EA046751B1 - LIPID NANOPARTICLE FOR DELIVERY OF NUCLEIC ACID TO A PATIENT OR INTO A CELL - Google Patents

Description

Cross reference to related application

The patent application for this invention claims priority to U.S. Patent Application No. 62/758088, filed November 9, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

State of the art

Lipid nanoparticles (LNPs) are effective drug delivery systems for bioactive compounds such as therapeutic nucleic acids, proteins, and peptides that are otherwise impermeable to cells. Nucleic acid drugs that contain large nucleic acid molecules, such as messenger RNA (mRNA) transcribed in vitro, as well as smaller polynucleotides that interact with messenger RNA or the gene, must be delivered to the appropriate cellular compartment, to be effective. For example, double-stranded nucleic acids such as double-stranded RNA (dsRNA) molecules, including, for example, siRNA, suffer from physicochemical properties that make them impermeable to cells. Once delivered to the appropriate compartment, siRNAs block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). Typically, siRNAs are large in size with a molecular weight of 12 to 17 kDa and are highly anionic due to their phosphate backbone containing up to 50 negative charges. In addition, two complementary strands of RNA form a rigid helix. These features contribute to the inappropriate medicinal properties of siRNAs. When administered intravenously, siRNA is rapidly cleared from the body with a typical half-life of only 10 min. In addition, siRNAs are rapidly degraded by nucleases present in blood and other fluids or tissues and have been shown to induce potent immune responses in vitro and in vivo. See, for example, Robbins et al., Oligonucleotides, 19:89 -102, 2009. mRNA molecules suffer from similar problems of impermeability, fragility and immunogenicity. (WO 2016/118697).

Lipid nanoparticle formulations have improved the delivery of nucleic acids in vivo. For example, such compositions have significantly reduced doses of siRNA required to achieve targeted blocking in vivo. See, Zimmermann et al., Nature, 441:111-114, 2006. Typically, such lipid nanoparticle drug delivery systems are multicomponent compositions containing cationic lipids, helper lipids, and polyethylene glycol-containing lipids. Positively charged cationic lipids bind to the anionic nucleic acid, while other components support stable self-assembly of lipid nanoparticles.

Efforts have been aimed at improving the delivery efficiency of lipid nanoparticle-based formulations. Many of these efforts have been aimed at developing more suitable cationic lipids. See, for example, Akinc et al., Nature Biotechnology, 26:561-569, 2008; Love et al., Proc. Natl. Acad. Sci. USA, 107:1864-1869, 2010; Baigude et al., Journal of Controlled Release, 107:276-287, 2005; Semple et al., Nature Biotechnology, 28:172–176, 2010. Despite these efforts, there remains a need for formulations containing lipid nanoparticles that provide high post-administration efficacy and allow for the administration of lower doses of nucleic acids.

The essence of the invention

The invention relates to certain specific lipid nanoparticles and pharmaceutical compositions containing lipid nanoparticles. Lipid nanoparticles and pharmaceutical compositions are particularly useful for delivering nucleic acid to a patient (eg, human) or cell.

Accordingly, in one embodiment, the invention provides a lipid nanoparticle of the invention, which is a lipid nanoparticle comprising:

(a) one or more nucleic acid molecules;

(b) cholesterol;

(c) DSPC;

(d) PEG-S-EMA; and (e) cationic lipid of the formula

or a salt thereof, where the mole percentage of total lipid for PEG-C-DMA, cationic lipid, cholesterol and DSPC is as follows:

PEG-C-DMA: about 1.5;

cationic lipids: about 50.0;

cholesterol: about 38.5;

DSPC: about 10.0.

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In one embodiment, the invention provides a lipid nanoparticle according to the invention, which is a lipid nanoparticle containing:

(a) one or more nucleic acid molecules;

(b) cholesterol;

(c) DSPC;

(d) PEG-C-DMA;

(e) cationic lipid of formula

or a salt thereof, where the mole percentage of total lipid for PEG-C-DMA, cationic lipid, cholesterol and DSPC is as follows:

PEG-C-DMA: 1.5;

cationic lipids: 50.0;

cholesterol: 38.5;

DSPC: 10.0.

The present invention also provides a pharmaceutical composition comprising the lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

The invention also provides a method for delivering a nucleic acid into a cell, comprising contacting the cell with a lipid nanoparticle of the invention. In general, the present invention relates to methods for introducing nucleic acids into a living cell in vivo or in vitro.

The invention also provides a method of treating a disease characterized by a genetic disorder that results in a deficiency of a functional protein, comprising administering to a subject with the disease a lipid nanoparticle of the invention, wherein the nucleic acid molecule is an mRNA encoding a functional protein or a protein having the same biological activity that and functional protein.

The invention also provides a method of treating a disease characterized by overexpression of a polypeptide, comprising administering to a subject with the disease a lipid nanoparticle of the invention, wherein the nucleic acid molecule is an siRNA that targets expression of the overexpressed polypeptide.

The invention also provides a lipid nanoparticle according to the invention for the therapeutic or prophylactic treatment of a disease characterized by a genetic disorder that results in a deficiency of a functional protein.

The present invention also provides a lipid nanoparticle of the invention for the therapeutic or prophylactic treatment of a disease characterized by overexpression of a polypeptide.

The invention also provides a method of treating a disease or disorder in an animal, comprising administering to the animal a therapeutically effective amount of a lipid nanoparticle according to the invention.

The invention also provides a method and intermediates that can be used to prepare lipid nanoparticles according to the present invention.

Detailed description of the invention

Definitions.

In the context of this document, the following terms have the meanings assigned to them unless otherwise noted.

The term about means ±5, ±4, ±3, ±2 or ±1%.

The term interfering RNA, or mRNA, or interfering RNA sequence refers to single-stranded RNA (i.e., mature miRNA) or double-stranded RNA (i.e., duplex RNA, such as miRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (for example, by promoting the degradation or inhibiting translation of mRNAs that are complementary to an interfering RNA sequence), if the interfering RNA is in the same cell as the target gene or sequence. Thus, interfering RNA refers to single-stranded RNA that is complementary to the target mRNA sequence or double-stranded RNA formed by two complementary strands or one self-complementary strand. The interfering RNA may have a high degree of similarity or be completely identical to the target gene or sequence, or may include a region of non-complementarity (ie, a non-complementary motif). The interfering RNA sequence may correspond to the full-length target gene or a subsequence thereof.

Interfering RNA includes small interfering RNA or siRNA, for example interfering RNA of about 15-60, 15-50 or 15-40 (duplex) nucleotides in length, more typically about

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15-30, 15-25 or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22 or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of a double-stranded siRNA is 15- 60, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides in length, preferably about 20-24, 21-22 or 21-23 nucleotides in length, and the length of the double-stranded siRNA is about 15- 60, 15-50, 15-40, 15-30, 15-25 or 19-25 base pairs in length, preferably about 18-22, 19-20 or 19-21 base pairs in length). The siRNA duplexes may contain 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate ends. Examples of siRNA include, but are not limited to, a double-stranded polynucleotide molecule assembled from two separate strand molecules, where one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single-stranded molecule, wherein the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed in vivo or in vitro to form an active double-stranded siRNA molecule.

Preferably, siRNAs are chemically synthesized. siRNAs can also be produced by digestion of longer double-stranded RNAs (eg, double-stranded RNAs larger than about 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process double-stranded RNA into biologically active siRNA (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003); Knight et al., Science, 293:2269-2271 (2001) and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, the double-stranded RNAs are at least 50 to about 100, 200, 300, 400, or 500 nucleotides in length. Double-stranded RNA can be 1000, 1500, 2000, 5000 nucleotides or more in length. This double-stranded RNA can encode a complete gene transcript or a partial gene transcript. In certain cases, miRNA may be encoded by a plasmid (eg, transcribed into sequences that automatically fold into duplexes with hairpin loops).

As used herein, the term non-complementary motif or region of non-complementarity refers to a portion of an interfering RNA sequence (eg, siRNA, miRNA, miRNA) that is not 100% complementary to its target sequence. The interfering RNA may have at least one, two, three, four, five, six or more regions of non-complementarity. Regions of non-complementarity may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides. The motifs or regions of non-complementarity may contain one nucleotide or may contain two, three, four, five or more nucleotides.

An effective amount or therapeutically effective amount of a nucleic acid, such as a nucleic acid (eg, interfering RNA or microRNA), is an amount sufficient to produce a desired effect, for example, inhibiting expression of a target sequence compared to the normal level of expression found in the absence of interfering RNA ; or microRNA-directed expression of such an amount of protein that causes the desired biological effect in the organism in which the protein is expressed. Inhibition of the expression of the target gene or target sequence is achieved when the value obtained with interfering RNA relative to the control is about 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20 , 15, 10, 5 or 0%. In other embodiments, the expressed protein is the active form of a protein that is typically expressed in a particular cell type in the body, and the therapeutically effective amount of miRNA is an amount that produces an amount of the encoded protein that is at least 50% (e.g., at least 60 %, or at least 70%, or at least 80%, or at least 90%) of the amount of protein that is normally expressed in a healthy human cell type. Suitable assays for measuring the expression of a target gene or target sequence include, for example, examining protein or RNA levels using methods known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, studies of enzymatic activity, as well as phenotyping methods known to specialists in this field.

By the terms reduce, decrease, reduce or decrease the immune response by interfering RNA, we mean a detectable decrease in the immune response to a given interfering RNA (eg, modified interfering RNA). The degree of reduction of the immune response by the modified interfering RNA can be determined relative to the level of the immune response in the presence of unmodified interfering RNA. The detectable reduction may be around 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80.

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85, 90, 95, 100% or more lower than the immune response detected in the presence of unmodified interfering RNA. A decrease in the immune response to interfering RNA is typically measured by a decrease in cytokine production (eg, IFNy, IFNa, TNFa, IL-6, or IL-12) by an immunoreactive cell in vitro or by a decrease in cytokine production in the serum of a mammalian subject following administration of interfering RNA.

By the terms reduce, decrease, decrease, or reduce the immune response by a microRNA, we mean a detectable decrease in the immune response to a given microRNA (eg, a modified microRNA). The extent to which the modified miRNA reduces the immune response can be determined relative to the level of immune response in the presence of the unmodified miRNA. The detectable reduction may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more lower than immune response detected in the presence of unmodified miRNA. Reduced immune response to miRNAs is typically measured by decreased production of cytokines (eg, IFNy, IFNa, TNFa, IL-6, or IL-12) by an immunoreactive cell in vitro or by decreased production of cytokines in the serum of mammalian subjects following administration of miRNAs.

As used herein, the term immunoreactive cell refers to a cell, preferably a mammalian cell, that produces a detectable immune response upon contact with immunostimulatory interfering RNA, such as unmodified siRNA. Typical immunoreactive cells include, for example, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like. Detectable immune responses include, for example, the production of cytokines or growth factors such as TNF-a, IFN-a, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5 , IL-6, IL-10, IL-12, IL-13, TGF and combinations thereof.

Essential identity refers to a sequence that hybridizes to a reference sequence under stringent conditions or to a sequence that has a given percentage of identity to a given region of the reference sequence.

The expression stringent hybridization conditions refers to conditions under which a nucleic acid hybridizes to its target sequence, usually in a complex mixture of nucleic acids, but without other sequences. The stringent conditions depend on the sequence and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. A detailed guide to nucleic acid hybridization can be found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, Overview of principles of hybridization and the strategy of nucleic acid assays (1993). Typically, stringent conditions are selected to be about 5-10°C below the melting temperature ( _Tm ) for a particular sequence at a particular ionic strength and pH. _Tm is the temperature (at a certain ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (since the target sequences are present in excess, at _Tm , 50% probes are occupied in an equilibrium state). Harsh conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least twice the background, preferably 10 times the background hybridization.

