, but are not limited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, miR-381-5p.
  • MicroRNA binding sites from any lung specific microRNA can be introduced to or removed from the signal-sensor polynucleotide to regulate the expression of the signal-sensor polynucleotide in the lung.
  • Lung specific microRNAs binding sites can be engineered alone or further in combination with immune cells (e.g. APCs) microRNA binding sites in order to prevent an immune reaction against protein expression in the lung.
  • immune cells e.g. APCs
  • MicroRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p and miR-92b-5p.
  • microRNA binding sites from any heart specific microRNA can be introduced to or removed from the signal-sensor polynucleotides to regulate the expression of the signal-sensor polynucleotides in the heart.
  • Heart specific microRNAs binding sites can be engineered alone or further in combination with immune cells (e.g. APCs) microRNA binding sites in order to prevent an immune reaction against protein expression in the heart.
  • immune cells e.g. APCs
  • MicroRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p, miR-30
  • microRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, miR-657.
  • miR-132-3p
  • microRNA binding sites from any CNS specific microRNA can be introduced to or removed from the signal-sensor polynucleotides to regulate the expression of the signal-sensor polynucleotide in the nervous system.
  • Nervous system specific microRNAs binding sites can be engineered alone or further in combination with immune cells (e.g. APCs) microRNA binding sites in order to prevent an immune reaction against protein expression in the nervous system.
  • immune cells e.g. APCs
  • MicroRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p and miR-944.
  • MicroRNA binding sites from any pancreas specific microRNA can be introduced to or removed from the signal-sensor polynucleotide to regulate the expression of the signal-sensor polynucleotide in the pancreas.
  • Pancreas specific microRNAs binding sites can be engineered alone or further in combination with immune cells (e.g. APCs) microRNA binding sites in order to prevent immune reaction against protein expression in the pancreas.
  • immune cells e.g. APCs
  • MicroRNAs that are known to be expressed in the kidney further include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p and miR-562.
  • MicroRNA binding sites from any kidney specific microRNA can be introduced to or removed from the signal-sensor polynucleotide to regulate the expression of the signal-sensor polynucleotide in the kidney.
  • Kidney specific microRNAs binding sites can be engineered alone or further in combination with immune cells (e.g. APCs) microRNA binding sites in order to prevent immune reaction against protein expression in the kidney.
  • immune cells e.g. APCs
  • MicroRNAs that are known to be expressed in the muscle further include, but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p and miR-25-5p.
  • MicroRNA binding sites from any muscle specific microRNA can be introduced to or removed from the signal-sensor polynucleotide to regulate the expression of the signal-sensor polynucleotide in the muscle.
  • Muscle specific microRNAs binding sites can be engineered alone or further in combination with immune cells (e.g. APCs) microRNA binding sites in order to prevent an immune reaction against protein expression in the muscle.
  • MicroRNAs are differentially expressed in different types of cells, such as endothelial cells, epithelial cells and adipocytes.
  • microRNAs that are expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p, miR-
  • MicroRNA binding sites from any endothelial cell specific microRNA can be introduced to or removed from the signal-sensor polynucleotide in order to modulate the expression of the signal-sensor polynucleotide in the endothelial cells in various conditions.
  • microRNAs that are expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells; let-7 family, miR-133a, miR-133b, miR-126 specific in lung epithelial cells; miR-382-3p, miR-382-5p specific in renal epithelial cells and miR-762 specific in corneal epithelial cells. MicroRNA binding sites from any epithelial cell specific
  • a large group of microRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal J A and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436; Goff L A et al., PLoS One, 2009, 4:e7192; Morin R D et al., Genome Res, 2008, 18, 610-621; Yoo J K et al., Stem Cells Dev.
  • MicroRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p, miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR
  • the binding sites of embryonic stem cell specific microRNAs can be included in or removed from the 3-UTR of the signal-sensor polynucleotide to modulate the development and/or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a degenerative condition (e.g. degenerative diseases), or to stimulate the senescence and apoptosis of stem cells in a disease condition (e.g. cancer stem cell).
  • a degenerative condition e.g. degenerative diseases
  • apoptosis of stem cells e.g. cancer stem cell
  • microRNA expression studies have been conducted, and are described in the art, to profile the differential expression of microRNAs in various cancer cells/tissues and other diseases. Some microRNAs are abnormally over-expressed in certain cancer cells and others are under-expressed. For example, microRNAs are differentially expressed in cancer cells (WO2008/154098, US2013/0059015, US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224); pancreatic cancers and diseases (US2009/0131348, US2011/0171646, US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation (U.S. Pat. No.
  • microRNA sites that are over-expressed in certain cancer and/or tumor cells can be removed from the 3-UTR of the signal-sensor polynucleotide encoding the oncology-related polypeptide, restoring the expression suppressed by the over-expressed microRNAs in cancer cells, thus ameliorating the corresponsive biological function, for instance, transcription stimulation and/or repression, cell cycle arrest, apoptosis and cell death.
  • normal cells and tissues, wherein microRNA expression is not up-regulated, will remain unaffected.
  • MicroRNA can also regulate complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176).
  • binding sites for microRNAs that are involved in such processes may be removed or introduced, in order to tailor the expression of the signal-sensor polynucleotides expression to biologically relevant cell types or to the context of relevant biological processes.
  • the signal-sensor polynucleotides are defined as auxotrophic signal-sensor polynucleotides.
  • Table 9 is a non-exhaustive listing of miRs and miR binding sites (miR BS) and their sequences which may be used with the present invention.
  • microRNAs are differentially expressed in different tissues and cells, and often associated with different types of diseases (e.g. cancer cells). The decision of removal or insertion of microRNA binding sites, or any combination, is dependent on microRNA expression patterns and their profilings in cancer cells.
  • HCC represents hepatocellular carcinoma
  • ALL stands for acute lymphoblastsic leukemia
  • RCC renal cell carcinoma
  • CLL chrominc lymphocytic leukemia
  • MALT stands for mucosa-associated lymphoid tissue.
  • MicroRNAs that are enriched in specific types of immune cells are listed in Table 11. Furthermore, novel microRNAs are discovered in the immune cells in the art through micro-array hybridization and microtome analysis (Jima D D et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, the content of each of which is incorporated herein by reference in its entirety).
  • HCC represents hepatocellular carcinoma
  • ALL stands for acute lymphoblastsic leukemia
  • CLL stands for chrominc lymphocytic leukemia.
  • MicroRNAs associated diseases functions/targets hsa-let-7a-2-3p 2508 3529 embryonic stem inflammatory, tumor cells, lung, various cancers suppressor, myeloid cells (lung, cervical, target to c-myc breast, pancreatic, etc) hsa-let-7a-3p 2509 3530 embryonic stem inflammatory, tumor cell, lung, various cancers suppressor, myeloid cells (lung, cervical, target to c-myc breast, pancreatic, etc) hsa-let-7a-5p 2510 3531 embryonic stem inflammatory, tumor cells, lung, various cancers suppressor, myeloid cells (lung, cervical, target to c-myc breast, pancreatic, etc) hsa-let-7c 2513 3534 dendritic cells various cacners tumor (cervical, pancreatic, suppressor lung, esopphageal, apoptosis etc) (
  • a signal-sensor polynucleotide such as a primary construct or a mRNA molecule
  • the terms “modification” or, as appropriate, “modified” refer to modification with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties.
  • the term “modification” refers to a modification as compared to the canonical set of 20 amino acids.
  • the modifications may be various distinct modifications.
  • the coding region, the flanking regions and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • a modified signal-sensor polynucleotide, primary construct, or mmRNA introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified signal-sensor polynucleotide, primary construct, or mmRNA.
  • the signal-sensor polynucleotides, primary constructs, and mmRNA can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone).
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
  • modifications e.g., one or more modifications
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the signal-sensor polynucleotides, primary constructs, and mmRNA of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
  • an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc, and/or 3) termination or reduction in protein translation.
  • an immune response is induced.
  • the invention provides a modified nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
  • the present disclosure provides signal-sensor polynucleotides comprising a nucleoside or nucleotide that can disrupt the binding of a major groove interacting, e.g. binding, partner with the polynucleotide (e.g., where the modified nucleotide has decreased binding affinity to major groove interacting partner, as compared to an unmodified nucleotide).
  • the signal-sensor polynucleotides, primary constructs, and mmRNA can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.).
  • the signal-sensor polynucleotides, primary constructs, or mmRNA may include one or more messenger RNAs (mRNAs) and one or more modified nucleoside or nucleotides (e.g., mmRNA molecules). Details for these signal-sensor polynucleotides, primary constructs, and mmRNA follow.
  • the signal-sensor polynucleotides, primary constructs, and mmRNA of the invention includes a first region of linked nucleosides encoding an oncology-related polypeptide of interest, a first flanking region located at the 5′ terminus of the first region, and a second flanking region located at the 3′ terminus of the first region.
  • the signal-sensor polynucleotide, primary construct, or mmRNA are constructed according to the methods and modifications of International Application PCT/US12/058519 filed Oct. 3, 2012 (M9), the contents of which are incorporated herein by reference in their entirety.
  • the signal-sensor polynucleotides, primary constructs, and mmRNA can optionally include 5′ and/or 3′ flanking regions, which are described herein.
  • mmRNA Signal-Sensor Modified RNA
  • the present invention also includes the building blocks, e.g., modified ribonucleosides, modified ribonucleotides, of modified signal-sensor mRNA (mmRNA) molecules.
  • these building blocks can be useful for preparing the signal-sensor polynucleotides, primary constructs, or mmRNA of the invention.
  • Such building blocks are taught in co-pending International Application PCT/US12/058519 filed Oct. 3, 2012 (M9), the contents of which are incorporated herein by reference in their entirety.
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • organic base e.g., a purine or pyrimidine
  • nucleotide is defined as a nucleoside including a phosphate group.
  • the nucleosides and nucleotides described herein are generally chemically modified on the major groove face.
  • modified nucleotides include an amino group, a thiol group, an alkyl group, a halo group, or any described herein.
  • the modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides).
  • the modified nucleosides and nucleotides can include a modified nucleobase.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil.
  • nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine.
  • These nucleobases can be modified or wholly replaced to provide signal-sensor polynucleotides, primary constructs, or mmRNA molecules having enhanced properties.
  • the nucleosides and nucleotides described herein can be chemically modified. In some embodiments, chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
  • the modified nucleotides which may be incorporated into a signal-sensor polynucleotide, primary construct, or mmRNA molecule, can be modified on the internucleoside linkage (e.g., phosphate backbone).
  • internucleoside linkage e.g., phosphate backbone
  • the phrases “phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • the ⁇ -thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • Phosphorothioate linked signal-sensor polynucleotides, primary constructs, or mmRNA molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
  • a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-p
  • internucleoside linkages that may be employed according to the present invention, including internucleoside linkages which do not contain a phosphorous atom, are described herein below.
  • the signal-sensor polynucleotides, primary constructs, and mmRNA of the invention can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein or in International Application PCT/US12/058519 filed Oct. 3, 2012 (M9), the contents of which are incorporated herein by reference in their entirety.
  • the signal-sensor polypeptides, primary constructs, and mmRNA molecules for use in accordance with the invention may be prepared according to any useful technique, as described herein.
  • the modified nucleosides and nucleotides used in the synthesis of signal-sensor polynucleotides, primary constructs, and mmRNA molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, a skilled artisan would be able to optimize and develop additional process conditions. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
  • HPLC high performance liquid chromatography
  • Preparation of signal-sensor polynucleotides, primary constructs, and mmRNA molecules of the present invention can involve the protection and deprotection of various chemical groups.
  • the need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art.
  • the chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
  • Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature.
  • a given reaction can be carried out in one solvent or a mixture of more than one solvent.
  • suitable solvents for a particular reaction step can be selected.
  • Resolution of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of numerous methods known in the art.
  • An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid.
  • Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids.
  • Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine).
  • an optically active resolving agent e.g., dinitrobenzoylphenylglycine
  • Suitable elution solvent composition can be determined by one skilled in the art.