Typical stringent hybridization conditions may be: 50% formamide, 5xSSC and 1% SDS, incubated at 42°C, or 5xSSC, 1% SDS, incubated at 65°C, washed in 0.2xSSC and 0.1% SDS at 65°C. For PCR, a temperature of about 36°C is typical for amplification under reduced stringency conditions, although annealing temperatures can vary from about 32°C to 48°C depending on primer length. For high-stringency PCR amplification, a temperature of about 62°C is typical, although high-stringency annealing temperatures can range from about 50 to about 65°C, depending on the length and specificity of the primer. Typical cycling conditions for both high and low stringency amplifications include a denaturation phase at 90-95°C for 30 s - 2 min, an annealing phase of 30 s - 2 min, and an extension phase at approximately 72°C for 1 -2 minutes. Protocols and guidelines for amplification reactions under higher and lower stringency conditions are presented, for example, in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).

Nucleic acids that do not hybridize with each other under stringent conditions are still essentially identical if the polypeptides they encode are essentially identical. This occurs, for example, when a copy of a nucleic acid is made using the maximum codon degeneracy allowed by the genetic code. In such cases, the nucleic acids are typically hybridized under moderately stringent hybridization conditions. Examples of moderately stringent hybridization conditions include hybridization in a buffer containing 40% formamide, 1 M NaCl, 1% SDS at 37°C, and washing in 1xSSC at 45°C. Positive hybridization is at least twice the background hybridization. Those skilled in the art will readily appreciate that it is possible to use al

- 4 046751 alternative hybridization and washing conditions to provide conditions of similar stringency. Additional guidance on determining hybridization parameters is provided in numerous references, such as Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms substantially identical or substantial identity, in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides (i.e., at least about 60%, preferably at least about 65. 70, 75, 80, 85, 90, or 95% identity in a defined region), when compared and aligned for best fit in the comparison window or designated region, measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition, when given in context, also applies similarly to complementary sequence. Preferably, significant identity exists in a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparisons, typically one sequence serves as a reference sequence to which the sequences of interest are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the sequence are indicated and the software parameters of the algorithm for working with sequences are indicated. You can use the default program settings or specify alternative settings. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences compared to the reference sequence based on software parameters.

The term comparison window as used herein includes reference to a segment of any of a plurality of contiguous positions selected from the group consisting of from about 5 to about 60, typically from about 10 to about 45, more commonly from about 15 to about 30, in which the sequence can be compared to a reference sequence at the same number of contiguous positions after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, using a local homology algorithm according to Smith and Waterman, Adv. Appl. Math., 2:482 (1981), homologous alignment algorithm according to Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), similarity search according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin, USA) or manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds. (1995 supplement)).

A preferred example of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used with the parameters described herein to determine the percent identity of nucleic acid sequences of the invention. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

The BLAST algorithm also performs statistical analysis of the similarity of two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity made by the BLAST algorithm is to determine the lowest cumulative probability (P(N)), which indicates the probability at which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the lowest overall probability when comparing the nucleic acid of interest to the reference nucleic acid is less than about 0.2, more preferably about 0.01, and most preferably less than about 0.001.

The term nucleic acid as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides, in single- or double-stranded form, and includes DNA and RNA. The DNA may be in the form of, for example, antisense molecules, plasmid DNA, precondensed DNA, PCR product, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA or derivatives and combinations of these groups. The RNA may be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogues or modified framework residues or units that are synthetic, naturally occurring and non-naturally occurring, and that have similar binding properties as the nucleic acid in question. Examples of such analogues include, but are not limited to, thiophosphates, phosphoramidates, methylphosphonates, chiral

- 5 046751 tylphosphonates, 2'-O-methylribonucleotides and peptide nucleic acids (PNA). Unless specifically limited, the term covers nucleic acids containing known analogues of natural nucleotides that have binding properties similar to those of the reference nucleic acids. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs (single nucleotide polymorphisms), and complementary sequences, as well as explicitly stated sequence. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991) ); Ohtsuka et al., J. Biol. Chem., 260:26052608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

Nucleotides contain the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are connected to each other by phosphate groups.

Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce new reactive groups, such as, without limitation, amines, alcohols, thiols, carboxylates and alkyl halides.

The term gene refers to a nucleic acid sequence (eg, DNA or RNA) that includes part or all of the coding sequences necessary to create a polypeptide or precursor polypeptide.

A gene product, as used herein, refers to a gene product, such as an RNA transcript or polypeptide.

The term lipid refers to a group of organic compounds that include, but are not limited to, fatty acid esters and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into three classes: (1) simple lipids, which include fats and oils as well as waxes; (2) complex lipids, which include phospholipids and glycolipids; and (3) lipid derivatives such as steroids.

As used herein, the term LNP refers to a lipid-nucleic acid particle or a nucleic acid-lipid particle (eg, a stable nucleic acid-lipid particle). LNP is a particle composed of lipids (e.g., cationic lipid, non-cationic lipid, and a conjugated lipid that prevents particle aggregation) and a nucleic acid, wherein the nucleic acid (e.g., siRNA, aiRNA, microRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA or plasmid, including plasmids from which interfering RNA or mRNA is transcribed) is encapsulated within a lipid. In one embodiment, the nucleic acid is at least 50% encapsulated in a lipid; in one embodiment, the nucleic acid is at least 75% encapsulated in a lipid; in one embodiment, the nucleic acid is at least 90% encapsulated in a lipid; and in one embodiment, the nucleic acid is completely encapsulated in a lipid. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid conjugate (eg, a PEG-lipid conjugate). LNPs are extremely useful for systemic use because they can exhibit an increased lifetime in the circulation following intravenous (IV) injection, they can accumulate at distal sites (eg, sites physically separated from the injection site), and they can mediate expression transfected gene or suppression of target gene expression at these distal sites.

The lipid particles of the invention (e.g., LNPs) typically have an average diameter of about 40 to about 150 nm, about 50 to about 150 nm, about 60 to about 130 nm, about 70 to about 110 nm, or about 70 to about 90 nm and is essentially non-toxic. In addition, the nucleic acids, when present in the lipid particles of the present invention, are resistant to nuclease degradation in aqueous solution. Nucleic acid and lipid-based particles and the process for their preparation are disclosed, for example, in US Patent Publications Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

As used herein, the term encapsulated lipid can refer to a lipid particle that provides a nucleic acid (eg, interfering RNA or mRNA) with complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid is completely encapsulated in a lipid particle (eg, to form an LNP or other nucleic acid-lipid particle).

The term cationic lipid refers to a compound of formula CL1 or a salt thereof:

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The term hydrophobic lipid refers to compounds having non-polar groups, which include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups that are optionally substituted with one or more aromatic, cycloaliphatic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

The term fusogenic refers to the ability of a lipid particle, such as LNP, to fuse with cell membranes. The membranes can be either the plasma membrane or membranes surrounding organelles such as the endosome, nucleus, etc.

As used herein, the term aqueous solution refers to a composition containing wholly or partially water.

As used herein, the term organic lipid solution refers to a composition containing, in whole or in part, an organic solvent having a lipid.

Distal region as used herein refers to a physically separated region that is not limited to the adjacent capillary bed, but includes regions widely distributed throughout the body.

Serum stable to nucleic acid and lipid based particles such as LNP means that the particle does not undergo significant degradation after exposure to a serum or nuclease assay that significantly degrades free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNase assay, or an RNase assay.

Systemic delivery as used herein refers to the delivery of lipid particles that results in widespread biodistribution of a nucleic acid, such as interfering RNA or mRNA, throughout the body. Some methods of administration may result in systemic delivery of certain agents but not others. Systemic delivery means that a suitable, preferably therapeutic, amount of agent is applied to most parts of the body. To achieve broad biodistribution, a residence time in the blood is usually required so that the agent is not rapidly degraded or cleared (e.g., by first-pass organs (liver, lungs, etc.) or by rapid, nonspecific cell binding) before reaching the disease site, distal to the injection site. Systemic delivery of lipid particles can be accomplished by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment of the invention, systemic delivery of lipid particles is accomplished via intravenous delivery.

Local delivery as used herein refers to the delivery of a nucleic acid, such as interfering RNA or mRNA, directly to a target site in the body. For example, the agent may be delivered locally by direct injection into a disease site such as a tumor, or another target site such as an inflammatory site, or a target organ such as the liver, heart, pancreas, kidney, or the like.

The term mammal refers to any species of mammal such as human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock and the like.

The term cancer refers to any member of the class of diseases that are characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue or solid, as well as cancers of all stages and grades, including pre- and post-metastatic cancer. Examples of various types of cancer include, but are not limited to, lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small bowel cancer, stomach cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, kidney cancer (eg, renal cell carcinoma), central nervous system cancer, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head and neck cancers, osteogenic sarcomas, and blood cancers. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (eg, caused by metastasis of certain other cell types other than liver cancer), and hepatoblastoma. The term tumor as used herein includes one or more cancer cells.

Description of Embodiments

In one embodiment, one or more nucleic acid molecules comprise siRNA.

In one embodiment, one or more nucleic acid molecules comprise mRNA.

In one embodiment, the lipid nanoparticle has a mass ratio (total lipid):(nucleic acid) that is greater than about 17.

In one embodiment, the lipid nanoparticle has a mass ratio (total lipid):(nucleic acid) that is greater than about 18.

In one embodiment, the lipid nanoparticle has a mass ratio (total

- 7 046751 pid): (nucleic acid), which is greater than about 19.

In one embodiment, the lipid nanoparticle has a mass ratio (total lipid):(nucleic acid) that is from about 22 to about 25.

In one embodiment, the PEG-C-DMA is PEG2000-C-0ML.

In one embodiment, the pharmaceutical composition is formulated for subcutaneous administration.

In certain embodiments, the nucleic acid is completely encapsulated in the lipid portion of the lipid particle such that the nucleic acid in the lipid particle is resistant in aqueous solution to enzymatic degradation, such as by a nuclease or protease. In certain other embodiments, the lipid particles are substantially non-toxic to mammals, such as humans.

In certain cases, the nucleic acid includes an interfering RNA molecule, such as, for example, siRNA, miRNA, microRNA, or mixtures thereof. In certain other instances, the nucleic acid includes single- or double-stranded DNA, RNA, or a DNA/RNA hybrid, such as, for example, an antisense oligonucleotide, ribozyme, plasmid, immunostimulatory oligonucleotide, or mixtures thereof. In certain other cases, the nucleic acid is one or more mRNA molecules (eg, a mixture).

In one embodiment, the nucleic acid comprises siRNA. In one embodiment, the siRNA molecule comprises a double-stranded region of about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length or 15. 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length). The siRNA molecules of the invention are capable of inhibiting the expression of a target sequence in vitro and/or in vivo.

In some embodiments, the siRNA molecule contains at least one modified nucleotide. In certain preferred embodiments, the siRNA molecule contains one, two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides in a double-stranded region. In certain cases, siRNA contains from about 1 to about 100% (for example, about 1.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%) modified nucleotides in the double-stranded region. In preferred embodiments, less than about 25% (for example, less than about 25, 20, 15, 10, or 5%) or from about 1 to about 25% (for example, about 1-25%, 5-25%, 10-25% , 15-25%, 20-25% or 10-20%) of the nucleotides in the double-stranded region contain modified nucleotides.