  • Modified nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568 (1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each of which are incorporated by reference in their entirety.
  • the signal-sensor polynucleotides, primary constructs, and mmRNA of the invention may or may not be uniformly modified along the entire length of the molecule.
  • one or more or all types of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotides X in a signal-sensor polynucleotide of the invention are modified, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • nucleotide modifications may exist at various positions in the signal-sensor polynucleotide, primary construct, or mmRNA.
  • nucleotide analogs or other modification(s) may be located at any position(s) of a signal-sensor polynucleotide, primary construct, or mmRNA such that the function of the signal-sensor polynucleotide, primary construct, or mmRNA is not substantially decreased.
  • a modification may also be a 5′ or 3′ terminal modification.
  • the signal-sensor polynucleotide, primary construct, or mmRNA may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
  • any one or more of A, G, U or C) or any intervening percentage e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 100%, and from 95% to 100%).
  • any intervening percentage e.g.,
  • the signal-sensor polynucleotide, primary construct, or mmRNA includes a modified pyrimidine (e.g., a modified uracil/uridine/U or modified cytosine/cytidine/C).
  • a modified pyrimidine e.g., a modified uracil/uridine/U or modified cytosine/cytidine/C.
  • the uracil or uridine (generally: U) in the signal-sensor polynucleotide, primary construct, or mmRNA molecule may be replaced with from about 1% to about 100% of a modified uracil or modified uridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from from from 20% to
  • the modified uracil or uridine can be replaced by a compound having a single unique structure or by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures, as described herein).
  • the cytosine or cytidine (generally: C) in the signal-sensor polynucleotide, primary construct, or mmRNA molecule may be replaced with from about 1% to about 100% of a modified cytosine or modified cytidine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 80%
  • At least 25% of the cytidines are replaced (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
  • At least 25% of the uracils are replaced (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
  • At least 25% of the cytidines are replaced, and at least 25% of the uracils are replaced (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
  • the present invention provides signal-sensor polynucleotides, primary constructs and mmRNA compositions and complexes in combination with one or more pharmaceutically acceptable excipients.
  • Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances.
  • additional active substances e.g. therapeutically and/or prophylactically active substances.
  • General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
  • compositions are administered to humans, human patients or subjects.
  • active ingredient generally refers to signal-sensor polynucleotides, primary constructs and mmRNA to be delivered as described herein.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
  • the signal-sensor polynucleotide, primary construct, and mmRNA of the invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the signal-sensor polynucleotide, primary construct, or mmRNA); (4) alter the biodistribution (e.g., target the polynucleotide, primary construct, or mmRNA to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
  • excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the signal-sensor polynucleotide, primary construct, or mmRNA); (4) alter the biodistribution (e.g., target the polynucleotide, primary construct, or mmRNA to specific tissues or cell types); (5)
  • excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with signal-sensor polynucleotide, primary construct, or mmRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
  • the signal-sensor polynucleotide, primary construct, or mmRNA of the present invention may be formulated using self-assembled nucleic acid nanoparticles.
  • the formulations of the invention can include one or more excipients, each in an amount that together increases the stability of the signal-sensor polynucleotide, primary construct, or mmRNA, increases cell transfection by the signal-sensor polynucleotide, primary construct, or mmRNA, increases the expression of polynucleotide, primary construct, or mmRNA encoded protein, and/or alters the release profile of signal-sensor polynucleotide, primary construct, or mmRNA encoded proteins.
  • the primary construct and mmRNA of the present invention may be formulated using self-assembled nucleic acid nanoparticles.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient may generally be equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage including, but not limited to, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
  • the formulations described herein may contain at least one signal-sensor mmRNA.
  • the formulations may contain 1, 2, 3, 4 or 5 signal-sensor mmRNA.
  • the formulation may contain modified mRNA encoding proteins selected from categories such as, proteins.
  • the formulation contains at least three signal-sensor modified mRNA encoding oncology-related proteins.
  • the formulation contains at least five signal-sensor modified mRNA encoding oncology-related proteins.
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md
  • any conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • the particle size of the lipid nanoparticle may be increased and/or decreased.
  • the change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the signal-sensor modified mRNA delivered to mammals.
  • compositions include, but are not limited to, inert diluents, surface active agents and/or emulsifiers, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the pharmaceutical formulations of the invention.
  • compositions of the present invention may comprise at least one adjuvant which may be a chemo-adjuvant.
  • adjuvant which may be a chemo-adjuvant.
  • Non-limiting examples of chemo-adjuvants and delivery systems which comprises a chemo-adjuvant are described in International Patent Publication No. WO2013134349, the contents of which is herein incorporated by reference in its entirety.
  • the chemo-adjuvant may be bonded to, non-covalently bonded to or encapsulated within a delivery vehicle described herein.
  • lipidoids The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of signal-sensor polynucleotides, primary constructs or mmRNA (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which are incorporated herein in their entireties).
  • the present disclosure describes their formulation and use in delivering single stranded signal-sensor polynucleotides, primary constructs, or mmRNA.
  • Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the signal-sensor polynucleotide, primary construct, or mmRNA, as judged by the production of an encoded protein, following the injection of a lipidoid formulation via localized and/or systemic routes of administration.
  • Lipidoid complexes of signal-sensor polynucleotides, primary constructs, or mmRNA can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.
  • nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety).
  • particle size Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety.
  • small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy.
  • Formulations with the different lipidoids including, but not limited to penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010)), C12-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.
  • TETA-5LAP penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride
  • C12-200 including derivatives and variants
  • MD1 penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride
  • lipidoid referred to herein as “98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879 and is incorporated by reference in its entirety.
  • the lipidoid referred to herein as “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670; both of which are herein incorporated by reference in their entirety.
  • the lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to signal-sensor polynucleotide, primary construct, or mmRNA.
  • formulations with certain lipidoids include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chain length).
  • formulations with certain lipidoids include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.
  • Combinations of different lipidoids may be used to improve the efficacy of signal-sensor polynucleotide, primary construct, or mmRNA directed protein production as the lipidoids may be able to increase cell transfection by the signal-sensor polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded oncology-related protein (see Whitehead et al., Mol. Ther. 2011, 19:1688-1694, herein incorporated by reference in its entirety).
  • the particle size of the lipid nanoparticle may be increased and/or decreased.
  • the change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of, the signal-sensor polynucleotide, primary construct, or mmRNA delivered to subjects.
  • Liposomes Liposomes, Lipoplexes, and Lipid Nanoparticles
  • the signal-sensor polynucleotide, primary construct, and mmRNA of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
  • pharmaceutical compositions of signal-sensor polynucleotide, primary construct, or mmRNA include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
  • Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
  • liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
  • compositions described herein may include, without limitation, liposomes such as those formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).
  • DOXIL® 1,2-dioleyloxy-N,N-dimethylaminopropane
  • compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al.
  • SPLP stabilized plasmid-lipid particles
  • SNALP stabilized nucleic acid lipid particle
  • the original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method.
  • the liposome formulations are composed of 3 to 4 lipid components in addition to the signal-sensor polynucleotide, primary construct, or mmRNA.
  • a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al.
  • DSPC disteroylphosphatidyl choline
  • PEG-S-DSG 10% PEG-S-DSG
  • DODMA 1,2-dioleyloxy-N,N-dimethylaminopropane
  • certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.
  • DSDMA 1,2-distearloxy-N,N-dimethylaminopropane
  • DODMA 1,2-dilinolenyloxy-3-dimethylaminopropane
  • compositions may include liposomes which may be formed to deliver signal-sensor mmRNA which may encode at least one immunogen.
  • the mmRNA may be encapsulated by the liposome and/or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub. Nos. WO2012031046, WO2012031043, WO201203091 and WO2012006378 herein incorporated by reference in their entireties).
  • the signal-sensor mmRNA which may encode an immunogen may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid which can interact with the signal-sensor mmRNA anchoring the molecule to the emulsion particle (see International Pub. No. WO2012006380).
  • the lipid formulation may include at least cationic lipid, a lipid which may enhance transfection and a least one lipid which contains a hydrophilic head group linked to a lipid moiety (International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; herein incorporated by reference in their entireties).
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA encoding an immunogen may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U.S. Pub. No. 20120177724, herein incorporated by reference in its entirety).
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid-polycation complex.
  • the formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size.
  • the liposome formulation was composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.
  • changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated by reference in its entirety).
  • the ratio of PEG in the LNP formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.
  • LNP formulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol.
  • the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
  • PEG-DSG 1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol
  • PEG-DPG 1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol
  • the cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.
  • the LNP formulations of the signal-sensor polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG 3% lipid molar ratio. In another embodiment, the LNP formulations of the signal-sensor polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG 1.5% lipid molar ratio.
  • the pharmaceutical compositions of the signal-sensor polynucleotides, primary constructs and/or mmRNA may include at least one of the PEGylated lipids described in International Publication No. 2012099755, herein incorporated by reference.
  • the pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
  • DiLa2 liposomes Marina Biotech, Bothell, Wash.
  • SMARTICLES® Marina Biotech, Bothell, Wash.
  • neutral DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • siRNA delivery for ovarian cancer Lianden et al. Cancer Biology & Therapy 2006 5(12)1708-1713
  • the liposome may be a liposomal nanostructure which has been formulated for treatment of cancers and other diseases or to control the cholesterol metabolism in cells.
  • the liposome nanostructure may also comprise a scavenger receptor type B-1 (SR-B1) in order to kill cancer cells.
  • SR-B1 scavenger receptor type B-1
  • Non-limiting examples of liposomal nanostructures, which may be used with the signal-sensor polynucleotides described herein, are described in International Publication No. WO2013126776, the contents of which are herein incorporated by reference in its entirety.
  • the liposomes described herein may comprise at least one immunomodulator such as, but not limited to, cytokines Formulations and methods of using the liposomes comprising at least one immunomodulator are described in International Publication No WO2013129935 and WO2013129936, the contents of each of which are herein incorporated by reference in their entirety.
  • the liposomes comprising at least one immunomodulator may be used in the treatment of cancer.
  • the liposomes comprising an immunomodulator may comprise a signal-sensor polynucleotide described herein.
  • the liposome comprising an immunomodulator may be used in a combination with at least one antibody such as the particulate or vesicular immunomodulators described in International Publication No WO2013129936, the contents of which are herein incorporated by reference in its entirety.
  • Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
  • Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity.
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
  • ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain.
  • the internal ester linkage may replace any carbon in the lipid chain.
  • the internal ester linkage may be located on either side of the saturated carbon.
  • an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
  • a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
  • the polymer may encapsulate the nanospecies or partially encapsulate the nanospecies.
  • the immunogen may be a recombinant oncology-related protein, a signal-sensor modified RNA and/or a primary construct described herein.
  • the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.
  • Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier.
  • Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
  • oral e.g., the buccal and esophageal membranes and tonsil tissue
  • ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
  • nasal, respiratory e.g., nasal, pharyngeal, tracheal and bronchial
  • Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosla tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm-500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487; Lai et al.
  • PEG polyethylene glycol
  • the transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT).
  • FRAP fluorescence recovery after photobleaching
  • MPT high resolution multiple particle tracking
  • the lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer.
  • the polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the polymeric material may be biodegradable and/or biocompatible.
  • Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (
  • the lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer, and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication 20100003337; herein incorporated by reference in their entireties).
  • the co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created.
  • the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; herein incorporated by reference in its entirety).
  • the vitamin of the polymer-vitamin conjugate may be vitamin E.
  • the vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
  • the lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, signal-sensor mmRNA, anionic protein (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin (34
  • the surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle.
  • the mucus penetrating lipid nanoparticles may comprise at least one signal-sensor mmRNA described herein.
  • the signal-sensor mmRNA may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the particle.
  • the signal-sensor mmRNA may be covalently coupled to the lipid nanoparticle.
  • Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
  • Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier.
  • Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
  • oral e.g., the buccal and esophageal membranes and tonsil tissue
  • ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
  • nasal, respiratory e.g., nasal, pharyngeal, tracheal and bronchial
  • Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosla tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm-500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487; Lai et al.
  • PEG polyethylene glycol
  • the transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT).
  • FRAP fluorescence recovery after photobleaching
  • MPT high resolution multiple particle tracking
  • the lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer.
  • the polymeric material may including, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the polymeric material may be biodegradable and/or biocompatible.
  • Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (
  • the lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer, and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication 20100003337; herein incorporated by reference in their entireties).
  • a co-polymer such as, but not limited to, a block co-polymer, and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication 20100003337; herein incorporated by reference in their entireties).
  • the vitamin of the polymer-vitamin conjugate may be vitamin E.
  • the vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
  • the lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, mmRNA, anionic protein (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin (34 dorn
  • the surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle.
  • the mucus penetrating lipid nanoparticles may comprise at least one signal-sensor polynucleotide, primary construct, or mmRNA described herein.
  • the signal-sensor polynucleotide, primary construct, or mmRNA may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the particle.
  • the signal-sensor polynucleotide, primary construct, or mmRNA may be covalently coupled to the lipid nanoparticle.
  • Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles.
  • formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
  • the nanoparticle may be for a dual modality therapy such as described by Mieszawska et al. (Bioconjugate Chemistry, 2013, 24 (9), pp 1429-1434; the contents of which is herein incorporated by reference in its entirety) comprising at least one therapeutic agent (e.g., a signal-sequence polynucleotide described herein).
  • the therapeutic agent or agents formulated in the lipid nanoparticle may be an anti-angiogenic and a cytotoxic agent (see e.g., the polymer-lipid nanoparticles taught by Mieszawska et al. Bioconjugate Chemistry, 2013, 24 (9), pp 1429-1434; the contents of which is herein incorporated by reference in its entirety).
  • the nanoparticle may comprise a LyP-1 peptide such as the nanocarrier composition described in International Patent Publication No. WO2013100869, the contents of which are herein incorporated by reference in its entirety.
  • the LyP-1 peptide may be contained in the nanoparticles disclosed herein, or may be a conjugate, derivative, analogue or pegylated form of the peptide.
  • a nanoparticle comprising the LyP-1 peptide may comprise a signal-sensor polynucleotide and may be used for cancer treatment and/or imaging.
  • the signal-sensor polynucleotide, primary construct, or mmRNA is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids (Ale
  • such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest.
  • lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated by reference in its entirety).
  • Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol.
  • the signal-sensor polynucleotide, primary construct, or mmRNA is formulated as a solid lipid nanoparticle.
  • a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers.
  • the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; herein incorporated by reference in its entirety).
  • Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of signal-sensor polynucleotide, primary construct, or mmRNA directed protein production as these formulations may be able to increase cell transfection by the signal-sensor polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded protein.
  • One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; herein incorporated by reference in its entirety).
  • the liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the signal-sensor polynucleotide, primary construct, or mmRNA.
  • the signal-sensor polynucleotide, primary construct, and mmRNA of the invention can be formulated using natural and/or synthetic polymers.
  • polymers which may be used for delivery include, but are not limited to, Dynamic POLYCONJUGATETM formulations from MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.
  • RONDELTM RNAi/Oligonucleotide Nanoparticle Delivery
  • PHASERXTM pH responsive co-block polymers
  • PLGA formulations include, but are not limited to, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
  • PLGA injectable depots e.g., ELIGARD® which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
  • NMP N-methyl-2-pyrrolidone
  • This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887).
  • the polymer complex On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer.
  • the polymer Through replacement of the N-acetylgalactosamine group with a mannose group, it was shown one could alter targeting from asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells.
  • Another polymer approach involves using transferrin-targeted cyclodextrin-containing polycation nanoparticles.
  • the polymer formulation can permit the sustained or delayed release of signal-sensor polynucleotide, primary construct, or mmRNA (e.g., following intramuscular or subcutaneous injection).
  • the altered release profile for the signal-sensor polynucleotide, primary construct, or mmRNA can result in, for example, translation of an encoded protein over an extended period of time.
  • the polymer formulation may also be used to increase the stability of the signal-sensor polynucleotide, primary construct, or mmRNA.
  • Biodegradable polymers have been previously used to protect nucleic acids other than mmRNA from degradation and been shown to result in sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci USA.
  • the pharmaceutical compositions may be sustained release formulations.
  • the sustained release formulations may be for subcutaneous delivery.
  • Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.). TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).
  • modified mRNA may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the signal-sensor modified mRNA in the PLGA microspheres while maintaining the integrity of the signal-sensor modified mRNA during the encapsulation process.
  • EVAc are non-biodegradable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine device; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters).
  • Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5° C. and forms a solid gel at temperatures greater than 15° C.
  • PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days.
  • GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.
  • Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464:1067-1070; herein incorporated by reference in its entirety).
  • GalNAc N-acetylgalactosamine
  • the signal-sensor mmRNA of the invention may be formulated with or in a polymeric compound.
  • the polymer may include at least one polymer such as, but not limited to, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, linear biodegradable copolymer, poly[ ⁇ -(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers or combinations thereof.
  • PEG
  • the signal-sensor mmRNA of the invention may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274 herein incorporated by reference in its entirety.
  • the formulation may be used for transfecting cells in vitro or for in vivo delivery of the signal-sensor mmRNA.
  • the signal-sensor mmRNA may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. 20090042829 and 20090042825 each of which are herein incorporated by reference in their entireties.
  • a polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. 20100260817 herein incorporated by reference in its entirety).
  • a pharmaceutical composition may include the signal-sensor mmRNA and the polyamine derivative described in U.S. Pub. No. 20100260817 (the contents of which are incorporated herein by reference in its entirety.
  • the signal-sensor mmRNA of the invention may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof.
  • the biodegradable cationic lipopolymer may be made my methods known in the art and/or described in U.S. Pat. No.
  • the poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. 20100004315, herein incorporated by reference in its entirety.
  • the biodegradable polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos.
  • the linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886.
  • the PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,217,912 herein incorporated by reference in its entirety.
  • the PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co-glycolides).
  • the biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 herein incorporated by reference in their entireties.
  • the multi-block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines.
  • LPEI linear polyethyleneimine
  • the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 herein incorporated by reference in their entireties.
  • a gene delivery composition may include a nucleotide sequence and a poloxamer.
  • the signal-sensor mmRNA of the present invention may be used in a gene delivery composition with the poloxamer described in U.S. Pub. No. 20100004313.
  • the polymer formulation of the present invention may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups.
  • the polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No. 20090042829 herein incorporated by reference in its entirety.
  • the cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-
  • the signal-sensor polynucleotide, primary construct, and mmRNA of the invention can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate.
  • Components may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so to delivery of the signal-sensor polynucleotide, primary construct and mmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug Deliv Rev.
  • Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver signal-sensor polynucleotides, primary constructs and mmRNA in vivo.
  • a lipid coated calcium phosphate nanoparticle which may also contain a targeting ligand such as anisamide, may be used to deliver the signal-sensor polynucleotide, primary construct and mmRNA of the present invention.
  • a lipid coated calcium phosphate nanoparticle was used (Li et al., J Contr Rel. 2010 142: 416-421; Li et al., J Contr Rel.
  • This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.
  • calcium phosphate with a PEG-polyanion block copolymer may be used to deliver signal-sensor polynucleotides, primary constructs and mmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006 111:368-370).
  • a PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle to deliver the signal-sensor polynucleotides, primary constructs and mmRNA of the present invention.
  • the PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
  • core-shell nanoparticles have additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-13001).
  • the complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle.
  • the core-shell nanoparticles may efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
  • a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG may be used to delivery of the signal-sensor polynucleotide, primary construct and mmRNA of the present invention.
  • a luciferase-expressing tumor it was determined that the lipid-polymer-lipid hybrid nanoparticle significantly suppressed luciferase expression, as compared to a conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031).
  • the signal-sensor polynucleotide, primary construct, and mmRNA of the invention can be formulated with peptides and/or proteins in order to increase transfection of cells by the polynucleotide, primary construct, or mmRNA.
  • peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations.
  • a non-limiting example of a cell penetrating peptide which may be used with the pharmaceutical formulations of the present invention includes a cell-penetrating peptide sequence attached to polycations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des.
  • compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space.
  • a cell penetrating agent e.g., liposomes
  • signal-sensor polynucleotides, primary constructs, and mmRNA of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, Mass.) and Permeon Biologics (Cambridge, Mass.) in order to enable intracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 2009 73:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all of which are herein incorporated by reference in its entirety).
  • Aileron Therapeutics Cambridge, Mass.
  • Permeon Biologics Cambridge, Mass.
  • the cell-penetrating polypeptide may comprise a first domain and a second domain.
  • the first domain may comprise a supercharged polypeptide.
  • the second domain may comprise a protein-binding partner.
  • protein-binding partner includes, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides.
  • the cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner.
  • the cell-penetrating polypeptide may be capable of being secreted from a cell where the signal-sensor polynucleotide, primary construct, or mmRNA may be introduced.
  • Formulations of the including peptides or proteins may be used to increase cell transfection by the signal-sensor polynucleotide, primary construct, or mmRNA, alter the biodistribution of the signal-sensor polynucleotide, primary construct, or mmRNA (e.g., by targeting specific tissues or cell types), and/or increase the translation of encoded protein.
  • the signal-sensor polynucleotide, primary construct, and mmRNA of the invention can be transfected ex vivo into cells, which are subsequently transplanted into a subject.
  • the pharmaceutical compositions may include red blood cells to deliver modified RNA to liver and myeloid cells, virosomes to deliver modified RNA in virus-like particles (VLPs), and electroporated cells such as, but not limited to, from MAXCYTE® (Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modified RNA. Examples of use of red blood cells, viral particles and electroporated cells to deliver payloads other than mmRNA have been documented (Godfrin et al., Expert Opin Biol Ther.
  • Cell-based formulations of the signal-sensor polynucleotide, primary construct, and mmRNA of the invention may be used to ensure cell transfection (e.g., in the cellular carrier), alter the biodistribution of the signal-sensor polynucleotide, primary construct, or mmRNA (e.g., by targeting the cell carrier to specific tissues or cell types), and/or increase the translation of encoded oncology-related protein.
  • nucleic acid into a cell
  • non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.
  • Sonoporation or cellular sonication
  • sound e.g., ultrasonic frequencies
  • Sonoporation methods are known to those in the art and are used to deliver nucleic acids in vivo (Yoon and Park, Expert Opin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr Pharm Biotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 2007 14:465-475; all herein incorporated by reference in their entirety).
  • Electroporation techniques are also well known in the art and are used to deliver nucleic acids in vivo and clinically (Andre et al., Curr Gene Ther. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 2010 10:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all herein incorporated by reference in their entirety).
  • signal-sensor polynucleotides, primary constructs or mmRNA may be delivered by electroporation as described in Example 12.
  • the intramuscular or subcutaneous localized injection of signal-sensor polynucleotide, primary construct, or mmRNA of the invention can include hyaluronidase, which catalyzes the hydrolysis of hyaluronan.
  • hyaluronidase catalyzes the hydrolysis of hyaluronan.
  • hyaluronidase By catalyzing the hydrolysis of hyaluronan, a constituent of the interstitial barrier, hyaluronidase lowers the viscosity of hyaluronan, thereby increasing tissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440; herein incorporated by reference in its entirety). It is useful to speed their dispersion and systemic distribution of encoded proteins produced by transfected cells.
  • the hyaluronidase can be used to increase the number of cells exposed to a signal-sensor polynucleotide, primary construct, or mmRNA
  • the signal-sensor polynucleotide, primary construct or mmRNA of the invention may be encapsulated within and/or absorbed to a nanoparticle mimic.
  • a nanoparticle mimic can mimic the delivery function organisms or particles such as, but not limited to, pathogens, viruses, bacteria, fungus, parasites, prions and cells.
  • the signal-sensor polynucleotide, primary construct or mmRNA of the invention may be encapsulated in a non-viron particle which can mimic the delivery function of a virus (see International Pub. No. WO2012006376 herein incorporated by reference in its entirety).
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the invention can be attached or otherwise bound to at least one nanotube such as, but not limited to, rosette nanotubes, rosette nanotubes having twin bases with a linker, carbon nanotubes and/or single-walled carbon nanotubes,
  • the signal-sensor polynucleotides, primary constructs or mmRNA may be bound to the nanotubes through forces such as, but not limited to, steric, ionic, covalent and/or other forces.
  • the nanotube can release one or more signal-sensor polynucleotides, primary constructs or mmRNA into cells.
  • the size and/or the surface structure of at least one nanotube may be altered so as to govern the interaction of the nanotubes within the body and/or to attach or bind to the signal-sensor polynucleotides, primary constructs or mmRNA disclosed herein.
  • the building block and/or the functional groups attached to the building block of the at least one nanotube may be altered to adjust the dimensions and/or properties of the nanotube.
  • the length of the nanotubes may be altered to hinder the nanotubes from passing through the holes in the walls of normal blood vessels but still small enough to pass through the larger holes in the blood vessels of tumor tissue.
  • At least one nanotube may also be coated with delivery enhancing compounds including polymers, such as, but not limited to, polyethylene glycol.
  • delivery enhancing compounds including polymers, such as, but not limited to, polyethylene glycol.
  • at least one nanotube and/or the signal-sensor polynucleotides, primary constructs or mmRNA may be mixed with pharmaceutically acceptable excipients and/or delivery vehicles.
  • the signal-sensor polynucleotides, primary constructs or mmRNA are attached and/or otherwise bound to at least one rosette nanotube.
  • the rosette nanotubes may be formed by a process known in the art and/or by the process described in International Publication No. WO2012094304, herein incorporated by reference in its entirety.
  • At least one signal-sensor polynucleotide, primary construct and/or mmRNA may be attached and/or otherwise bound to at least one rosette nanotube by a process as described in International Publication No.
  • rosette nanotubes or modules forming rosette nanotubes are mixed in aqueous media with at least one signal-sensor polynucleotide, primary construct and/or mmRNA under conditions which may cause at least one signal-sensor polynucleotide, primary construct or mmRNA to attach or otherwise bind to the rosette nanotubes.
  • the signal-sensor polynucleotides, primary constructs, and mmRNA of the invention include conjugates, such as a polynucleotide, primary construct, or mmRNA covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide).
  • conjugates such as a polynucleotide, primary construct, or mmRNA covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide).
  • the conjugates of the invention include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
  • the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer).
  • polyamino acids examples include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine.
  • PLL polylysine
  • poly L-aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer poly(L-lactide-co-glycolied) copolymer
  • divinyl ether-maleic anhydride copolymer divinyl ether-
  • polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • the conjugate of the present invention may function as a carrier for the signal-sensor mmRNA of the present invention.
  • the conjugate may comprise a cationic polymer such as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine which may be grafted to with poly(ethylene glycol).
  • the conjugate may be similar to the polymeric conjugate and the method of synthesizing the polymeric conjugate described in U.S. Pat. No. 6,586,524 herein incorporated by reference in its entirety.
  • the conjugates can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a cell or tissue targeting agent e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.
  • Targeting groups can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
  • Targeting groups may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers.
  • the ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.
  • the targeting group can be any ligand that is capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, apatamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.
  • the targeting group is an aptamer.
  • the aptamer can be unmodified or have any combination of modifications disclosed herein.
  • compositions of the present invention may include chemical modifications such as, but not limited to, modifications similar to locked nucleic acids.
  • LNA locked nucleic acid
  • Some embodiments featured in the invention include signal-sensor polynucleotides, primary constructs or mmRNA with phosphorothioate backbones and oligonucleosides with other modified backbones, and in particular —CH 2 —NH—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — [known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —N(CH 3 )—CH 2 —CH 2 — [wherein the native phosphodiester backbone is represented as —O—P(O) 2 —O—CH 2 —] of the above-referenced U.S.
  • the polynucleotides featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
  • Modifications at the 2′ position may also aid in delivery.
  • modifications at the 2′ position are not located in a polypeptide-coding sequence, i.e., not in a translatable region.
  • Modifications at the 2′ position may be located in a 5′UTR, a 3′UTR and/or a tailing region.
  • Modifications at the 2′ position can include one of the following at the 2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ).
  • n OCH 3 O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
  • the signal-sensor polynucleotides, primary constructs or mmRNA include one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties, or a group for improving the pharmacodynamic properties, and other substituents having similar properties.
  • the modification includes a 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • 2′-dimethylaminooxyethoxy i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples herein below
  • 2′-dimethylaminoethoxyethoxy also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE
  • 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 also described in examples herein below.
  • modifications include 2′-methoxy (2′-OCH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.
  • signal-sensor polynucleotides of the invention may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
  • the signal-sensor polynucleotide, primary construct, or mmRNA is covalently conjugated to a cell penetrating polypeptide.
  • the cell-penetrating peptide may also include a signal peptide sequence.
  • the conjugates of the invention can be designed to have increased stability; increased cell transfection; and/or altered the biodistribution (e.g., targeted to specific tissues or cell types).
  • Self-assembled nanoparticles have a well-defined size which may be precisely controlled as the nucleic acid strands may be easily reprogrammable.
  • the optimal particle size for a cancer-targeting nanodelivery carrier is 20-100 nm as a diameter greater than 20 nm avoids renal clearance and enhances delivery to certain tumors through enhanced permeability and retention effect.
  • Using self-assembled nucleic acid nanoparticles a single uniform population in size and shape having a precisely controlled spatial orientation and density of cancer-targeting ligands for enhanced delivery.
  • oligonucleotide nanoparticles were prepared using programmable self-assembly of short DNA fragments and therapeutic siRNAs.
  • nanoparticles are molecularly identical with controllable particle size and target ligand location and density.
  • the DNA fragments and siRNAs self-assembled into a one-step reaction to generate DNA/siRNA tetrahedral nanoparticles for targeted in vivo delivery. (Lee et al., Nature Nanotechnology 2012 7:389-393).
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington's The Science and Practice of Pharmacy 21 st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference) discloses various excipients
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
  • crospovidone cross-linked poly(vinyl-pyrrolidone)
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g.
  • polyoxyethylene lauryl ether [BRIJ®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g.
  • acacia sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate)(VEEGUM°, and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONETM, KATHONTM, and/or EUXYL®.
  • Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • the present disclosure encompasses the delivery of signal-sensor polynucleotides, primary constructs or mmRNA for any of therapeutic, pharmaceutical, diagnostic or imaging by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be delivered to a cell naked.
  • naked refers to delivering signal-sensor polynucleotides, primary constructs or mmRNA free from agents which promote transfection.
  • the polynucleotides, primary constructs or mmRNA delivered to the cell may contain no modifications.
  • the naked signal-sensor polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be formulated, using the methods described herein.
  • the formulations may contain signal-sensor polynucleotides, primary constructs or mmRNA which may be modified and/or unmodified.
  • the formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot.
  • the formulated signal-sensor polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.
  • compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, (into the base of the penis), intravaginal administration, intrauter
  • compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
  • Non-limiting routes of administration for the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention are described below.
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example,
  • oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the rate of drug release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g.
  • the dosage form may comprise buffering agents.
  • solution retarding agents e.g. paraffin
  • absorption accelerators e.g. quaternary ammonium compounds
  • wetting agents e.g. cetyl alcohol and glycerol monostearate
  • absorbents e.g. kaolin and bentonite clay
  • lubricants e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate
  • the dosage form may comprise buffering agents.
  • compositions containing the signal-sensor polynucleotides, primary constructs or mmRNA of the invention may be formulated for administration topically.
  • the skin may be an ideal target site for delivery as it is readily accessible. Gene expression may be restricted not only to the skin, potentially avoiding nonspecific toxicity, but also to specific layers and cell types within the skin.
  • the site of cutaneous expression of the delivered compositions will depend on the route of nucleic acid delivery.
  • Three routes are commonly considered to deliver signal-sensor polynucleotides, primary constructs or mmRNA to the skin: (i) topical application (e.g. for local/regional treatment and/or oncology-related applications); (ii) intradermal injection (e.g. for local/regional treatment and/or oncology-related applications); and (iii) systemic delivery (e.g. for treatment of dermatologic diseases that affect both cutaneous and extracutaneous regions).
  • Signal-sensor polynucleotides, primary constructs or mmRNA can be delivered to the skin by several different approaches known in the art.
  • the invention provides for a variety of dressings (e.g., wound dressings) or bandages (e.g., adhesive bandages) for conveniently and/or effectively carrying out methods of the present invention.
  • dressing or bandages may comprise sufficient amounts of pharmaceutical compositions and/or signal-sensor polynucleotides, primary constructs or mmRNA described herein to allow a user to perform multiple treatments of a subject(s).
  • the invention provides for the signal-sensor polynucleotides, primary constructs or mmRNA compositions to be delivered in more than one injection.
  • tissue such as skin
  • a device and/or solution which may increase permeability.
  • the tissue may be subjected to an abrasion device to increase the permeability of the skin (see U.S. Patent Publication No. 20080275468, herein incorporated by reference in its entirety).
  • the tissue may be subjected to an ultrasound enhancement device.
  • An ultrasound enhancement device may include, but is not limited to, the devices described in U.S. Publication No. 20040236268 and U.S. Pat. Nos. 6,491,657 and 6,234,990; herein incorporated by reference in their entireties. Methods of enhancing the permeability of tissue are described in U.S. Publication Nos. 20040171980 and 20040236268 and U.S. Pat. No. 6,190,315; herein incorporated by reference in their entireties.
  • a device may be used to increase permeability of tissue before delivering formulations of modified mRNA described herein.
  • the permeability of skin may be measured by methods known in the art and/or described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety.
  • a modified mRNA formulation may be delivered by the drug delivery methods described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety.
  • tissue may be treated with a eutectic mixture of local anesthetics (EMLA) cream before, during and/or after the tissue may be subjected to a device which may increase permeability.
  • EMLA local anesthetics
  • enhancers may be applied to the tissue before, during, and/or after the tissue has been treated to increase permeability.
  • Enhancers include, but are not limited to, transport enhancers, physical enhancers, and cavitation enhancers. Non-limiting examples of enhancers are described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety.
  • a device may be used to increase permeability of tissue before delivering formulations of modified mRNA described herein, which may further contain a substance that invokes an immune response.
  • a formulation containing a substance to invoke an immune response may be delivered by the methods described in U.S. Publication Nos. 20040171980 and 20040236268; herein incorporated by reference in their entireties.
  • Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, foams, powders, solutions, sprays, inhalants and/or patches.
  • an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
  • the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body.
  • dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium.
  • rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
  • Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
  • liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
  • Topically-administrable formulations may, for example, comprise from about 0.1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • the signal-sensor polynucleotides, primary construct and mmRNA of present invention may use various penetration enhancers to deliver the signal-sensor polynucleotides, primary construct and mmRNA to at least one area associated with one or more hyperproliferative diseases, disorders or conditions.
  • Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail. Combinations of penetration enhancer may also be encompassed by the scope of the present invention, for example, fatty acids/salts in combination with bile acids/salts. Other non-limiting examples of combinations of penetration enhancers include the combination of sodium salt of lauric acid, capric acid and UDCA.
  • surfactants are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of the signal-sensor polynucleotides, primary constructs and mmRNA through the mucosa is enhanced.
  • these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
  • fatty acids and their derivatives which act as penetration enhancers include, but are not limited to, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C 1 -C 10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee
  • bile salts include any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • the bile salts of the invention include, but are not limited to, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's
  • Chelating agents can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of signal-sensor polynucleotides, primary construct and mmRNA through the mucosa is enhanced.