In other embodiments, the siRNA molecule contains modified nucleotides, including, but not limited to, 2'-O-methyl(2'OMe)nucleotides, 2'-deoxy-2'-fluoro(2T')nucleotides, 2'-deoxynucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In preferred embodiments, the siRNA comprises 2'OMe nucleotides (e.g., 2'OMe purine and/or pyrimidine nucleotides), such as, for example, 2'OMe guanosine nucleotides, 2'OMe uridine nucleotides, 2'OMe adenosine nucleotides, 2 'OME-cytosine nucleotides and their mixtures. In certain cases, siRNA does not contain 2'OMe-cytosine nucleotides. In other embodiments, the siRNA comprises a hairpin structure.

siRNA may contain modified nucleotides on one strand (ie, sense or antisense) or on both strands of the double-stranded region of the siRNA molecule. Preferably, the uridine and/or guanosine nucleotides are modified at selective positions in the double-stranded region of the siRNA duplex. With respect to modifications of uridine nucleotides, at least one, two, three, four, five, six or more uridine nucleotides in the sense and/or antisense strand may be a modified uridine nucleotide, such as a 2'OMe-uridine nucleotide. In some embodiments, each uridine nucleotide in the sense and/or antisense strand is a 2'OMe-uridine nucleotide. With respect to modifications of guanosine nucleotides, at least one, two, three, four, five, six or more guanosine nucleotides in the sense and/or antisense strand may be a modified guanosine nucleotide, such as a 2'OMe-guanosine nucleotide. In some embodiments, each guanosine nucleotide in the sense and/or antisense strand is a 2'OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six, seven or more 5'-GU-3' motifs in the siRNA sequence may be modified, for example, by introducing nucleotide pairing errors to eliminate the 5'-GU-3' motifs. GU-3' and/or by introducing modified nucleotides such as 2'OMe nucleotides. The 5'-GU-3' motif can be on the sense strand, the antisense strand, or both strands of the siRNA sequence. The 5'-GU-3' motifs may be adjacent to each other or alternatively they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides.

In some preferred embodiments, the modified siRNA molecule is less immunostimulatory than the corresponding unmodified siRNA sequence. In such embodiments, the modified siRNA molecule with reduced immunostimulatory properties preferentially retains RNAi activity against the target sequence.

- 8 046751 activity. In another embodiment, the immunostimulatory properties of the modified siRNA molecule and its ability to suppress target gene expression can be balanced or optimized by introducing minimal and selective 2'OMe modifications to the siRNA sequence, such as, for example, in the double-stranded region of the siRNA duplex. In certain cases, the modified siRNA is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% has a reduced immunostimulatory effect than the corresponding unmodified siRNA. Those skilled in the art will appreciate that the immunostimulatory properties of a modified siRNA molecule and a corresponding unmodified siRNA molecule can be determined, for example, by measuring the levels of INF-α and/or IL-6 from about 2 to about 12 hours after systemic administration to a mammal or transfecting an immunoreactive mammalian cell using a suitable lipid-based delivery system (eg, such as the LNP delivery system disclosed herein).

In certain embodiments, the modified siRNA molecule has an IC ₅₀ (i.e., half-maximal inhibition concentration) less than or equal to ten times the value of the corresponding unmodified siRNA (i.e., the modified siRNA has an IC ₅₀ that is less than or equal to ten times the IC ₅₀ value of the corresponding unmodified siRNA ). In other embodiments, the modified siRNA has an IC ₅₀ value less than or equal to three times the value of the corresponding unmodified siRNA sequence. In still other embodiments, the modified siRNA has an IC ₅₀ value less than or equal to two times the value of the corresponding unmodified siRNA. Those skilled in the art will appreciate that a dose-response curve can be generated and IC ₅₀ values for the modified siRNA and the corresponding unmodified siRNA can be readily determined using methods known to those skilled in the art.

In yet another embodiment, the modified siRNA molecule is capable of inhibiting expression of the target sequence by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80. 85, 90, 95 or 100% relative to the corresponding unmodified siRNA sequence.

In some embodiments, the siRNA molecule does not contain modifications to the phosphate backbone, for example, in the sense and/or antisense strand of the double-stranded region. In other embodiments, the siRNA contains one, two, three, four or more modifications of the phosphate backbone, for example, in the sense and/or antisense strand of the double-stranded region. In preferred embodiments, the siRNA does not contain modifications to the phosphate backbone.

In additional embodiments, the siRNA does not contain 2'-deoxynucleotides, for example, in the sense and/or antisense strand of the double-stranded region. In still further embodiments, the siRNA comprises one, two, three, four or more 2'-deoxynucleotides, for example, in the sense and/or antisense strand of a double-stranded region. In preferred embodiments, the siRNA does not contain 2'-deoxynucleotides.

In certain cases, the nucleotide at the 3' end of the double-stranded region of the sense and/or antisense strand is not a modified nucleotide. In certain other cases, nucleotides near the 3' end (eg, within one, two, three or four nucleotides at the 3' end) of the double-stranded region of the sense and/or antisense strand are not modified nucleotides.

The siRNA molecules described herein may have 3' overhangs of one, two, three, four or more nucleotides on one or both sides of the double-stranded region, or may have no overhangs (i.e., blunt ends) on one or both sides of the double-stranded region. areas. Preferably, the siRNA has 3' overhangs of two nucleotides on each side of the double-stranded region. In certain cases, the 3' overhang on the antisense strand is complementary to the target sequence, and the 3' overhang on the sense strand is complementary to the complementary strand of the target sequence. Alternatively, the 3' overhangs are not complementary to the target sequence or its complementary strand. In some embodiments, the 3' overhangs contain one, two, three, four or more nucleotides, such as 2'-deoxy(2'H)nucleotides. In certain preferred embodiments, the 3' overhangs comprise deoxythymidine (dT) and/or uridine nucleotides. In other embodiments, one or more nucleotides in the 3' overhangs on one or both sides of the double-stranded region contain modified nucleotides. Non-limiting examples of modified nucleotides are described above and include 2OMe nucleotides, 2'-deoxy-2T' nucleotides, 2'-deoxynucleotides, 2'-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, one, two, three, four or more nucleotides in the 3' overhangs present on the sense and/or antisense strand of the siRNA contain 2'OMe nucleotides (e.g., purine and/or pyrimidine 2'OMe nucleotides) such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides and mixtures thereof.

siRNA may contain at least one or a mixture (for example, at least two, three, four, five, six, seven, eight, nine, ten or more) unmodified and/or modified

- 9 046751 sequences of miRNAs that suppress the expression of the target gene. The siRNA mixture may contain sequences that target the same region or domain (eg, hot spot) and/or different regions or domains of one or more target genes. In certain cases, one or more (eg, at least two, three, four, five, six, seven, eight, nine, ten or more) modified siRNAs that suppress expression of the target gene are present in the mixture. In certain other cases, one or more (eg, at least two, three, four, five, six, seven, eight, nine, ten or more) unmodified siRNA sequences that inhibit expression of the target gene are present in the mixture.

In some embodiments, the antisense strand of the siRNA molecule contains or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof. In other embodiments, the antisense strand of the siRNA molecule contains or consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In additional embodiments, the antisense strand of the siRNA molecule contains or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.

In additional embodiments, the sense strand of the siRNA molecule contains or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof. In additional embodiments, the sense strand of the siRNA molecule contains or consists of a sequence that is 100% identical to the target sequence or a portion thereof.

Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl 2'-hydroxyethyl ether, cholesteryl 4'-hydroxybutyl ether, and mixtures thereof. This document describes the synthesis of cholesteryl 2'-hydroxyethyl ether.

As used herein, DSPC means distearoylphosphatidylcholine.

In the lipid particles of the invention, the nucleic acid can be completely encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from enzymatic degradation. In preferred embodiments, the LNP containing a nucleic acid, such as interfering RNA (eg, siRNA) or mRNA, is completely encapsulated in the lipid portion of the particle, thereby protecting the nucleic acid from nuclease digestion. In certain cases, the nucleic acid in the LNP is substantially undegraded after exposure of the particle to nuclease at 37° C. for at least about 20, 30, 45, or 60 minutes. In certain other cases, the nucleic acid in the LNP is substantially undegraded after the particle is incubated in serum at 37°C for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7. 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the nucleic acid (e.g., a nucleic acid such as siRNA or mRNA ) forms a complex with the lipid part of the particle. One of the advantages of the compositions of this invention is that the lipid particle compositions are substantially non-toxic to mammals such as humans.

The term completely encapsulated indicates that the nucleic acid in the lipid particle does not undergo significant degradation after exposure to serum or nuclease or protease that would significantly degrade free DNA, RNA or protein. In a fully encapsulated system, preferably less than about 25% of the nucleic acid in the particle is degraded by a treatment that would typically degrade 100% of the free nucleic acid, more preferably less than about 10%, and most preferably less than about 5% of the nucleic acid is degraded in the particle. In the context of nucleic acid-based therapeutics, complete encapsulation can be determined using the Oligreen® assay. Oligreen® is an ultrasensitive fluorescent nucleic acid dye for the quantification of oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, CA). Completely encapsulated also indicates that the lipid particles are stable in the blood serum, i.e. they do not rapidly decompose into their constituent parts when administered in vivo.

In another aspect of the present invention, a lipid particle composition (eg, LNP) is provided that contains a plurality of lipid particles. In preferred embodiments, the nucleic acid (eg, nucleic acid) is completely encapsulated in the lipid portion of the lipid particle (eg, LNP), such that from about 30 to about 100%, from about 40 to about 100%, from about 50 to about 100% , from about 60 to about 100%, from about 70 to about 100%, from about 80 to about 100%, from about 90 to about 100%, from about 30 to about 95%, from about 40 to about 95%, from about 50 to about 95%, from about 60 to about 95%, from about 70 to about 95%, from about 80 to about 95%, from about 85 to about 95%, from about 90 to about 95%, from about 30 up to about 90%, from about 40 to about 90%, from about 50 to about 90%, from about 60 to about 90%, from about 70 to about 90%, from about 80 to about 90%, or at least about 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% (or any

- 10 046751 fraction or range) of lipid particles (eg LNP) contain an encapsulated active agent or therapeutic agent.

Typically, the lipid particles (eg, LNPs) of the invention have a lipid:active agent (eg, lipid:nucleic acid) ratio (wt/wt ratio) of about 1 to about 100. In some cases, a lipid:active agent (eg, lipid:nucleic acid) ratio of about 1 to about 100. In some cases, a lipid:active agent ratio (eg, lipid: nucleic acid) (weight/weight ratio) ranges from about 1 to about 50, from about 2 to about 25, from about 3 to about 20, from about 4 to about 15, or from about 5 to about 10.

Typically, lipid particles (eg, LNPs) of the invention have an average diameter of from about 40 nm to about 150 nm. In preferred embodiments, the lipid particles (e.g., LNPs) of the invention have an average diameter of from about 40 to about 130 nm, from about 40 to about 120 nm, from about 40 to about 100 nm, from about 50 to about 120 nm, from about 50 to about 100 nm, from about 60 to about 120 nm, from about 60 to about 110 nm, from about 60 to about 100 nm, from about 60 to about 90 nm, from about 60 to about 80 nm, from about 70 to about 120 nm, about 70 to about 110 nm, about 70 to about 100 nm, about 70 to about 90 nm, about 70 to about 80 nm, or less than about 120, 110, 100, 90 or 80 nm (or any fraction or range thereof).

The present invention also provides a pharmaceutical composition comprising lipid particles (eg, LNPs) described herein and a pharmaceutically acceptable carrier.

In a further aspect of the present invention, there is provided a method of introducing one or more active agents or therapeutics (eg, a nucleic acid) into a cell, comprising contacting the cell with a lipid particle (eg, LNP) described herein. In one embodiment, the cell is a mammalian cell, such as a human. In another embodiment, the present invention provides a method for in vivo delivery of one or more active agents or therapeutics (eg, a nucleic acid), comprising administering to a mammalian subject a lipid particle (eg, LNP) described herein. In a preferred embodiment, the route of administration includes, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. Preferably, the mammalian subject is human.

In one embodiment, at least about 5, 10, 15, 20, or 25% of the total administered dose of lipid particles (eg, LNP) is present in plasma at about 8, 12, 24, 36, or 48 hours after injection. In another embodiment, more than about 20, 30, 40, 60, 70, or 80% of the total administered dose of lipid particles (eg, LNP) is present in the plasma at about 8, 12, 24, 36, or 48 hours after injection. In certain cases, more than about 10% of the particle population is present in the plasma of the mammal approximately 1 hour after administration. In certain other cases, the presence of lipid particles (eg, LNP) can be detected at least about 1 hour after administration of the particle. In certain embodiments, the presence of a nucleic acid, such as interfering RNA (e.g., siRNA) or mRNA, is detected in cells at about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration (e.g., lungs, liver , tumor or source of inflammation). In other embodiments, inhibition of expression of the target sequence by a nucleic acid, such as interfering RNA (eg, siRNA), is detected at about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In still other embodiments, inhibition of expression of a target sequence by a nucleic acid, such as interfering RNA (eg, siRNA), occurs preferentially in tumor cells or cells at the site of inflammation. In additional embodiments, the presence or effect of a nucleic acid, such as interfering RNA (e.g., siRNA), in cells at a site proximal or distal to the injection site, or in lung, liver, or tumor cells is detected after about 12, 24, 48, 72 or 96 hours or approximately 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. In other embodiments, up-regulation of expression of a target nucleic acid sequence, such as mRNA or self-amplifying RNA, is detected at about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In still other embodiments, up-regulation of expression of a target nucleic acid sequence, such as mRNA or self-amplifying RNA, occurs preferentially in tumor cells or cells at the site of inflammation. In additional embodiments, the presence or effect of a nucleic acid, such as mRNA or self-amplifying RNA, in cells at a site proximal or distal to the injection site, or in lung, liver, or tumor cells is detected after about 12, 24, 48, 72, or 96 hours or approximately 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. In additional embodiments, the lipid particles (eg, LNPs) of the invention are administered parenterally or intraperitoneally. In embodiments, the lipid particles (eg, LNPs) of the invention are administered intramuscularly.

In some embodiments, lipid particles (eg, LNPs) of the invention are useful in methods for therapeutically delivering one or more nucleic acids containing an interfering RNA sequence (eg, siRNA). In particular, one object of this invention

- 11 046751 is to provide in vitro and in vivo methods for treating a disease or disorder in a mammal (for example, a rodent such as a mouse or a primate such as a human, chimpanzee or monkey) by inhibiting or disabling the transcription and/or translation of one or more representative interest of target nucleic acid sequences or genes. By way of non-limiting example, the methods of the invention are useful for in vivo delivery of interfering RNA (eg, siRNA) to the liver and/or tumor of a mammalian subject. In certain embodiments, the disease or disorder is associated with gene expression and/or overexpression, and gene expression or overexpression is reduced by interfering RNA (eg, siRNA). In certain other embodiments, a therapeutically effective amount of lipid particles (eg, LNP) may be administered to a mammal. In some cases, interfering RNA (eg, siRNA) is included in the LNP and the particles are administered to patients requiring such treatment. In other cases, cells are removed from the patient, interfering RNA (eg, siRNA) is delivered in vitro (eg, using the LNP described herein), and the cells are reintroduced to the patient.

A further aspect of the invention provides lipid particles (eg, LNPs) containing asymmetric interfering RNA (aiRNA) molecules that inhibit expression of a target gene, and methods of using such particles to inhibit expression of a target gene.

In one embodiment, the aiRNA molecule comprises a double-stranded region of about 10 to about 25 base pairs in length, wherein the aiRNA molecule comprises an antisense strand containing 5' and 3' overhangs, and wherein the aiRNA molecule is capable of repressing expression target gene.

In certain cases, the aiRNA molecule contains a double-stranded (duplex) region of about 12-20, 12-19, 12-18, 13-17 or 14-17 (base pairs) nucleotides in length, most often 12, 13, 14, 15, 16, 17, 18, 19 or 20 (base pairs) nucleotides in length. In certain other cases, the 5' and 3' overhangs on the antisense strand contain sequences that are complementary to the target RNA sequence, and may additionally contain non-target sequences. In some embodiments, each of the 5' and 3' overhangs on the antisense strand contains or consists of one, two, three, four, five, six, seven, or more nucleotides.

In other embodiments, the aiRNA molecule contains modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2T' nucleotides, 2'-deoxynucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In a preferred embodiment, the aiRNA molecule contains 2'OMe nucleotides. By way of non-limiting example, 2'OMe nucleotides may be selected from the group consisting of 2'OMe guanosine nucleotides, 2'OMe uridine nucleotides, and mixtures thereof.

In a related aspect, the present invention provides lipid particles (eg, LNPs) containing microRNA molecules (miRNAs) that suppress the expression of a target gene, as well as methods of using such particles to suppress the expression of a target gene.

In one embodiment, the miRNA molecule comprises from about 15 to about 60 nucleotides in length, and the miRNA molecule is capable of repressing the expression of a target gene.

In certain cases, the microRNA molecule is about 15-50, 15-40, or 15-30 nucleotides in length, more typically about 15-25 or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length. In a preferred embodiment, the miRNA molecule is a mature miRNA molecule targeting the RNA sequence of interest.

In other embodiments, the microRNA molecule contains modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'nucleotides, 2'deoxynucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In a preferred embodiment, the microRNA molecule contains 2'OMe nucleotides. By way of non-limiting example, 2'OMe nucleotides may be selected from the group consisting of 2'OMe guanosine nucleotides, 2'OMe uridine nucleotides, and mixtures thereof.

In some embodiments, the lipid particles (eg, LNPs) of the invention are useful in methods for therapeutically delivering one or more mRNA molecules. In particular, one object of the present invention is to provide in vitro and in vivo methods for treating a disease or disorder in a mammal (e.g., a rodent such as a mouse, or a primate such as a human, chimpanzee, or monkey) through the expression of one or more target proteins . By way of non-limiting example, the methods of the invention are useful for in vivo delivery of one or more mRNA molecules to a mammalian subject. In certain other embodiments, a therapeutically effective amount of lipid particles (eg, LNP) may be administered to a mammal. In some cases, one or more mRNA molecules are included in the LNP, and the particles are administered to patients requiring such treatment. In other cases, cells are removed from the patient, one or more mRNA molecules are delivered in vitro (eg, using the LNP described herein), and the cells are reintroduced to the patient.

- 12 046751

In other embodiments, the mRNA molecule contains modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2F nucleotides, 2'deoxynucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In a related aspect, the present invention provides lipid particles (eg, LNPs) containing microRNA molecules (miRNAs) that suppress the expression of a target gene, as well as methods of using such particles to suppress the expression of a target gene.

As such, the lipid particles of the invention (eg, LNPs) are advantageous and suitable for use in administering active agents or therapeutic agents, such as a nucleic acid (eg, interfering RNA, such as siRNA, aiRNA, and/or microRNA; or mRNA) to a subject (eg, a mammal such as a human) because they are stable in circulation, have the size necessary for pharmacodynamic behavior to access extravascular sites, and are capable of reaching target cell populations.

As used herein, the terms polynucleotide and oligonucleotide refer to a polymer or nucleotide oligomer, or nucleoside monomers, consisting of naturally occurring bases, sugars and intersugar (framework) units. The terms polynucleotide and oligonucleotide also include polymers or oligomers containing non-naturally occurring monomers or fragments thereof that act in a similar manner. Such modified or substituted oligonucleotides are often preferred over natural forms due to their properties, such as, for example, enhanced cellular uptake, reduced immunogenicity and increased stability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribonucleotide consists of a five-atom sugar called deoxyribose covalently linked to a phosphate at the 5' and 3' carbon atoms of the sugar to form an alternating, straight-chain polymer. The ribooligonucleotide consists of a similar repeating structure, where the five-atom sugar is ribose.

The nucleic acid that is present in the lipid-nucleic acid particle of the present invention includes any known form of nucleic acid. The nucleic acids used herein may be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA are described herein and include, for example, structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA are described herein and include, for example, siRNA and other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature microRNA and triplex oligonucleotides.

Nucleic acids can vary in length, usually depending on the specific form of the nucleic acid. For example, in particular embodiments, plasmids or genes can be from about 1,000 to about 100,000 nucleotide residues in length. In specific embodiments, the oligonucleotides can be from about 10 to about 100 nucleotides in length. In various related embodiments, the oligonucleotides, both single-stranded, double-stranded and triple-stranded, can have a length of from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides or about 20 to about 30 nucleotides in length.

In specific embodiments, an oligonucleotide (or strand thereof) of the invention specifically hybridizes to or is complementary to a target polynucleotide sequence. As used herein, the terms specifically hybridizable and complementary indicate a sufficient degree of complementarity such that stable and specific binding occurs between the target DNA or RNA and the oligonucleotide. It is understood that an oligonucleotide does not need to be 100% complementary to its target nucleic acid sequence for specific hybridization. In preferred embodiments, an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to a target sequence interferes with the normal function of the target sequence, causing loss of its utility or expression, and there is a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions where which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatments, or, in the case of in vitro assays, under the conditions under which the assays are performed. Thus, an oligonucleotide may contain 1, 2, 3 or more base substitutions relative to the region of the gene or mRNA sequence it targets or to which it specifically hybridizes.

siRNA.

siRNA as a component of the nucleic acid and lipid particles of the present invention is capable of suppressing the expression of a target gene of interest. Each siRNA duplex strand is typically about 15 to about 60 nucleotides long, preferably about 15 to about 30 nucleotides long.

- 13 046751 dov in length. In certain embodiments, the siRNA comprises at least one modified nucleotide. The modified siRNA is typically less immunostimulatory than the corresponding unmodified siRNA sequence and retains RNAi activity against the target gene of interest. In some embodiments, the modified siRNA comprises at least one purine or pyrimidine 2'OMe nucleotide, such as 2'OMeguanosine, 2'OMe-uridine, 2'OMe-adenosine, and/or 2'OMe-cytosine nucleotide. In preferred embodiments, one or more uridine and/or guanosine nucleotides are modified. Modified nucleotides may be present on one strand (ie, sense or antisense) or on both strands of the siRNA. The miRNA sequences may have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)) or may not have protrusions (i.e., have blunt ends).

The modified siRNA is typically from about 1 to about 100% (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%) modified nucleotides in the double-stranded region of the siRNA duplex. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more nucleotides in the double-stranded region of the siRNA contain modified nucleotides.

In some embodiments, less than about 25% (e.g., less than about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 , 6, 5, 4, 3, 2 or 1%) of the nucleotides in the double-stranded region of the siRNA contain modified nucleotides.

In other embodiments, from about 1 to about 25% (e.g., from about 1-25%, 2-25%, 3-25%, 4-25%, 5-25%, 6-25%, 7-25% , 8-25%, 9-25%, 10-25%, 11-25%, 12-25%, 13-25%, 14-25%, 15-25%, 16-25%, 17-25% , 18-25%, 19-25%, 20-25%, 21-25%, 22-25%, 23-25%, 24-25%, etc.) or from about 1 to about 20% ( for example, from about 1-20%, 2-20%, 3-20%, 4-20%, 5-20%, 6-20%, 7-20%, 8-20%, 9-20%, 10 -20%, 11-20%, 12-20%, 13-20%, 14-20%, 15-20%, 16-20%, 17-20%, 18-20%, 19-20%, 1 -19%, 2-19%, 3-19%, 4-19%, 5-19%, 6-19%, 7-19%, 8-19%, 9-19%, 10-19%, 11 -19%, 12-19%, 13-19%, 14-19%, 15-19%, 16-19%, 17-19%, 18-19%, 1-18%, 2-18%, 3 -18%, 4-18%, 5-18%, 6-18%, 7-18%, 8-18%, 9-18%, 10-18%, 11-18%, 12-18%, 13 -18%, 14-18%, 15-18%, 16-18%, 17-18%, 1-17%, 2-17%, 3-17%, 4-17%, 5-17%, 6 -17%, 7-17%, 8-17%, 9-17%, 10-17%, 11-17%, 12-17%, 13-17%, 14-17%, 15-17%, 16 -17%, 1-16%, 2-16%, 3-16%, 4-16%, 5-16%, 6-16%, 7-16%, 8-16%, 9-16%, 10 -16%, 11-16%, 12-16%, 13-16%, 14-16%, 15-16%, 1-15%, 2-15%, 3-15%, 4-15%, 5 -15%, 6-15%, 7-15%, 8-15%, 9-15%, 10-15%, 11-15%, 12-15%, 13-15%, 14-15%, etc. .d.) nucleotides in the double-stranded region of miRNA contain modified nucleotides.