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
  • Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., citric acid
  • salicylates e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen e.g., laureth-9 and N-amino acyl derivatives
  • non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of signal-sensor polynucleotides, primary construct and mmRNA through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).
  • This class of penetration enhancers include, but are not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • Agents that enhance uptake of signal-sensor polynucleotides, primary construct and mmRNA at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of signal-sensor polynucleotides, primary construct and mmRNA.
  • agents may be utilized to enhance the penetration of the administered signal-sensor polynucleotides, primary construct and mmRNA, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • the composition is formulated in depots for extended release.
  • a specific organ or tissue a “target tissue” is targeted for administration.
  • the signal-sensor polynucleotides, primary constructs or mmRNA are spatially retained within or proximal to a target tissue.
  • retention is determined by measuring the amount of the nucleic acid present in the composition that enters one or more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the nucleic acids administered to the subject are present intracellularly at a period of time following administration.
  • intramuscular injection to a mammalian subject is performed using an aqueous composition containing a ribonucleic acid and a transfection reagent, and retention of the composition is determined by measuring the amount of the ribonucleic acid present in the muscle cells.
  • aspects of the invention are directed to methods of providing a composition to a target tissue of a mammalian subject, by contacting the target tissue (containing one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue.
  • the composition contains an effective amount of a signal-sensor polynucleotides, primary constructs or mmRNA such that the polypeptide of interest is produced in at least one target cell.
  • the compositions generally contain a cell penetration agent, although “naked” nucleic acid (such as nucleic acids without a cell penetration agent or other agent) is also contemplated, and a pharmaceutically acceptable carrier.
  • the amount of an oncology-related protein produced by cells in a tissue is desirably increased.
  • this increase in oncology-related protein production is spatially restricted to cells within the target tissue.
  • the composition includes a plurality of different signal-sensor polynucleotides, primary constructs or mmRNA, where one or more than one of the signal-sensor polynucleotides, primary constructs or mmRNA encodes an oncology-related polypeptide of interest.
  • the composition also contains a cell penetration agent to assist in the intracellular delivery of the composition.
  • a determination is made of the dose of the composition required to produce the oncology-related polypeptide of interest in a substantial percentage of cells contained within the predetermined volume of the target tissue (generally, without inducing significant production of the oncology-related polypeptide of interest in tissue adjacent to the predetermined volume, or distally to the target tissue). Subsequent to this determination, the determined dose is introduced directly into the tissue of the mammalian subject.
  • the invention provides for the signal-sensor polynucleotides, primary constructs or mmRNA to be delivered in more than one injection or by split dose injections.
  • the invention may be retained near target tissue using a small disposable drug reservoir or patch pump.
  • patch pumps include those manufactured and/or sold by BD® (Franklin Lakes, N.J.), Insulet Corporation (Bedford, Mass.), SteadyMed Therapeutics (San Francisco, Calif.), Medtronic (Minneapolis, Minn.), UniLife (York, Pa.), Valeritas (Bridgewater, N.J.), and SpringLeaf Therapeutics (Boston, Mass.).
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm.
  • Such compositions are suitably in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
  • Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm.
  • Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1% to 20% (w/w) of the composition.
  • a propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
  • compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension.
  • Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate.
  • Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.
  • Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 ⁇ m to 500 ⁇ m. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient.
  • Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein.
  • Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention.
  • the signal-sensor polynucleotides, primary constructs or mmRNA described herein can be used in a number of different scenarios in which delivery of a substance (the “payload”) to a biological target is desired, for example delivery of detectable substances for detection of the target, or delivery of a therapeutic agent.
  • Detection methods can include, but are not limited to, both imaging in vitro and in vivo imaging methods, e.g., immunohistochemistry, bioluminescence imaging (BLI), Magnetic Resonance Imaging (MRI), positron emission tomography (PET), electron microscopy, X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging, fluorescence reflectance imaging, fluorescence microscopy, fluorescence molecular tomographic imaging, nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging, photoacoustic imaging, lab assays, or in any situation where tagging/staining/imaging is required.
  • imaging in vitro and in vivo imaging methods e.g., immunohistochemistry, bioluminescence imaging (BLI), Magnetic Resonance Imaging (MRI), positron emission tomography (PET), electron microscopy, X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging
  • the signal-sensor polynucleotides, primary constructs or mmRNA can be designed to include both a linker and a payload in any useful orientation.
  • a linker having two ends is used to attach one end to the payload and the other end to the nucleobase, such as at the C-7 or C-8 positions of the deaza-adenosine or deaza-guanosine or to the N-3 or C-5 positions of cytosine or uracil.
  • the signal-sensor polynucleotide of the invention can include more than one payload (e.g., a label and a transcription inhibitor), as well as a cleavable linker.
  • the modified nucleotide is a modified 7-deaza-adenosine triphosphate, where one end of a cleavable linker is attached to the C7 position of 7-deaza-adenine, the other end of the linker is attached to an inhibitor (e.g., to the C5 position of the nucleobase on a cytidine), and a label (e.g., Cy5) is attached to the center of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless in FIG. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporated herein by reference).
  • an inhibitor e.g., to the C5 position of the nucleobase on a cytidine
  • a label e.g., Cy5
  • the resulting signal-sensor polynucleotide having a cleavable linker attached to a label and an inhibitor (e.g., a polymerase inhibitor).
  • an inhibitor e.g., a polymerase inhibitor.
  • the linker e.g., with reductive conditions to reduce a linker having a cleavable disulfide moiety
  • the label and inhibitor are released. Additional linkers and payloads (e.g., therapeutic agents, detectable labels, and cell penetrating payloads) are described herein.
  • the signal-sensor polynucleotides, primary constructs or mmRNA described herein can be used in reprogramming induced pluripotent stem cells (iPS cells), which can directly track cells that are transfected compared to total cells in the cluster.
  • iPS cells induced pluripotent stem cells
  • a drug that may be attached to the signal-sensor polynucleotides, primary constructs or mmRNA via a linker and may be fluorescently labeled can be used to track the drug in vivo, e.g. intracellularly.
  • Other examples include, but are not limited to, the use of signal-sensor polynucleotides, primary constructs or mmRNA in reversible drug delivery into cells.
  • the signal-sensor polynucleotides, primary constructs or mmRNA described herein can be used in intracellular targeting of a payload, e.g., detectable or therapeutic agent, to specific organelle.
  • exemplary intracellular targets can include, but are not limited to, the nuclear localization for advanced mRNA processing, or a nuclear localization sequence (NLS) linked to the mRNA containing an inhibitor.
  • NLS nuclear localization sequence
  • the signal-sensor polynucleotides, primary constructs or mmRNA described herein can be used to deliver therapeutic agents to cells or tissues, e.g., in living animals.
  • the signal-sensor polynucleotides, primary constructs or mmRNA described herein can be used to deliver highly polar chemotherapeutics agents to kill cancer cells.
  • the signal-sensor polynucleotides, primary constructs or mmRNA attached to the therapeutic agent through a linker can facilitate member permeation allowing the therapeutic agent to travel into a cell to reach an intracellular target.
  • the signal-sensor polynucleotides, primary constructs or mmRNA can be attached to the polynucleotides, primary constructs or mmRNA a viral inhibitory peptide (VIP) through a cleavable linker.
  • VIP viral inhibitory peptide
  • the signal-sensor polynucleotides, primary constructs or mmRNA can be attached through the linker to an ADP-ribosylate, which is responsible for the actions of some bacterial toxins, such as cholera toxin, diphtheria toxin, and pertussis toxin.
  • ADP-ribosylate which is responsible for the actions of some bacterial toxins, such as cholera toxin, diphtheria toxin, and pertussis toxin.
  • toxin proteins are ADP-ribosyltransferases that modify target proteins in human cells.
  • cholera toxin ADP-ribosylates G proteins modifies human cells by causing massive fluid secret
  • the payload may be a therapeutic agent such as a cytotoxin, radioactive ion, chemotherapeutic, or other therapeutic agent.
  • a cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S.
  • Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium.
  • iodine e.g., iodine 125 or iodine 131
  • strontium 89 phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium.
  • therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and
  • the payload may be a detectable agent, such as various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18 F, 67 Ga, 81m Kr, 82 Rb, 111 In, 123 I, 133 Xe, 201 Tl, 125 I, 35 S, 14 C, 3 H, or 99m Tc (e.g., as pertechnetate (technetate(VII), TcO 4 ⁇ )), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles
  • fluorescent materials
  • optically-detectable labels include for example, without limitation, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives (e.g., acridine and acridine isothiocyanate); 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanosine; 4′,6-
  • the detectable agent may be a non-detectable pre-cursor that becomes detectable upon activation (e.g., fluorogenic tetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))).
  • fluorogenic tetrazine-fluorophore constructs e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X
  • enzyme activatable fluorogenic agents e.g., PROSENSE® (VisEn Medical)
  • ELISAs enzyme linked immunosorbent assays
  • IA enzyme immunoassays
  • RIA radioimmunoassays
  • Western blot analysis In vitro assays in which the enzyme labeled compositions can be used include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence, enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.
  • the signal-sensor polynucleotides, primary constructs or mmRNA may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
  • Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • the signal-sensor nucleic acids or mmRNA may be used in combination with a pharmaceutical agent for the treatment of cancer or to control hyperproliferative cells.
  • a combination therapy for the treatment of solid primary or metastasized tumor is described using a pharmaceutical composition including a DNA plasmid encoding for interleukin-12 with a lipopolymer and also administering at least one anticancer agent or chemotherapeutic.
  • the signal-sensor nucleic acids and mmRNA of the present invention that encodes anti-proliferative molecules may be in a pharmaceutical composition with a lipopolymer (see e.g., U.S. Pub. No.
  • the present invention provides methods comprising administering modified mRNAs and their encoded proteins or complexes in accordance with the invention to a subject in need thereof.
  • Nucleic acids, proteins or complexes, or pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof may be administered to a subject using any amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition relating to working memory deficits).
  • a disease, disorder, and/or condition e.g., a disease, disorder, and/or condition relating to working memory deficits.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.
  • the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose.
  • a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.
  • the mmRNA of the present invention are administered to a subject in split doses.
  • the mmRNA may be formulated in buffer only or in a formulation described herein.
  • a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous.
  • Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art including, but not limited to,
  • compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed include, but are not limited to, are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the absorption of the active ingredient may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility.
  • the rate of absorption of the signal-sensor polynucleotide, primary construct or mmRNA then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally administered signal-sensor polynucleotide, primary construct or mmRNA may be accomplished by dissolving or suspending the signal-sensor polynucleotide, primary construct or mmRNA in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the signal-sensor polynucleotide, primary construct or mmRNA in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of the signal-sensor polynucleotide, primary construct or mmRNA to polymer and the nature of the particular polymer employed, the rate of signal-sensor polynucleotide, primary construct or mmRNA release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the signal-sensor polynucleotide, primary construct or mmRNA in liposomes or microemulsions which are compatible with body tissues.
  • Formulations described herein as being useful for pulmonary delivery may also be use for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 ⁇ m to 500 ⁇ m.
  • Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
  • Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • compositions described herein can be characterized by one or more of bioavailability, therapeutic window and/or volume of distribution.
  • the signal-sensor polynucleotides, primary constructs or mmRNA when formulated into a composition with a delivery agent as described herein, can exhibit an increase in bioavailability as compared to a composition lacking a delivery agent as described herein.
  • bioavailability refers to the systemic availability of a given amount of signal-sensor polynucleotides, primary constructs or mmRNA administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (C max ) of the unchanged form of a compound following administration of the compound to a mammal.
  • AUC is a determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis).
  • the AUC for a particular compound can be calculated using methods known to those of ordinary skill in the art and as described in G. S. Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, herein incorporated by reference.
  • the C max value is the maximum concentration of the compound achieved in the serum or plasma of a mammal following administration of the compound to the mammal.
  • the C max value of a particular compound can be measured using methods known to those of ordinary skill in the art.