In additional embodiments, for example, when one or both strands of the siRNA are selectively modified with uridine and/or guanosine nucleotides, the resulting modified siRNA may contain less than about 30% modified nucleotides (e.g., less than about 30, 29, 28, 27, 26, 25 , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% modified nucleotides) or from about 1 to about 30% modified nucleotides (for example, from about 1-30%, 2-30%, 3-30%, 4-30%, 5-30%, 6-30%, 7-30 %, 8-30%, 9-30%, 10-30%, 11-30%, 12-30%, 13-30%, 14-30%, 15-30%, 16-30%, 17-30 %, 18-30%, 19-30%, 20-30%, 21-30%, 22-30%, 23-30%, 24-30%, 25-30%, 26-30%, 27-30 %, 28-30% or 29-30% modified nucleotides).

Selection of miRNA sequences.

Suitable miRNA sequences can be identified by any methods known in the art. Typically, the methods described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001), are combined with the directed modification rules set forth in Reynolds et al. ., Nature Biotech., 22(3):326-330 (2004).

Typically, the 3' nucleotide sequence of the AUG start codon of a transcript from a target gene of interest is scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, where N = C, G, or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). Nucleotides located immediately adjacent to the 3' dinucleotide sequences are identified as potential siRNA sequences (ie, target sequence or sense strand sequence). Typically, 19, 21, 23, 25, 27, 29, 31, 33, 35 or more nucleotides immediately adjacent to the 3' dinucleotide sequences are identified as potential miRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and the 19 nucleotides immediately adjacent to the 3' dinucleotide of the AA or NA are identified as potential siRNA sequences. miRNA sequences are typically located at different positions along the length of the target gene. To further improve the silencing efficiency of miRNA sequences, candidate miRNA sequences can be analyzed to identify sites that do not contain regions of homology to other coding sequences, for example, in the target cell or organism. For example, a suitable miRNA sequence of about 21 base pairs will typically have no more than 16-17 contiguous base pairs of homology with coding sequences in the target cell or organism. If miRNA sequences are to be expressed with

- 14 046751 RNA Pol III promoter, select miRNA sequences that do not contain more than 4 contiguous A or T.

Once a potential siRNA sequence has been identified, a complementary sequence (ie, antisense strand sequence) can be designed. The potential miRNA sequence can also be analyzed using a variety of criteria known in the art. For example, to improve the efficiency of expression silencing, miRNA sequences can be analyzed using a targeted modification algorithm to identify sequences that have one or more of the following characteristics: (1) a G/C content of about 25 to about 60% G/C; (2) at least 3 A/U in positions 15-19 of the sense chain; (3) absence of internal repetitions; (4) And in position 19 of the semantic chain; (5) A in position 3 of the semantic chain; (6) U at position 10 of the semantic chain; (7) absence of G/C at position 19 of the sense strand and (8) absence of G at position 13 of the sense strand. SiRNA design tools, which include algorithms that assign appropriate values for each of these characteristics and are useful for miRNA selection, can be found, for example, at: http://boz094.ust.hk/RNAi/siRNA. One of ordinary skill in the art will appreciate that sequences with one or more of the above characteristics may be selected for further analysis and testing as potential siRNA sequences

In addition, potential miRNA sequences with one or more of the following criteria can often be excluded as miRNAs: (1) sequences containing a stretch of 4 or more of the same base in a series; (2) sequences containing Gs homopolymers (i.e., to reduce possible nonspecific effects due to the structural characteristics of these polymers; (3) sequences containing three-base motifs (e.g., GGG, CCC, AAA, or TTT); (4) ) sequences containing stretches of 7 or more G/Cs in a row; and (5) sequences containing direct repeats of 4 or more bases in candidates resulting in internal folded structures. However, those skilled in the art will recognize that sequences with one or more of. the above characteristics can still be selected for further analysis and testing as potential miRNA sequences

In some embodiments, potential miRNA sequences can be further analyzed based on the asymmetry of the miRNA duplex, as described, for example, in Khvorova et al., Cell, 115:209-216 (2003) and Schwarz et al., Cell, 115:199- 208 (2003). In other embodiments, candidate miRNA sequences can be further analyzed based on secondary structure at the target site, as described, for example, in Luo et al., Biophys. Res. Commun., 318:303–310 (2004). For example, secondary structure at a target site can be modeled using the Mfold algorithm (available at http://www.bioinfo.rpi.edu/applications/mfold/ma/forml.cgi) to select miRNA sequences that provide accessibility to the site -target with less secondary structure in the form of base pairing and stem-loop structures.

Once a potential miRNA sequence has been identified, the sequence can be analyzed for any immunostimulatory properties, for example using an in vitro cytokine assay or an in vivo animal model. Motifs in the sense and/or antisense strand of an siRNA sequence, such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU- 3', etc.) may also indicate whether the sequence may be immunostimulatory. Once an siRNA molecule is found to be immunostimulatory, it can be modified to reduce its immunostimulatory properties, as described herein. As a non-limiting example, the siRNA sequence may be contacted with an immunoreactive mammalian cell under conditions such that the cell produces a detectable immune response to determine whether the siRNA is an immunostimulatory siRNA or a non-immunostimulatory siRNA. The immunoreactive mammalian cell may be derived from a naïve mammal (ie, a mammal that has not previously been exposed to the gene product of the siRNA sequence). The immunoreactive mammalian cell may be, for example, a peripheral blood mononuclear cell (PBMC), a macrophage, or the like. The detectable immune response may include the production of a cytokine or growth factor, such as, for example, TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or a combination thereof. A siRNA molecule identified as immunostimulatory can then be modified to reduce its immunostimulatory properties by replacing at least one of the sense and/or antisense strand nucleotides with modified nucleotides. For example, less than about 30% (eg, less than about 30, 25, 20, 15, 10, or 5%) of the nucleotides in the double-stranded region of the siRNA duplex can be replaced with modified nucleotides, such as 2'OMe nucleotides. The modified siRNA can then be contacted with an immunoreactive mammalian cell as described above to confirm that its immunostimulatory properties have been reduced or suppressed.

Suitable in vitro assays for detecting immune response include, but are not limited to, the double monoclonal antibody sandwich immunoassay developed by David et al.

- 15 046751 (US patent No. 4376110); monoclonal-polyclonal antibody sandwich assay (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); Western blot Gordon et al. (US Patent No. 4452901); immunoprecipitation of labeled ligand (Brown et al., J. Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assay (ELISA), as described, for example, in Raines et al., J. Biol. Chem., 257:5154-5160 (1982); immunocytochemical methods, including the use of fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980)); and neutralizing activity (Bowen-Pore et al., Proc. Natl. Acad. Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassays described above, a number of other immunoassays are available, including those described in US Pat. Nos. 3,817,827, 3,850,752, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876. The information reported by these references is incorporated herein in its entirety. by reference for all purposes.

A non-limiting example of an in vivo model for detecting an immune response includes an in vivo murine cytokine induction assay as described, for example, in Judge et al., Mol. Ther., 13:494-505 (2006). In certain embodiments, the assay may be performed as follows: (1) siRNA may be administered by standard intravenous injection into a lateral tail vein; (2) blood can be collected by cardiac puncture approximately 6 hours after administration and plasma isolated for cytokine analysis; and (3) cytokines can be quantified using sandwich ELISA kits according to the manufacturer's instructions (eg, mouse and human IFN-α (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF-α (eBioscience; San Diego, CA) and murine IL-6, TNF-α, and IFN-γ (BD Biosciences; San Diego, CA)).

Monoclonal antibodies that specifically bind cytokines and growth factors are commercially available from a variety of sources and can be prepared using methods known in the art (see, for example, Kohler et al., Nature, 256:495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999). The production of monoclonal antibodies has been described previously and can be performed by any methods known in the art (Buhring et al., in Hybridoma, Vol. 10, No. 1, p. 77-78 (1991)). In some methods, monoclonal antibodies are labeled (eg, with any composition determined by spectroscopic, photochemical, biochemical, electrical, optical or chemical methods) to facilitate detection.

Preparation of siRNA molecules.

siRNA can be present in several forms, including, for example, as one or more isolated duplexes of small interfering RNA (siRNA), as extended double-stranded RNA (dsRNA), or as siRNA, or dsRNA transcribed from a transcription cassette in plasmid DNA . The miRNA sequences may have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)) or may not have protrusions (i.e., have blunt ends).

The RNA population can be used to generate long RNA precursors, or long RNA precursors that have significant or complete identity to a selected target sequence can be used to create siRNA. RNA can be isolated from cells or tissue, synthesized and/or cloned by methods well known to those skilled in the art. The RNA may be a mixed population (derived from cells or tissue, transcribed from cDNA subtracted, selected, etc.) or may represent a single target sequence. RNA may be natural (eg, isolated from tissue or cell samples), synthesized in vitro (eg, using T7 or SP6 polymerase and PCR products or cloned cDNA), or chemically synthesized.

To produce long dsRNA for synthetic RNAs, complement is also transcribed in vitro and hybridized to produce dsRNA. If a naturally occurring RNA population is used, RNA complements are also provided (eg, to generate dsRNA for E. coli RNase III or Dicer digestion), for example, by transcribing a cDNA of the appropriate RNA population or by using RNA polymerases. The RNA precursors then hybridize to form double-stranded RNAs for cleavage. The dsRNAs can be administered directly to the subject or they can be digested in vitro before administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, constructing and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as well as PCR methods (see US patent No. 4683195 and 4683202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression sequence libraries are also well known to those skilled in the art. Additional basic texts covering general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual ( ^2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The information provided through these links is incorporated herein by reference in its entirety for all purposes.

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Preferably, the siRNAs are chemically synthesized. Oligonucleotides that contain the siRNA molecules of the present invention can be synthesized using any of the methods known in the art, such as those described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:26772684 (1995) and Wincott et al., Methods Mol. Bio., 74:59 (1997). Oligonucleotide synthesis uses common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5' end and phosphoramidites at the 3' end. By way of non-limiting example, a small-scale synthesis process can be performed using a synthesizer from Applied Biosystems using a 0.2 µM scale-up protocol. Alternatively, synthesis on a 0.2 μM scale can be accomplished using a 96-well plate synthesizer from Protogene (Palo Alto, CA). However, larger or smaller scale syntheses are also within the scope of this invention. Suitable reagents for the synthesis of oligonucleotides, methods for deprotecting RNA, and methods for purifying RNA are known to those skilled in the art.

SiRNA molecules can also be synthesized using a tandem synthesis technique, in which both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker, which is subsequently cleaved to produce individual fragments or strands that hybridize to form an siRNA duplex. The linker may be a polynucleotide linker or a non-nucleotide linker. Tandem siRNA synthesis can be easily adapted to both multi-well/multiplate synthesis platforms and large-scale synthesis platforms using batch reactors, synthesis columns, etc. Alternatively, siRNA molecules can be composed of two different oligonucleotides, one containing the sense strand and the other containing the antisense strand of the siRNA. For example, each chain can be synthesized separately and joined together by hybridization or ligation, after synthesis and/or deprotection. In certain other cases, siRNA molecules can be synthesized as a single contiguous oligonucleotide fragment where self-complementary sense and antisense regions hybridize to form a siRNA duplex having a hairpin secondary structure.

Modification of miRNA sequences.

In certain aspects, siRNA molecules comprise a duplex having two strands and at least one modified nucleotide in the double-stranded region, where each strand is from about 15 to about 60 nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory than the corresponding unmodified siRNA sequence, but retains the ability to inhibit expression of the target sequence. In preferred embodiments, the degree of chemical modifications introduced into the siRNA molecule strikes a balance between reducing or eliminating the immunostimulatory properties of the siRNA and maintaining RNAi activity. As a non-limiting example, an siRNA molecule that targets a gene of interest may be minimally modified (e.g., less than about 30, 25, 20, 15, 10, or 5% modified) at selective uridine and/or guanosine nucleotides in the siRNA duplex to eliminating the immune response generated by siRNA while maintaining its ability to suppress the expression of the target gene.