  • the phrases “increasing bioavailability” or “improving the pharmacokinetics,” as used herein mean that the systemic availability of a first signal-sensor polynucleotide, primary construct or mmRNA, measured as AUC, C max , or C min in a mammal is greater, when co-administered with a delivery agent as described herein, than when such co-administration does not take place.
  • the bioavailability of the signal-sensor polynucleotide, primary construct or mmRNA can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • the signal-sensor polynucleotides, primary constructs or mmRNA when formulated into a composition with a delivery agent as described herein, can exhibit an increase in the therapeutic window of the administered signal-sensor polynucleotide, primary construct or mmRNA composition as compared to the therapeutic window of the administered signal-sensor polynucleotide, primary construct or mmRNA composition lacking a delivery agent as described herein.
  • therapeutic window refers to the range of plasma concentrations, or the range of levels of therapeutically active substance at the site of action, with a high probability of eliciting a therapeutic effect.
  • the therapeutic window of the signal-sensor polynucleotide, primary construct or mmRNA when co-administered with a delivery agent as described herein can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • the signal-sensor polynucleotides, primary constructs or mmRNA when formulated into a composition with a delivery agent as described herein, can exhibit an improved volume of distribution (V dist ), e.g., reduced or targeted, relative to a composition lacking a delivery agent as described herein.
  • the volume of distribution (V dist ) relates the amount of the drug in the body to the concentration of the drug in the blood or plasma.
  • volume of distribution refers to the fluid volume that would be required to contain the total amount of the drug in the body at the same concentration as in the blood or plasma: V dist equals the amount of drug in the body/concentration of drug in blood or plasma.
  • the volume of distribution would be 1 liter.
  • the volume of distribution reflects the extent to which the drug is present in the extravascular tissue.
  • a large volume of distribution reflects the tendency of a compound to bind to the tissue components compared with plasma protein binding.
  • V dist can be used to determine a loading dose to achieve a steady state concentration.
  • the volume of distribution of the signal-sensor polynucleotide, primary construct or mmRNA when co-administered with a delivery agent as described herein can decrease at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.
  • the biological effect of the signal-sensor modified mRNA delivered to the animals may be categorized by analyzing the protein expression in the animals.
  • the protein expression may be determined from analyzing a biological sample collected from a mammal administered the signal-sensor modified mRNA of the present invention.
  • the expression protein encoded by the signal-sensor modified mRNA administered to the mammal of at least 50 pg/ml may be preferred.
  • a protein expression of 50-200 pg/ml for the protein encoded by the signal-sensor modified mRNA delivered to the mammal may be seen as a therapeutically effective amount of protein in the mammal.
  • Mass spectrometry is an analytical technique that can provide structural and molecular mass/concentration information on molecules after their conversion to ions.
  • the molecules are first ionized to acquire positive or negative charges and then they travel through the mass analyzer to arrive at different areas of the detector according to their mass/charge (m/z) ratio.
  • Mass spectrometry is performed using a mass spectrometer which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis.
  • ionization of the sample may be performed by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization.
  • ESI electrospray ionization
  • APCI atmospheric pressure chemical ionization
  • FAB fast atom bombardment
  • LIMS liquid secondary ionization
  • MALDI matrix assisted laser desorption/ionization
  • field ionization field desorption
  • thermospray/plasmaspray ionization and particle beam ionization.
  • the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio (i.e., m/z).
  • Suitable analyzers for determining mass-to-charge ratios include quadropole analyzers, ion traps analyzers, and time-of-flight analyzers.
  • the ions may be detected using several detection modes. For example, selected ions may be detected (i.e., using a selective ion monitoring mode (SIM)), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
  • SIM selective ion monitoring mode
  • MRM multiple reaction monitoring
  • SRM selected reaction monitoring
  • LC-MS/MRM Liquid chromatography-multiple reaction monitoring
  • MRM multiple reaction monitoring
  • a biological sample which may contain at least one protein encoded by at least one modified mRNA of the present invention may be analyzed by the method of MRM-MS.
  • the quantification of the biological sample may further include, but is not limited to, isotopically labeled peptides or proteins as internal standards.
  • the biological sample once obtained from the subject, may be subjected to enzyme digestion.
  • digest means to break apart into shorter peptides.
  • the phrase “treating a sample to digest proteins” means manipulating a sample in such a way as to break down proteins in a sample.
  • enzymes include, but are not limited to, trypsin, endoproteinase Glu-C and chymotrypsin.
  • a biological sample which may contain at least one protein encoded by at least one modified mRNA of the present invention may be digested using enzymes.
  • a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein using electrospray ionization.
  • Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to aid in the transfer of ions from the solution to the gaseous phase before they are analyzed by mass spectrometry.
  • Samples may be analyzed using methods known in the art (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12).
  • the ionic species contained in solution may be transferred into the gas phase by dispersing a fine spray of charge droplets, evaporating the solvent and ejecting the ions from the charged droplets to generate a mist of highly charged droplets.
  • the mist of highly charged droplets may be analyzed using at least 1, at least 2, at least 3 or at least 4 mass analyzers such as, but not limited to, a quadropole mass analyzer.
  • the mass spectrometry method may include a purification step.
  • the first quadrapole may be set to select a single m/z ratio so it may filter out other molecular ions having a different m/z ratio which may eliminate complicated and time-consuming sample purification procedures prior to MS analysis.
  • a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein in a tandem ESIMS system (e.g., MS/MS).
  • the droplets may be analyzed using a product scan (or daughter scan) a precursor scan (parent scan) a neutral loss or a multiple reaction monitoring.
  • a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS).
  • MALDI matrix-assisted laser desorption/ionization
  • MALDIMS matrix-assisted laser desorption/ionization mass spectrometry
  • MALDI provides for the nondestructive vaporization and ionization of both large and small molecules, such as proteins.
  • the analyte is first co-crystallized with a large molar excess of a matrix compound, which may also include, but is not limited to, an ultraviolet absorbing weak organic acid.
  • Non-limiting examples of matrices used in MALDI are ⁇ -cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.
  • Laser radiation of the analyte-matrix mixture may result in the vaporization of the matrix and the analyte.
  • the laser induced desorption provides high ion yields of the intact analyte and allows for measurement of compounds with high accuracy.
  • Samples may be analyzed using methods known in the art (e.g., Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894).
  • mass analyzers used in the MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or a Fourier transform mass analyzer.
  • the analyte-matrix mixture may be formed using the dried-droplet method.
  • a biologic sample is mixed with a matrix to create a saturated matrix solution where the matrix-to-sample ratio is approximately 5000:1.
  • An aliquot (approximately 0.5-2.0 uL) of the saturated matrix solution is then allowed to dry to form the analyte-matrix mixture.
  • the analyte-matrix mixture may be formed using the thin-layer method.
  • a matrix homogeneous film is first formed and then the sample is then applied and may be absorbed by the matrix to form the analyte-matrix mixture.
  • the analyte-matrix mixture may be formed using the thick-layer method.
  • a matrix homogeneous film is formed with a nitro-cellulose matrix additive. Once the uniform nitro-cellulose matrix layer is obtained the sample is applied and absorbed into the matrix to form the analyte-matrix mixture.
  • the analyte-matrix mixture may be formed using the sandwich method.
  • a thin layer of matrix crystals is prepared as in the thin-layer method followed by the addition of droplets of aqueous trifluoroacetic acid, the sample and matrix. The sample is then absorbed into the matrix to form the analyte-matrix mixture.
  • the signal-sensor polynucleotides, primary constructs and mmRNA of the present invention are designed, in preferred embodiments, to provide for avoidance or evasion of deleterious bio-responses such as the immune response and/or degradation pathways, overcoming the threshold of expression and/or improving protein production capacity, improved expression rates or translation efficiency, improved drug or protein half life and/or protein concentrations, optimized protein localization, to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, secretion efficiency (when applicable), accessibility to circulation, and/or modulation of a cell's status, function and/or activity.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine.
  • signal-sensor polynucleotide, primary construct or mmRNA described herein can be administered to a subject, wherein the signal-sensor polynucleotide, primary construct or mmRNA is translated in vivo to produce a therapeutic or prophylactic oncology-related polypeptide in the subject.
  • compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals are provided.
  • the active therapeutic agents of the invention include signal-sensor polynucleotides, primary constructs or mmRNA, cells containing polynucleotides, primary constructs or mmRNA or polypeptides translated from the signal-sensor polynucleotides, primary constructs or mmRNA.
  • combination therapeutics containing one or more signal-sensor polynucleotide, primary construct or mmRNA containing translatable regions that encode for a protein or proteins that boost a mammalian subject's immunity along with a protein that induces antibody-dependent cellular toxicity.
  • a recombinant polypeptide in a cell population using the signal-sensor polynucleotide, primary construct or mmRNA described herein.
  • Such translation can be in vivo, ex vivo, in culture, or in vitro.
  • the cell population is contacted with an effective amount of a composition containing the signal-sensor nucleic acid that has at least one nucleoside modification, and a translatable region encoding the recombinant oncology-related polypeptide.
  • the population is contacted under conditions such that the signal-sensor nucleic acid is localized into one or more cells of the cell population and the recombinant oncology-related polypeptide is translated in the cell from the signal-sensor nucleic acid.
  • an “effective amount” of the composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the nucleic acid (e.g., size, and extent of modified nucleosides), and other determinants.
  • an effective amount of the composition provides efficient protein production in the cell, preferably more efficient than a composition containing a corresponding unmodified nucleic acid. Increased efficiency may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid), increased protein translation from the nucleic acid, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified nucleic acid), or reduced innate immune response of the host cell.
  • aspects of the invention are directed to methods of inducing in vivo translation of a recombinant polypeptide in a mammalian subject in need thereof.
  • an effective amount of a composition containing a nucleic acid that has at least one structural or chemical modification and a translatable region encoding the recombinant polypeptide is administered to the subject using the delivery methods described herein.
  • the nucleic acid is provided in an amount and under other conditions such that the nucleic acid is localized into a cell of the subject and the recombinant polypeptide is translated in the cell from the nucleic acid.
  • the cell in which the nucleic acid is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of nucleic acid administration.
  • the administered signal-sensor polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that provide a functional activity which is substantially absent in the cell, tissue or organism in which the recombinant oncology-related polypeptide is translated.
  • the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
  • the administered signal-sensor polynucleotide, primary construct or mmRNA directs production of one or more recombinant oncology-related polypeptides that increases (e.g., synergistically) a functional activity which is present but substantially deficient in the cell in which the recombinant oncology-related polypeptide is translated.
  • the administered signal-sensor polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that replace an oncology-related polypeptide (or multiple oncology-related polypeptides) that is substantially absent in the cell in which the recombinant oncology-related polypeptide is translated.
  • Such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof.
  • the recombinant oncology-related polypeptide increases the level of an endogenous oncology-related protein in the cell to a desirable level; such an increase may bring the level of the endogenous oncology-related protein from a subnormal level to a normal level or from a normal level to a super-normal level.
  • the recombinant oncology-related polypeptide functions to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell.
  • the activity of the endogenous oncology-related protein is deleterious to the subject; for example, due to mutation of the endogenous oncology-related protein resulting in altered activity or localization.
  • the recombinant oncology-related polypeptide antagonizes, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell.
  • antagonized biological moieties include lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a nucleic acid, a carbohydrate, a protein toxin such as shiga and tetanus toxins, or a small molecule toxin such as botulinum, cholera, and diphtheria toxins. Additionally, the antagonized biological molecule may be an endogenous protein that exhibits an undesirable activity, such as a cytotoxic or cytostatic activity.
  • the recombinant oncology-related proteins described herein may be engineered for localization within the cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
  • modified signal-sensor mRNAs and their encoded oncology-related polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions described herein.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be used in the treatment, management, characterization and/or diagnosis of cancer, a cancer-related and/or a cancer treatment-related disorder, side effect and/or condition.