Examples of modified nucleotides suitable for use in the invention include, but are not limited to, ribonucleotides containing 2'-O-methyl(2'OMe), 2'-deoxy-2'fluoro(2T), 2'-deoxy, 5 -C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino or 2'-C-allyl group. Modified nucleotides having a Northern conformation such as those described, for example, in Saenger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984) are also suitable for use in siRNA molecules. Such modified nucleotides include, but are not limited to, locked nucleic acid (LNA) nucleotides (e.g., 2'-O,4'-C-methylene-N-ribofuranosyl)nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides, 2'-methylthioethyl nucleotides, 2'-deoxy-2'fluoro(2 ^g B) nucleotides, 2'-deoxy-2'-chloro (2'C1) nucleotides and 2'-azidonucleotides. In certain cases, the siRNA molecules described herein contain one or more G-Clamp nucleotides. A G-fixing base nucleotide refers to a modified cytosine analogue in which the modification imparts the ability to hydrogen bond to the Watson-Crick and Hoogsten faces of the complementary guanine nucleotide in the duplex (see, e.g., Lin et al., J. Am. Chem. Soc. ., 120:8531-8532 (1998)). In addition, nucleotides having a nucleotide base analogue such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole (see, for example, Loakes, Nucl. Acids Res., 29:2437-2447 (2001)). In certain embodiments, the siRNA molecules may further comprise one or more chemical modifications, such as cap terminals, phosphate backbone modifications, and the like.

Examples of terminal cap fragments include, but are not limited to, inverted deoxyabase residues, glycerol modifications, 4',5'-methylene nucleotides, 1-(β-erythrofuranosyl)nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides,

- 17 046751 α-nucleotides, modified basic nucleotides, threopentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide fragments, 3 '-3'-inverted base fragments, 3'-2'-inverted nucleotide fragments, 3'-2'-inverted base fragments, 5'-5'-inverted nucleotide fragments, 5'-5'-inverted base fragments, 3 '-5'-inverted deoxyabase moieties, 5'-aminoalkylphosphate, 1,3-diamino-2-propylphosphate, 3-aminopropylphosphate, 6-aminohexylphosphate,

1,2-Aminododecylphosphate, hydroxypropylphosphate, 1,4-butanediolphosphate, 3'-phosphoramidate, 5'-phosphoramidate, hexylphosphate, aminohexylphosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioate, 5'-phosphorothioate, phosphorodithioate and bridged or non-bridged methylphosphonate or 5'-mercaptofragments (see, for example, US Pat. No. 5,998,203; Beaucage et al., Tetrahedron, 49:1925 (1993)). Non-limiting examples of modifications of the phosphate backbone (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholine, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formoacetal, ethal and alkylsilyl substitutions ( see, for example, Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994). Such chemical modifications can occur at the 5' end and/or 3' end of the sense strand, antisense strand, or both strands of the siRNA. The information provided through these references is incorporated herein by reference in its entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNA molecule may further comprise a 3'-terminal overhang containing from about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxyribonucleotides and/or any combination of modified and unmodified nucleotides. Additional examples of modified nucleotides and types of chemical modifications that can be introduced into siRNA molecules are described, for example, in British Patent No. GB 2397818 B and in US Patent Publications No. 20040192626, 20050282188 and 20070135372, the disclosures of which are incorporated herein by reference in their entirety. all purposes.

The siRNA molecules described herein may optionally contain one or more non-nucleotides in one or both siRNA strands. As used herein, the term non-nucleotide refers to any group or compound that can be included in a nucleic acid chain in place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exert their activity. A group or compound is abasic in the sense that it does not contain a recognized nucleotide base such as adenosine, guanine, cytosine, uracil or thymine, and therefore does not have a base at the 1' position.

In other embodiments, chemical modification of the siRNA involves attaching a conjugate to the siRNA molecule. The conjugate can be attached to the 5' and/or 3' end of the sense and/or antisense strand of the siRNA through a covalent attachment, such as, for example, a biodegradable linker. The conjugate can also be attached to siRNA, for example, through a carbamate group or other linking group (see, for example, US Patent Publications No. 20050074771, 20050043219 and 20050158727). In certain cases, a conjugate is a molecule that facilitates the delivery of siRNA into a cell. Examples of conjugate molecules suitable for attachment to siRNA include, but are not limited to, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (eg , folic acid, folic acid analogues and their derivatives), sugars (for example, galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular absorption and combinations thereof (see, for example, US Pat. Nos. 20030130186, 20040110296, and 20040249178; US Pat. No. 6,753,423). Other examples include lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid, small molecule, oligosaccharide, carbohydrate cluster, intercalator, minor groove binder, cleavage agent, and crosslinker conjugate molecules described in U.S. Patent Publications No. 20050119470 and 20050107325. Still other examples include 2'-O-alkylamine, 2'-β-alkoxyalkylamine, polyamine, C5 cationically modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic amino acid conjugate molecules described in US Patent Publication No. 20050153337 Additional examples include the hydrophobic group, membrane active compound, cell permeable compound, cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in US Patent Publication No. 20040167090. Additional examples include the conjugate molecules described in US Patent Publication No. 20050239739. The type of conjugate used and the degree of conjugation to the siRNA molecule can be assessed to improve pharmacokinetic profiles, bioavailability and/or stability

- 18 046751 siRNA while maintaining RNAi activity. Thus, one of ordinary skill in the art can screen siRNA molecules with various conjugates attached to identify molecules having improved properties and full RNAi activity using any of a variety of well-known in vitro cell cultures or in vivo animal models. The disclosures of the above patent documents are incorporated herein by reference in their entirety for all purposes.

Target genes.

In certain embodiments, the nucleic acid component (e.g., siRNA) of the nucleic acid and lipid-based particles described herein can be used to suppress or disable translation (ie, expression) of a gene of interest. Genes of interest include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (eg, liver diseases and disorders), genes associated with tumorigenesis and cell transformation (eg, cancer), angiogenic genes, immunomodulator genes such as genes associated with inflammatory and autoimmune responses, ligand receptor genes and genes associated with neurodegenerative disorders. In certain embodiments, the gene of interest is expressed in hepatocytes.

Genes associated with viral infection and survival include genes expressed by the virus for binding, entry, and replication within the cell. Of particular interest are viral sequences associated with chronic viral diseases. Viral sequences of particular interest include those of filoviruses such as Ebola virus and Marburg virus (see, for example, Geisbert et al., J. Infect. Dis., 193:1650-1657 (2006)); arenaviruses such as Lassa virus, Junin virus, Machupo virus, Guanarito virus and Sabia virus (Buchmeier et al., Arenaviridae: the viruses and their replication, In: FIELDS VIROLOGY, Knipe et al. (eds.), ^4th ed. , Lippincott-Raven, Philadelphia, (2001)); influenza viruses such as influenza A, B and C viruses (see, for example, Steinhauer et al., Annu Rev. Genet., 36:305-332 (2002) and Neumann et al., J. Gen Virol., 83 :2635-2662 (2002)); hepatitis viruses (see, for example, Hamasaki et al., FEBS Lett., 543:51 (2003); Yokota et al., EMBO Rep., 4:602 (2003); Schlomai et al., Hepatology, 37:764 (2003); Proc. Natl. Sci. USA, 100:2783; VIROLOGY, Knipe et al. (eds.), ^4th ed., Lippincott-Raven, Philadelphia (2001)); human immunodeficiency virus (HIV) (Banerjea et al., Mol. Ther., 8:62 (2003); Song et al., J. Virol., 77:7174 (2003); Stephenson, JAMA, 289:1494 (2003 ); Qin et al., Proc. Acad. USA, 100:183) herpes viruses (Jia et al., J. Virol., 77:3301 (2003)) and human papillomaviruses (HPV) (Hall et al., J. Virol., 77:6066 (2003)); Jiang et al., Oncogene, 21:6041 (2002)).

Examples of filovirus nucleic acid sequences whose expression can be inhibited include, but are not limited to, nucleic acid sequences encoding structural proteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase proteins (L-pol)) and membrane-bound proteins (e.g. , VP40, glycoprotein (GP), VP24). The complete genome sequences of the Ebola virus are reported, for example, in Genbank under accession numbers NC-002549; AY769362; NC-006432; NC-004161; AY729654; AY354458; AY142960; AB050936; AF522874; AF499101; AF272001 and AF086833. The sequences of Ebola virus VP24 are reported, for example, in Genbank under accession numbers U77385 and AY058897. The sequences of the Ebola virus L-pol are presented, for example, in Genbank under accession number X67110. The sequences of Ebola virus VP40 are reported, for example, in Genbank under accession number AY058896. The sequences of the Ebola virus NP are reported, for example, in Genbank under accession numbers AY058895. The sequences of Ebola virus GP are reported, for example, in Genbank under accession number AY058898; Sanchez et al., Virus Res., 29:215-240 (1993); Will et al., J. Virol., 67:12031210 (1993); Volchkov et al., FEBS Lett, 305:181-184 (1992) and in US patent No. 6713069. Additional sequences of the Ebola virus are set forth, for example, in Genbank under accession numbers L11365 and X61274 Complete genomic sequences of the Marburg virus are set forth, for example, in Genbank under accession numbers NC-001608; AY430365; AY430366; and AY358025. The sequences of the Marburg GP virus are reported, for example, in Genbank under accession numbers AF005734; AF005733 and AF005732. The sequences of Marburg virus VP35 are reported, for example, in Genbank under accession numbers AF005731 and AF005730. Additional Marburg virus sequences are provided, for example, in Genbank under accession numbers X64406; Z29337; AF005735 and Z12132. Non-limiting examples of siRNA molecules targeting Ebola virus and Marburg virus nucleic acid sequences include those described in US Patent Publication No. 20070135370, the disclosure of which is incorporated herein by reference for all purposes.

Examples of influenza virus nucleic acid sequences whose expression can be inhibited include, but are not limited to, nucleic acid sequences encoding nucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins (NS1 and NS2), RNA polymerase (PA, PB1 , PB2), neuraminidase (NA) and hemagglutinin (HA). Influenza A NP sequences are reported, for example, in Genbank under accession numbers: NC-004522; AY818138; AB166863; AB188817; AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493; AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651500; AY651501; AY651502; AY651503; AY651504; AY651505;

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AY651506; AY651507; AY651509; AY651528; AY770996; AY790308; AY818138 and AY818140.

Influenza A RA sequences are presented, for example, in Genbank under accession numbers: AY818132; AY790280; AY646171; AY818132; AY818133; AY646179; AY818134; AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611; AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615; AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995 and AY724786.

Non-limiting examples of siRNA molecules targeting influenza virus nucleic acid sequences include those described in US Patent Publication No. 20070218122, the disclosure of which is incorporated herein by reference for all purposes.

Examples of hepatitis virus nucleic acid sequences whose expression can be inhibited include, but are not limited to, nucleic acid sequences involved in transcription and translation (eg, En1, En2, X, P) and nucleic acid sequences encoding structural proteins (eg , core proteins, including protein C and C-related proteins, capsid and envelope proteins, including proteins S, M and/or L or fragments thereof) (see, for example, FIELDS VIROLOGY, supra). Examples of hepatitis C virus (HCV) nucleic acid sequences whose expression can be inhibited include, but are not limited to, 5' untranslated region (5'-UTR), 3' untranslated region (3'-UTR), initiation codon region translation polyprotein, internal ribosome entry site (IRES) sequence and/or nucleic acid sequences encoding core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protease/helicase, NS4A protein, NS4B protein, NS5A protein and/or RNA-dependent RNA polymerase NS5B. The HCV genome sequences are reported, for example, in Genbank accession numbers NC-004102 (HCV genotype la), AJ238799 (HCV genotype 1b), NC-009823 (HCV genotype 2), NC-009824 (HCV genotype 3), NC-009825 ( HCV genotype 4), NC-009826 (HCV genotype 5) and NC-009827 (HCV genotype 6). The nucleic acid sequences of the hepatitis A virus are set forth, for example, in Genbank accession number NC-001489; hepatitis B virus nucleic acid sequences are set forth, for example, in Genbank accession number NC-003977; hepatitis D virus nucleic acid sequences are set forth, for example, in Genbank accession number NC-001653; The nucleic acid sequences of the hepatitis E virus are set forth, for example, in Genbank accession number NC-001434; and the nucleic acid sequences of the hepatitis G virus are reported, for example, in Genbank under accession number NC-001710. Inhibition of the expression of sequences encoding genes associated with viral infection and survival can conveniently be used in combination with the administration of conventional agents used to treat a viral disease. Non-limiting examples of siRNA molecules targeting hepatitis virus nucleic acid sequences include those described in US Patent Publications No. 20060281175, 20050058982 and 20070149470; US Patent No. 7,348,314 and US Provisional Application No. 61/162,127, filed March 20, 2009, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Genes associated with metabolic diseases and disorders (eg, disorders that target the liver and liver diseases and disorders) include, for example, genes expressed in dyslipidemia (eg, liver X receptors such as LXRa and LXRe (accession no. in Genbank NM-007121), farnesoid X receptors (FXR) (Genbank accession number NM-005123), sterol regulatory element binding protein (SREBP), site-1 protease (SIP), 3-hydroxy-3-methylglutaryl coenzyme -A reductase (HMG-coenzyme A reductase), apolipoprotein B (ApoB) (Genbank accession number NM-000384), apolipoprotein CIII (ApoC3) (Genbank accession numbers NM-000040 and NG-008949 REGION: 5001.8164) and apolipoprotein E (ApoE) (Genbank accession numbers NM-000041 and NG-007084 REGION: 5001.8612)); and in diabetes (eg, glucose-6phosphatase) (see, for example, Forman et al., Cell, 81:687 (1995); Seol et al., Mol. Endocrinol., 9:72 (1995), Zavacki et al. al., Proc. Natl. Sci. USA, 94:7909; Sakai et al., Cell, 85:1037-1046 (1996); 12778-12785 (1997); Willy et al., Genes Dev., 9:1033-1045 (1995); , Nature, 383:728-731 (1996); and Peet et al., Cell, 93:693-704 (1998)). One of ordinary skill in the art will appreciate that genes associated with metabolic diseases and disorders (e.g., diseases and disorders that target the liver and liver diseases and disorders) include genes that are expressed in the liver itself as well as genes expressed in other organs and tissues. Inhibition of the expression of sequences encoding genes associated with metabolic diseases and disorders can conveniently be used in combination with the administration of conventional agents used to treat the disease or disorder. Non-limiting examples of siRNA molecules targeting the ApoB gene include those described in US Patent Publication No. 20060134189, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the ApoC3 gene include those described in US Provisional Application No. 61/147235, filed January 26, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Examples of gene sequences associated with tumorigenesis and cell transformation (eg

- 20 046751 measures, cancer or other neoplasia) include mitotic kinesins such as Eg5 (KSP, KIF11; Genbank accession number NM-004523); serine/threonine kinases such as polo-like kinase 1 (PLK-1) (Genbank accession number NM-005030; Barr et al., Nat. Rev. Mol. Cell. Biol., 5:429-440 (2004) ); tyrosine kinases such as WEE1 (Genbank accession numbers NM-003390 and NM-001143976); apoptosis inhibitors such as XIAP (Genbank accession number NM-001167); COP9 signanosome subunits such as CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM-006837); CSN6, CSN7A, CSN7B and CSN8; ubiquitin ligases such as COP1 (RFWD2; Genbank accession numbers NM-022457 and NM-001001740); and histone deacetylases such as HDAC1, HDAC2 (Genbank accession numbers NM-001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc. Non-limiting examples of siRNA molecules targeting the Eg5 and XIAP genes include those described in US Patent Application No. 11/807872, filed October 29, 2007, the disclosure of which is incorporated herein by reference for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in US Patent Publications Nos. 20050107316 and 20070265438 and US Patent Application No. 12/343,342, filed October 23, 2008, the entire disclosure of which is incorporated herein by reference for all purposes. Non-limiting examples of siRNA molecules targeting the CSN5 gene include those described in US Provisional Application No. 61/045251, filed April 15, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Additional examples of gene sequences associated with tumorigenesis and cell transformation include translocation sequences such as the MLL, BCR-ABL fusion genes (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003); Wu et al., Cancer Res. 63:1515 (2003)), cyclins (Li et al., Cancer Res. ., 63:3593 (2003); Zou et al., Genes Dev., 16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291 (2003)), telomerase genes (Kosciolek et al., Mol. Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (eg, EGFR/ErbB1 (Genbank accession numbers NM-005228, NM-201282, NM-201283 and NM-201284; see also Nagy et al. Exp. Cell Res., 285:39-49 (2003), ErbB2/HER-2 (Genbank accession numbers NM-004448 and NM- 001005862), ErbB3 (Genbank accession numbers NM-001982 and NM-001005915), and ErbB4 (Genbank accession numbers NM-005235 and NM-001042599); and mutated sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002). Non-limiting examples of siRNA molecules targeting the EGFR gene include those described in US patent application No. 11/807872, filed October 29, 2007, the disclosure of which is incorporated herein by reference in its entirety. for all purposes.

Suppression of the expression of sequences encoding DNA repair enzymes is used in combination with the administration of chemotherapeutic agents. (Collis et al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associated with tumor migration are also target sequences of interest, such as integrins, selectins, and metalloproteinases. The above examples are not exclusive. Those skilled in the art will appreciate that any whole or partial gene sequence that facilitates or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration may be included as a template sequence.

Angiogenic genes can promote the formation of new blood vessels. Of particular interest is vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis., 9:210 (2003)) or VEGFR. SiRNA sequences targeting VEGFR are set out, for example, in GB 2396864; US Patent Publication No. 20040142895 and CA 2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Antiangiogenic genes are able to suppress neovascularization. These genes are particularly useful for treating those cancers in which angiogenesis plays a role in the pathological progression of the disease. Examples of antiangiogenic genes include, but are not limited to, endostatin (see, e.g., US Pat. No. 6,174,861), angiostatin (see, e.g., US Pat. No. 5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J. Pathol., 188: 369-377 (1999)), the descriptions of which are incorporated herein by reference in their entirety for all purposes. Immunomodulator genes are genes that modulate one or more immune responses. Examples of immunomodulator genes include, but are not limited to, cytokines such as growth factors (eg, TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (eg, IL-2, IL-4, IL-12 (Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL-20, etc.) , interferons (eg, IFN-α, IFN-β, IFN-γ, etc.) and TNF. The Fas and Fas ligand genes are also immunomodulator target sequences of interest (Song et al., Nat. Med., 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present invention, for example Tec family kinases such as Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)).

- 21 046751

Cellular receptor ligands include ligands that are capable of binding to cell surface receptors (e.g., insulin receptor, erythropoietin (EPO) receptor, G protein-coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to modulate (e.g., inhibit, activate, etc..) a physiological pathway in which the receptor is involved (e.g., glucose modulation, blood cell development, mitogenesis, etc.) Examples of cellular receptor ligands include but but not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin, insulin, glucagon, G protein-coupled receptor ligands, etc. Templates encoding trinucleotide repeat expansions (eg, CAG repeats) are used to suppress the expression of pathogenic sequences in neurodegenerative disorders caused by trinucleotide repeat expansions, such as spinobulbular muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet. , 11:175 (2002)).

Some other target genes that can be targeted by nucleic acid (e.g., siRNA) to suppress or repress gene expression include, but are not limited to, aortic smooth muscle alpha actin-2 (ASTA2), alcohol dehydrogenase 1A (ADH1A), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), afamin (AFM), angiotensinogen (AGT), serine pyruvate aminotransferase (AGXT), alpha-2-N-glycoprotein (AHSG), aldokereductase family member C4 1 (AKR1C4), serum albumin (ALB ), alpha-1-microglobulin/bikunin precursor (AMBP), angiopoietin-like protein 3 (ANGPTL3), amyloid P component of serum (APCS), apolipoprotein A-II (APOA2), apolipoprotein B-100 (APOB), apolipoprotein C3 ( APOC3), apolipoprotein C-IV (APOC4), apolipoprotein F (APOF), beta-2-glycoprotein 1 (APON), aquaporin-9 (AQP9), bile acid KoA: amino acid N-acyltransferase (BAAT), C4b-binding protein beta chain (C4BPB), putative characterized protein encoded by LINC01554 (C5orf27), complement factor 3 (C3), complement factor 5 (C5), complement factor C6 (C6), complement factor C8 alpha chain (C8A), complement factor C8 beta chain (C8B), complement factor C8 gamma chain (C8G), complement factor C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor-related protein 1 H (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor tissue (CTGF), chemokine 2 with C-X-C motif (CXCL2), cytochrome P450 1A2 (CYP1A2), cytochrome P450 2A6 (CYP2A6), cytochrome P450 2C8 (CYP2C8), cytochrome P450 2C9 (CYP2C9), cytochrome P450, family 2, subfamily D, member 6 (CYP2D6), cytochrome P450 2E1 (CYP2E1), phylloquinone omegahydroxy CYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one-12-alpha-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (F12), coagulation factor II (thrombin) (F2), coagulation factor IX (F9), fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), gamma chain fibrinogen (FGG), fibrinogen-like protein 1 (FGL1), flavin monooxygenase 3 (FMO3), flavin monooxygenase 5 (FMO5), group-specific component (vitamin D binding protein) (GC), growth hormone receptor (GHR), glycine- N-methyltransferase (GNMT), hyaluronan binding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP), hydroxy acid oxidase (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC), haptoglobin-associated protein; haptoglobin (HPR), hemopexin (HPX), histidine-rich glycoprotein (HRG), hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), hydroxysteroid (17-beta) dehydrogenase 13 (HSD17B13), inter-alpha trypsin inhibitor heavy chain H1 (ITIH1), inter-alphatrypsin inhibitor heavy chain H2 (ITIH2), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), prekallikrein (KLKB1), lactate dehydrogenase A ( LDHA), liver expressed antimicrobial peptide 2 (LEAP2), leukocyte cell-derived chemotaxin 2 (LECT2), lipoprotein (a) (LPA), mannan-binding lectin serine protease 2 (MASP2), S-adenosylmethionine synthase isoform type 1 ( MAT1A), NADPH oxidase 4 (NOX4), poly [ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1), paraoxonase 3 (PON3), vitamin K-dependent protein C (PROC), retinol dehydrogenase 16 (RDH16) , serum amyloid A4, constitutive (SAA4), serine dehydratase (SDS), alpha-1-antitrypsin (SERPINA1), serpin A11 (SERPINA11), kallistatin (SERPINA4), corticosteroid binding globulin (SERPINA6), antithrombin-III (SERPINC1), heparin cofactor 2 (SERPIND1), serpin H family member 1 (SERPINH1), solute carrier family 5 member 2 (SLC5A2), sodium/bile acid cotransporter (SLC10A1), solute carrier family member 5 13 (SLC13A5), carrier family member 1 solute carrier family 22 (SLC22A1), solute carrier family 25 member 47 (SLC25A47), solute carrier family 2, glucose transporter type 2 (SLC2A2), Sodium-linked neutral amino acid transporter 4 (SLC38A4), member of organic anion transporter 1B1 family ( SLCO1B1), sphingomyelin phosphodiesterase 1 (SMPD1), bile salt sulfotransferase (SULT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase (TDO2), UDP-glucuronosyltransferase family 2, polypeptide B10 (UGT2B10), UDP-glucuronosyltransferase family 2 , polypeptide B15 (UGT2B15), UDP-glucuronosyltransferase family 2, polypeptide B4 (UGT2B4) and vitronectin

- 22 046751 (VTN).

In addition to being useful for inhibiting the expression of any of the genes described above for therapeutic purposes, certain nucleic acids (e.g., siRNAs) described herein are also useful in research and development, as well as diagnostic, prophylactic, prognostic, clinical, and other healthcare applications . By way of non-limiting example, certain nucleic acids (eg, siRNA) can be used in target validation studies aimed at testing whether a gene of interest could potentially be a therapeutic target. Certain nucleic acids (eg, siRNAs) can also be used in target identification studies aimed at discovering genes as potential therapeutic targets.

CRISPR.