  • Such disease, disorders and conditions include, but are not limited to, adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be used in the treating, managing or manipulating at least one cancer-related or cancer treatment-related disorder, side effect or condition such as chemo brain, peripheral neuropathy, fatigue, depression, nausea and vomiting, pain, anemia, lymphedema, infections, second cancers caused by cancer treatment, sexual side effects, reduced fertility or infertility, ostomies, insomnia and hair loss.
  • side effect or condition such as chemo brain, peripheral neuropathy, fatigue, depression, nausea and vomiting, pain, anemia, lymphedema, infections, second cancers caused by cancer treatment, sexual side effects, reduced fertility or infertility, ostomies, insomnia and hair loss.
  • the signal-sensor polynucleotides, primary constructs and/or mmRNA may be used to reduce the effect of at least one symptom of cancer in a subject.
  • the symptom may include, but is not limited to, weakness, aches and pains, fever, fatigue, weight loss, blood clots, increased blood calcium levels, low white blood cell count, short of breath, dizziness, headaches, hyperpigmentation, jaundice, erthema, pruritis, excessive hair growth, change in bowel habits, change in bladder function, long-lasting sores, white patches inside the mouth, white spots on the tongue, unusual bleeding or discharge, thickening or lump on parts of the body, indigestion, trouble swallowing, changes in warts or moles, change in new skin and nagging cough or hoarseness.
  • the signal-sensor polynucleotides may be investigated in any number of cancer or normal cell lines.
  • Non-limiting examples of cell lines which may be useful in these investigations include those from ATCC (Manassas, Va.) including MRC-5, A549, T84, NCI-H2126 [H2126], NCI-H1688 [H1688], WI-38, WI-38 VA-13 subline 2RA, WI-26 VA4, C3A [HepG2/C3A, derivative of Hep G2 (ATCC HB-8065)], THLE-3, H69AR, NCI-H292 [H292], CFPAC-1, NTERA-2 cl.D1 [NT2/D1], DMS 79, DMS 53, DMS 153, DMS 114, MSTO-211H, SW 1573 [SW-1573, SW1573], SW 1271 [SW-1271, SW1271], SHP-77, SNU-398, SNU-449, SNU-182, SNU-475, SNU
  • the signal-sensor polynucleotides described herein may be investigated in human lung adenocarcinoma.
  • a signal-sensor polynucleotide encoding constitutively active caspase 3 fully modified with 5-methylcytidine and 1-methylpseudouridine or fully modified with 1-methylpseudouridine may be delivered to cultured human lung adenocarcinoma A549 cells (see e.g., the experiment outlined in Example 53).
  • a signal-sensor polynucleotide encoding constitutively active caspase 6 fully modified with 5-methylcytidine and 1-methylpseudouridine or fully modified with 1-methylpseudouridine may be delivered to cultured human lung adenocarcinoma A549 cells (see e.g., the experiment outlined in Example 53).
  • the signal-sensor polynucleotides described herein may be investigated in human hepatocellular carcinoma.
  • a signal-sensor polynucleotide encoding constitutively active caspase 3 fully modified with 5-methylcytidine and 1-methylpseudouridine or fully modified with 1-methylpseudouridine may be delivered to human hepatocellular carcinoma Hep3B cells (see e.g., the experiment outlined in Example 54).
  • the signal-sensor polynucleotides may be investigated in an animal model.
  • the animal model may be for lung cancer such as the lung cancer model of Fukazawa et al (Anticancer Research, 2010; 30: 4193-4200) where a congenic mouse is created by crossing a ubiquitously expressing dominant negative Myc (Omomyc) mouse with a KRAS mutation-positive lung cancer model mouse.
  • Omomyc ubiquitously expressing dominant negative Myc
  • lung tumors caused by the expression of mutated KRAS regresses in the congenic mouse, indicating that Omomyc caused tumor cell death of KRAS mutation-positive lung cancer.
  • the signal-sensor polynucleotides may be investigated in a transgenic animal model.
  • the transgenic animal model is a LSL-KRAS G12D : TRE Omomyc:CMV rtTA triple transgenic model which involves the use of an adenovirus expressing Cre recombinase which is administered via inhalation to induce oncogene expression via excision of the floxed STOP codon, and ubiquitous Omomyc expression is controlled via doxycycline.
  • the model is reported in Soucek et al. (Nature, 1-5 (2008)).
  • mice of Soucek may be crossed with the LSLKRAS G12D single transgenic mice (Jackson Laboratories) and may be used for inhalation delivered or otherwise lung-delivered studies of signal-sensor polynucleotides expressing MYC inhibitor D or other oncology related polypeptide described herein.
  • the signal-sensor polynucleotides may be investigated in a mouse-in-mouse model such as, but not limited to a model which is akin to the p53 ⁇ / ⁇ :c-Myc overexpressing HCC model of Zender (Cell. 2006 Jun. 30; 125(7): 1253-1267).
  • the signal-sensor polynucleotides may be investigated in a Nongermline genetically engineered mouse model (NGEMM).
  • NGEMM Nongermline genetically engineered mouse model
  • the design of mouse-in-mouse model may involve starting with the WT or tumor suppressor deleted (such as p53 ⁇ / ⁇ ) 129 Sv/Ev Mm ES cell clone; introduction of liver activated protein (LAP) promoter directed tetracycline transactivator (tTA) and tetO-luciferase for liver specific imaging; freezing the resulting LAP-tTA: tetO-luciferase clones to be used for c-Myc as well as other liver relevant programs oncogene; adding tetO driven oncogene, e.g.
  • LAP liver activated protein
  • tTA tetracycline transactivator
  • tO-luciferase for liver specific imaging
  • tetOcMyc Freeze resulting LAP-tTA: tetO-luciferase: tetO-MYC clones; injecting resulting ES clones into C57Bl/6 blastocytes and implant in pseudo pregnant mothers whereby the resulting chimeric animals are the tumor model upon removal of doxycycline (i.e. Tet-Off).
  • Tet-Off doxycycline
  • the type of model will ideally evince inducible nodules of c-Myc-driven, luciferase-expressing HCC surrounded by normal hepatocytes.
  • the signal-sensor polynucleotides may be investigated in Orthotopic HCC models using the HEP3B cell lines in mice (Crown Bio).
  • any of the animal models described above may be used to investigate a signal-sensor polynucleotide encoding MYC inhibitor D.
  • the study may also include a signal-sensor polynucleotide encoding a negative control such as, but not limited to, an untranslatable mRNA for MYC inhibitor D and a vehicle only delivery.
  • the animal may be evaluated for gene expression, tumor status and/or for any of the hallmarks that are generally associated with cancer phenotypes or genotypes.
  • any of the animal models described above may be used to investigate a signal-sensor polynucleotide encoding dominant negative hTERT.
  • the study may also include a signal-sensor polynucleotide encoding a negative control such as, but not limited to, an untranslatable mRNA for dominant negative hTERT and a vehicle only delivery.
  • the animal may be evaluated for gene expression, tumor status and/or for any of the hallmarks that are generally associated with cancer phenotypes or genotypes.
  • any of the animal models described above may be used to investigate a signal-sensor polynucleotide encoding dominant negative survivin.
  • the study may also include a signal-sensor polynucleotide encoding a negative control such as, but not limited to, an untranslatable mRNA for dominant negative survivin and a vehicle only delivery.
  • the animal may be evaluated for gene expression, tumor status and/or for any of the hallmarks that are generally associated with cancer phenotypes or genotypes.
  • signal-sensor polynucleotides may include at least one miRNA-binding site in the 3′UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells in an animal model described herein.
  • a strong apoptotic signal and at least one miR-122a binding site is encoded by the signal-sensor polynucleotide where the at least one miR-122a binding site is located in the 3′UTR.
  • apoptosis inducing factor short isoform (AIFsh) and at least one miR-122a binding site is encoded by the signal-sensor polynucleotide where the at least one miR-122a binding site is located in the 3′UTR.
  • HSV1-tk and at least one miR-122a binding site is encoded by the signal-sensor polynucleotide where the at least one miR-122a binding site is located in the 3′UTR.
  • signal-sensor polynucleotides may include three miRNA-binding sites in the 3′UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells in an animal model described herein.
  • a strong apoptotic signal and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • apoptosis inducing factor short isoform (AIFsh) and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • constitutively active (C.A.) caspase 6 and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • HSV1-tk and three miR-122a binding sites are encoded by the signal-sensor polynucleotide where the three miR-122a binding sites are located in the 3′UTR.
  • Brain cancer is the growth of abnormal cells in the tissues of the brain usually related to the growth of malignant brain tumors. Brain tumors grow and press on the nearby areas of the brain which can stop that part of the brain from working the way it should. Brain cancer rarely spreads into other tissues outside of the brain.
  • the grade of tumor based on how abnormal the cancer cells look under a microscope, may be used to tell the difference between slow- and fast-growing tumors. Grade I tumors grow slowly, rarely spreads into nearby tissues, has cells that look like normal cells and the entire tumor may be removable by surgery. Grade II tumors also grow slowly but may spread into nearby tissue and may recur. Grade III tumors grow quickly, is likely to spread into nearby tissue and the tumor cells look very different from normal cells.
  • Symptoms of brain cancer may include, but are not limited to, morning headache or headache that goes away after vomiting, frequent nausea and vomiting, vision, hearing, and speech problems, loss of balance and trouble walking, weakness on one side of the body, unusual sleepiness or change in activity level, unusual changes in personality or behavior, seizures.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with brain cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with brain cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with brain cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • breast cancer forms in the tissues of the breast, of both men and women, such as, but not limited to, the ducts and the lobules.
  • the most common type of breast cancer is ductal carcinoma which begins in the cells of the ducts.
  • Lobular cancer which begins in the lobes or lobules, is often found in both breasts.
  • An uncommon type of breast cancer, inflammatory breast cancer causes the breast to be warm, red and swollen.
  • Hereditary breast cancer makes up approximately 5-10% of all breast cancer and altered genes are common in some ethnic groups making that ethnic group more susceptible to breast cancer.
  • Symptoms of breast cancer include, but are not limited to, a lumpm or thickening in or near the breast or in the underarm area, change in the size or shape of the breast, dimple or puckering in the skin of the breast, inward turned nipple of the breast, fluid from the nipple which is not breast milk, scaly, red or swollen skin on the breast, nipple, or areola, and dimples in the breast that look like the skin of orange (peau d′orange).
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with breast cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with breast cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with breast cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Cervical cancer forms in the tissues of the cervic and is usually slow-growing.
  • the cause of cervical cancer usually related to the human papillomavirus (HPV) infection.
  • HPV human papillomavirus
  • cervical cancer may not show any signs, possible symptoms may include, but are not limited to, vaignal bleeding, unusual vaginal discharge, pelvic pain and pain during sexual intercourse.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with cervical cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with cervical cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with cervical cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Esophageal cancer is cancer that forms in the tissues lining the esophagus.
  • Squamous cell carcinoma is cancer that forms in the thin, flat cells lining the esophagus (also called epidermoid carcinoma).
  • Cancer that begins in the glandular (secretory) cells which produce and release fluids such as mucus is called adneocarcinoma.
  • Common symptoms associated with esophageal cancer include, but are not limited to, painful or difficult swallowing, weight loss, pain behind the breastbone, hoarseness and cough, and indigestion and heartburn.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with esophageal cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with esophageal cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with esophageal cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide.
  • Familial cancer syndrome describes the genetic predisposition of a subject to develop cancer. 5-10% of all cancers are hereditary and are passed on through specific in specific genes passed from one blood relative to another. Subjects that inherit one of these gene changes may have a higher likelihood of developing cancer within their lifetime.
  • Familial cancer syndrome includes disorder such as, but not limited to, Ataxia Telangiectasia, Basal Cell Nevus Syndrome, Nevoid Basal Cell Carcinoma Syndrome, Gorlin Syndrome, Beck-with Wiedemann Syndrome, Birt-Hogg-Dube Syndrome, Bloom Syndrome, hereditary breast and/or ovarian cancer, Carney Complex, Types I and II, Familial Chordoma, Colon Cancer, Hereditary Nonpolyposis-Lynch Syndrome, Costello Syndrome, Facio-Cutaneous-Skeletal Syndrome, Cowden Syndrome, Dyskeratosis Congenita, Tylosis with Esophaeal Cancer, Keratosis Palmaris et Plantaris with Esophageal Cancer, Howel-Evans Syndrome, Herediatary Multiple Exostosis, Fanconi Anemia, Hereditary Diffuse Gastric Cancer, Gastrointestinal Stromal Tumor, Multiple Gastrointestinal Stromal Tumor, Familial Hyperparathyroidism, Acute Mye
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with Familial cancer syndrome by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with Familial cancer syndrome by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with Familial cancer syndrome by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Leukemia is a form of cancer that starts in blood-forming tissue such as the bone marrow which can cause a large number of blood cells to be produced and enter the blood stream. Leukemia can also spread to the central nervous system and cause brain and spinal cord cancer. Types of leukemia include, but are not limited to, adult acute lymphoblastic, childhood acute lymphoblastic, aduct acute myeloid, chronic lymphocytic, chronic myelogenous and hairy cell.
  • Non-limiting examples of symptoms of leukemia include weakness or feeling tired, fever, easy bruising or bleeding, petechiae, shortness of breath, weight loss or loss of appetite, pain in the bones or stomach, pain or feeling of fullness below the ribs, and painless lumps in the neck, underarm, stomach or groin.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with leukemia by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with leukemia by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with leukemia by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • liver cancer There are two types of liver cancer, primary liver cancer which forms in the tissue of the liver and secondary liver cancer, or metastatic liver cancer, that spreads to the liver from another part of the body. Possible symptoms of liver cancer include, but are not limited to, a hard lump on the right side just below the rib cage, discomfort in the upper abdomen on the right side, pain around the right shoulder blade, unexplained weight loss, jaundice, unusual tiredness, nausea and loss of appetite.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with liver cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with liver cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with liver cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the c-myc protein is a multifunctional bHLHZip transcription factor with critical roles in normal cellular processes and aberrantly regulated in the majority of human cancers.
  • c-, N- and L-Myc are family members that can dimerize with partners such as Max, Mad and Miz-1.
  • the protein is implicated in the transactivation and repression of a vast number of proposed transcriptional targets and recent work has demonstrated a role for Myc as a “transcriptional amplifier” of otherwise transactivated genes in developing cancers. It has a well established function in cancer cell proliferation, growth, biosynthetic metabolism, ribogenesis and translation and possibly a non-redundant node through which oncogenic signals must navigate.
  • MYC inhibitor D (also known as Omomyc) is a unique dominant-negative 90 a.a. protein comprised of the human c-Myc oligomerization domain with 4 introduced mutations E57T, E64I, R70Q, R71N (Soucek et al., Oncogene, 1998; 17, 2463-2472). Importantly, it exhibits selectivity in binding and inhibitory capability: binding c-Myc, N-Myc, Max and Miz-1. It also prevents E-box mediated transactivation while retaining Miz-1 directed transrepression.
  • MYC inhibitor D has been specifically exhibited in vivo where transgenic expression of OMOMYC blocked MycERTAM induced keratinocyte proliferation (Soucek et al., CDD 2004; 11, 1038-1045); transgenic Omomyc prevented the establishment and induced the regression of forming and mature lung tumors, respectively, in the LSL-KrasG12D mouse model with reversible toxicity (Soucek et al., Nature 2008, 455, 679-683); transgenic Omomyc prevents tumor formation and regresses established tumors in the RIP 1-TAG2 model of pancreatic neuroendocrine cancer with controllable side effects, and further shows a role for cancer cell Myc in the maintenance of a permissive tumor microenvironment (Sodir et al., Genes and Development 2011, 25, 907-916); and it was reported “that Omomyc induces cell death of KRAS-mutated human lung adenocarcinoma A549 cells in vitro and
  • the present invention includes signal-sensor polynucleotides encoding MYC inhibitor D as the oncology-related polypeptide; with or without a sensor sequence for the treatment of hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • the studies of HCC may be performed in any of the subclasses of HCC cell lines as described by Hoshida et al (Cancer Research 2009; 69: 7385-7392). These include S2 cells which have higher TGF-beta and WNT signaling and demonstrate and associated with a greater risk of early recurrence, S2 which exhibit increased myc and AKT expression and the highest level of alpha feto-protein or S3 which retain the hepatocyte like phenotype.
  • S1 and S2 types have also been shown to exhibit increased E2F1 and decreased p53 expression; while S2 alone has shown decreased levels of interferon.
  • S1 cell lines include SNU-387, SNU-423, SNU-449, SNU-475, SNU-182, SK-Hep1, HLE, HLF, and Focus, whereas S2 cell lines include Huh-1, Huh-6, Huh-7, HepG2, Hep3B, Hep3B-TR, Hep40, and PLC/PRF/5 cells.
  • Lung cancer forms in the tissues of the lung usually in the cells lining the air passages and is classified as either small cell lung cancer or non-small cell lung cancer.
  • small cell lung cancer There are two types of small cell lung cancer, small cell carcinoma and combined small cell carcinoma.
  • the types of on-small cell lung cancer are squamous cell carcinoma (cancer begins in the squamous cells), large cell carcinoma (cancer may begin in several types of cells) and adenocarcinoma (cancer begins in the cells that line the alveoli and in cells that make mucus).
  • Symptoms of lung cancer include, but are not limited to, chest discomfort or pain, cough that does not go away or gets worse over time, trouble breathing, wheezing, blood in the sputum, hoarseness, loss of appetite, weight loss for no known reason, feeling very tired, trouble swallowing and swelling in the face and/or veins in the neck.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with lung cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with lung cancer by administering to said subject an isolated polynucleotide encoding a polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with lung cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Lymphoma is cancer that beings in the cells of the immune system.
  • Subjects who have Hodgkin lymphoma have a cell called Reed-Sternberg cell and non-Hodgkin lymphoma includes a large group of cancers of immune system cells.
  • Examples of Lymphoma include, but are not limited to, painless, swollen lymph nodes in the neck, underarm or groin, fever for no known reason, drenching night sweats, weight loss for no known reason, itchy skin and fatigue.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with lymphoma by administering to said subject an isolated polynucleotide encoding a polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with lymphoma by administering to said subject an isolated polynucleotide encoding a polypeptide of interest.
  • the polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with lymphoma by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Ovarian cancer is cancer which forms in the tissues of the ovary which are either ovarian epithelial carcinomas (begins on the surface of the ovary) or malignant germ cell tumors (cancer that begins in the egg cells).
  • Symptoms of ovarian cancer include, but are not limited to, pain or swelling in the abdomen, pain in the pelvis, gastrointestinal problems such as gas, bloating, or constipation and vaginal bleeding after menopause.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with ovarian cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or signal-sensor mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with ovarian cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or signal-sensor mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with ovarian cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Prostate that forms in the tissue of the prostate mainly affects older men.
  • Non-limiting examples of prostate cancer include, but are not limited to, weak or interrupted flow of urine, frequent urination, trouble urinating, pain or burning during urination, blood in the urine or semen, pain in the back, hips or pelvis that does not go away and painful ejaculation.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with prostate cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with prostate cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with prostate cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Testicular cancer forms in the tissues of one or both testicles and is most common in young or middle-aged men. Most testicular cancers being in germ cells and are called testicular germ cell tumors. There are two types of testicular germ cell tumors called seminomas and nonseminomas. Common symptoms of testicular cancer include, but are not limited to, a painless lump or swelling in either testicle, change in how the testicle feels, dull ache in the lower abdomen or the groin, sudden build-up of fluid in the scrotum and pain or discomfort in a testicle or in the scrotum.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with testicular cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with testicular cancer by administering to said subject an isolated signal-sensor polynucleotide encoding an oncology-related polypeptide of interest.
  • the polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with testicular cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Throat cancer forms in the tissues of the pharynx and includes cancer of the nasopharynx (nasopharyngeal cancer), oropharynx (oropharyngeal cancer), hypopharynx (hypopharyngeal cancer), and larynx (laryngeal cancer).
  • Common symptoms of throat cancer include, but are not limited to, a sore throat that does not go away, ear pain, lump in the neck, painful or difficulty swallowing, change or hoarseness in the voice, trouble breathing or speaking, nosebleeds, trouble hearing, pain or ringing in the ear, headaches, dull pain behind the breast bone, cough and weight loss for no reason.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to treat a disease, disorder and/or condition in a subject who has been diagnosed or may be diagnosed with throat cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce, eliminate, or prevent tumor growth in a subject who has been diagnosed or may be diagnosed with throat cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • the signal-sensor polynucleotides, primary constructs or mmRNA of the present invention may be used to reduce and/or ameliorate at least one symptom of cancer in a subject who has been diagnosed or may be diagnosed with throat cancer by administering to said subject an isolated polynucleotide encoding an oncology-related polypeptide of interest.
  • Hypoxia-inducible factors control cellular adaptation to oxygen deprivation. Cancer cells engage HIFs to sustain their growth in adverse conditions, thus promoting a cellular reprogramming that includes metabolism, proliferation, survival and mobility. HIFs overexpression in human cancer biopsies correlates with high metastasis and mortality.
  • HIFs regulate genes related to metabolism such as GLUT1, GLUT3, ALDOA, ENO1, GAPDH, HK1, HK2, PFKL, PGK1, PKM2, LDHA, proliferation such as IGF-2, TGFA, VEGFA, survival such as TERT, NANOG, OCT4 and cell migration-invasion such as ZEB1, ZEB2, SNAI2, MMP14, MMP9, AMF, MET, PTHrP. (Keith, et al Nat Rev Cancer 2012; 12:9-22).
  • one or more signal-sensor polynucleotides may be administered to the cancer cell to investigate the destabilization of cancer.
  • the selection of the sequence, dose or administrative route is optionally informed by diagnostic evaluation of the cell, tumor, tissue or organism including, but not limited to, expression profiling of the cancer, metabolic evaluation (hypoxic, acidotic), apoptotic vs. survival profiling, cell cycle vs. senescent profiling, immune sensitivities, and/or evaluation of stromal factors.
  • the signal-sensor polynucleotides may encode either or both of the oncology related polypeptides, CITED4 and SHARP1.
  • the signal-sensor polynucleotides are then administered where the administration of either or both results in the inhibition of the transcriptome of HIF-1alpha in cancer cells. Suppression of HIF1-alpha gene regulated expression occurs upon administration with higher suppression when both polynucleotides are administered together. Reporter constructs such as luciferase under HIF1-alpha are used in the manner similar to the methods disclosed in van de Sluis et al, (J Clin Invest. 2010; 120(6):2119-2130).
  • additional experiments can be conducted using a cancer cell line where CITED4 and SHARP1 are themselves down regulated either under hypoxic conditions.
  • a positive result would demonstrate that specifically targeting the metabolic profile (in this case hypoxic-adaptations of CITED4 and SHAPR1) with replacement of native proteins via signal-sensor polynucleotides can directly impact the transcriptome and survival advantage of cancer cells with this profile. Further, the data could show that the relative impact of signal-sensor polynucleotide vs. vehicle under hypoxic conditions was more significant for cancer cells than for normal cells.
  • the cancer cells have a disproportionate survival advantage based on their CITED4+SHARP1 down regulation
  • a normal cell is to overproduction of it. It is understood that a cancer cell will likely be experiencing hypoxic conditions and that a normal cell under normoxic conditions might be able to tolerate CITED4 and SHARP1 over expression because the normal cell is not dependent on HIF1alpha transctiptome for survival advantage.
  • in vivo experiments are performed according to the design of the in vitro experiments where the animal model is one evincing metastasis in the cancer setting because HIF-1alpha appears to confer the largest portion of its advantage in metastasis.
  • Animals are administered the signal-sensor polynucleotide compared to no treatment or a control polynucleotide. Animal cells, tissues and/or organs are then evaluated for alterations in gene expression profiles or transcriptome levels.

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AU2013374345A1 (en) 2015-08-06
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