Targeted genome editing has evolved from a niche technology to one used by many biological researchers. This progress is largely fueled by the advent of clustered regularly interspaced short palindromic repeat (CRISPR) technology (see, for example, Sander et al., Nature Biotechnology, 32(4), 347-355, including supporting information (2014) and international publications No. WO 2016/197132 and WO 2016/197133). Accordingly, this paper presents improvements (e.g., lipid nanoparticles and their formulations) that can be used in combination with CRISPR technology to treat diseases such as HBV. In terms of targets for CRISPR use, the guide RNA (gRNA) used in CRISPR technology can be designed to target specifically identified sequences, such as target genes, such as those of the HBV genome. Examples of such target sequences are given in international publication WO 2016/197132. In addition, International Publication No. WO 2013/151665 (for example, see Table 6; this document is specifically incorporated by reference, particularly including Table 6 and the associated sequence listing) describes approximately 35,000 mRNA sequences claimed in the context of an expression construct mRNA. In certain embodiments of the present invention, CRISPR technology is used to target the expression of any of these sequences. In certain embodiments of the present invention, CRISPR technology may also be used to target the expression of a target gene discussed herein.

aiRNA.

Like siRNA, asymmetric interfering RNA (aiRNA) can recruit the RNA-inducible silencing complex (RISC) and lead to the efficient suppression of the expression of multiple genes in mammalian cells by mediating sequence-specific cleavage of the target sequence between nucleotides 10 and 11 relative to the 5' end of the antisense chains (Sun et al., Nat. Biotech., 26:1379-1382 (2008)). Typically, an aiRNA molecule contains a short RNA duplex having a sense and an antisense strand, with the duplex containing overhangs at the 3' and 5' ends of the antisense strand. aiRNA is usually asymmetrical because the sense strand is shorter at both ends compared to the complementary antisense strand. In some aspects, aiRNA molecules can be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules. As a non-limiting example, miRNA sequences can be selected and generated using the miRNA sequence selection methods described above.

In another embodiment, aiRNA duplexes of varying lengths (e.g., about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19 or base pairs) can be designed with overhangs at the 3' and 5' ends of the antisense strand to target the mRNA of interest. In certain cases, the sense strand of an aiRNA molecule is about 10-25, 12-20, 12-19, 12-18, 13-17 or 14-17 nucleotides in length, most often 12, 13, 14, 15, 16, 17. 18, 19 or 20 nucleotides in length. In certain other cases, the length of the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21 -22 or 21-23 nucleotides in length.

In some embodiments, the 5' overhang on the antisense strand contains one, two, three, four or more non-target nucleotides (eg, AA, UU, dTdT, etc.). In other embodiments, the 3' overhang on the antisense strand contains one, two, three, four or more non-target nucleotides (eg, AA, UU, dTdT, etc.). In certain aspects, the aiRNA molecules described herein may contain one or more modified nucleotides, for example, in the double-stranded region and/or in antisense overhangs. As a non-limiting example, the miRNA sequences may contain one or more of the modified nucleotides described above for miRNA sequences. In a preferred embodiment, the aiRNA molecule contains 2'OMe nucleotides, such as, for example, 2'OMe guanosine nucleotides, 2'OMe uridine nucleotides, or mixtures thereof.

In certain embodiments, the aiRNA molecules may contain an antisense strand that corresponds to the antisense strand of the siRNA molecule, such as one of the siRNA molecules, opi

- 23 046751 included in this document. In other embodiments, aiRNA molecules can be used to suppress the expression of any of the above target genes, such as, for example, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumorigenesis and cell transformation , angiogenic genes, immunomodulator genes such as genes associated with inflammatory and autoimmune responses, ligand receptor genes and genes associated with neurodegenerative disorders.

MicroRNA.

Typically, microRNAs (miRNAs) are single-stranded RNA molecules about 2123 nucleotides in length that regulate gene expression. microRNAs are encoded by genes from whose DNA they are transcribed, but microRNAs are not translated into protein (non-coding RNA); instead, each primary transcript (pre-miRNA) is converted into a short stem-loop structure called pre-miRNA, and finally into a functional mature miRNA. Mature microRNA molecules are partially or fully complementary to one or more messenger RNA (mRNA) molecules, and their main function is to suppress gene expression. The identification of microRNA molecules is described, for example, in Lagos-Quintana et al., Science, 294:853-858; Lau et al., Science, 294:858-862 and Lee et al., Science, 294:862-864.

Genes encoding microRNAs are much longer than the processed mature miRNA molecule. miRNAs are first transcribed as primary transcripts or pre-miRNAs with a cap and polyadenyl tail and are processed into short, ~70-nucleotide stem-loop structures known as pre-miRNAs in the cell nucleus. This processing is performed in animals by a protein complex known as the microprocessor complex, consisting of the Drosha nuclease and the double-stranded RNA-binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)).

These pre-miRNAs are then processed to form mature miRNAs in the cytoplasm through interaction with the endonuclease Dicer, which also initiates the formation of the RNA-inducible silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001). Either the sense strand or antisense strand of DNA can function as a template, giving rise to miRNA.

When Dicer cleaves the stem-loop structure of pre-miRNA, two complementary short RNA molecules are produced, but only one is integrated into the RISC complex. This strand is known as the guide strand and is selected by the argonaute protein, a catalytically active RNase in the RISC complex, based on the stability of the 5' end (Preall et al., Curr. Biol., 16:530-535 (2006)). The remaining strand, known as the antiguide or chaperone strand, is degraded as a substrate of the RISC complex (Gregory et al., Cell, 123:631-640 (2005)). Once integrated into the active RISC complex, miRNAs pair with their complementary mRNA molecules and cause degradation of the target mRNA and/or translational silencing of expression.

Mammalian microRNA molecules are usually complementary to a site in the 3'-UTR of the target mRNA sequence. In certain cases, miRNA annealing to target mRNA inhibits protein translation by blocking the protein translation machinery. In certain other cases, annealing of a miRNA to a target mRNA facilitates the cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNAs can also target methylation of genomic sites that correspond to the target mRNA. Typically, a miRNA functions in association with a set of proteins that are collectively called miRNAs.

In certain aspects, microRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15- 30, 15-25 or 19-25 nucleotides in length, preferably about 20-24, 21-22 or 21-23 nucleotides in length. In certain other aspects, miRNA molecules may contain one or more modified nucleotides. As a non-limiting example, miRNA sequences may contain one or more modified nucleotides described above for miRNA sequences. In a preferred embodiment, the microRNA molecule contains 2'OMe nucleotides, such as, for example, 2'OMe guanosine nucleotides, 2'OMe uridine nucleotides, or mixtures thereof.

In some embodiments, microRNA molecules can be used to suppress the expression of any of the above target genes, such as, for example, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumorigenesis and cell transformation , angiogenic genes, immunomodulator genes such as genes associated with inflammatory and autoimmune responses, ligand receptor genes and genes associated with neurodegenerative disorders.

In other embodiments, one or more agents that block the activity of a microRNA directed to an mRNA of interest are administered using a lipid particle of the invention (eg, a nucleic acid-lipid particle). Examples of blocking agents include, but are not limited to, space blocking oligonucleotides, blocked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blocking agents can bind directly to the miRNA or to the miRNA binding site on the target mRNA.

- 24 046751

Antisense oligonucleotides.

In one embodiment, the nucleic acid is an antisense oligonucleotide directed to a target gene or sequence of interest. The terms antisense oligonucleotide or antisense include oligonucleotides complementary to the target polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a selected sequence. Antisense RNA oligonucleotides prevent the translation of complementary RNA strands by binding to RNA. Antisense DNA oligonucleotides can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid can be cleaved by the enzyme RNase H. In a particular embodiment, the antisense oligonucleotides contain from about 10 to about 60 nucleotides, more preferably from about 15 to about 30 nucleotides. The term also includes antisense oligonucleotides, which may not be exactly complementary to the desired target gene. Thus, the invention can be used in cases where off-target specific activities are detected by an antisense sequence or where an antisense sequence containing one or more mismatches with the target sequence is most preferred for a particular use.

Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis and therefore can be used to specifically inhibit protein synthesis of a target gene. The effectiveness of antisense oligonucleotides in inhibiting protein synthesis is well known. For example, polygalactauronase synthesis and acetylcholine muscarinic receptor type 2 are inhibited by antisense oligonucleotides targeting their respective mRNA sequences (see US Pat. Nos. 5,739,119 and 5,759,829). Additionally, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multidrug resistance gene (MDR1), ICAM-1, E-selectin, STK-1, striatal receptor GABAA, and human EGF (see Jaskulski et al., Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Cornmun., 1:225-32 (1989); Brain Res Mol Brain Res., 57:310-20 (1998); US Patents 5801154, 5789573, 5718709 and 5610288). Moreover, antisense constructs have also been described that inhibit and can be used to treat various abnormal cell proliferations, such as cancer (see US Pat. Nos. 5,747,470; 5,591,317 and 5,783,683). The information provided through these links is incorporated herein by reference in its entirety for all purposes.

Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce an antisense oligonucleotide targeting any polynucleotide sequence. The selection of antisense oligonucleotide sequences specific for a given target sequence is based on analysis of the selected target sequence and determination of secondary structure, T _m , binding energy and relative stability. Antisense oligonucleotides may be selected based on their relative inability to form dimers, hairpins, or other secondary structures that may reduce or inhibit specific binding to target mRNA in the host cell. The most preferred mRNA target regions include regions at or near the translation initiation codon AUG and those sequences that are substantially complementary to the 5' regions of the mRNA. These secondary structure analyzes and target site selection can be performed, for example, using OLIGO (Molecular Biology Insights) primer analysis software version 4 and/or BLASTN 2.0.5 algorithm software. (Altschul et al., Nucleic. Acids Res., 25:3389-402 (1997)).

Ribozymes.

According to another embodiment of the invention, the nucleic acid and lipid particles are coupled to ribozymes. Ribozymes are RNA-protein complexes having specific catalytic domains that have endonuclease activity (see, Kim et al., Proc. Natl. Acad. Sci. USA, 84:8788-92 (1987) and Forster et al., Cell , 49:211-20 (1987)). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (see, Cech et al., Cell, 27:487-96 (1981); Michel et al., J Mol. Biol., 216:585-610 (1990); Reinhold-Hurek et al., Nature, 357:173-6 (1992). This specificity is explained by the requirement that the substrate bind through specific base-pairing interactions to the internal guide sequence (IGS) of the ribozyme prior to the chemical reaction.

At least six major types of naturally occurring enzymatic RNA molecules are currently known. Each can catalyze the hydrolysis of trans-phosphodiester bonds of RNA (and thus can cleave other RNA molecules) under physiological conditions. Typically, enzymatic nucleic acids first bind to the target RNA. This binding occurs through the target binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzyme portion of the molecule that cleaves the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds the target RNA at

- 25 046751 means of complementary base pairing, and after binding to the correct site it acts enzymatically, cutting the target RNA. Strategic cleavage of such target RNA disrupts its ability to direct the synthesis of the encoded protein. After an enzymatic nucleic acid has bound and cleaved its target RNA, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymatic nucleic acid molecule can be obtained, for example, in the form of a hammer head, hairpin, hepatitis 8 virus, group I intron or RNase P (in combination with an RNA guide sequence) or a Neurospora VS RNA motif. Specific examples of hammerhead motifs are described, for example, in Rossi et al., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifs are described, for example, in EP 0360257, Hampel et al., Biochemistry, 28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990) and US Patent No. 5,631,359. An example of the hepatitis virus 5 motif is described, for example, in Perrotta et al., Biochemistry, 31:11843-52 (1992). An example of an RNase P motif is described, for example, in Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of the Neurospora VS RNA ribozyme motif are described, for example, in Saville et al., Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA, 88:882630 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). An example of a group I intron is described, for example, in US Pat. No. 4,987,071. Important characteristics of the enzymatic nucleic acid molecules used in accordance with the invention are that they have a specific substrate binding site that is complementary to one or more regions of the DNA or RNA of the target gene, and that they have nucleotide sequences in or around this substrate binding site that give the molecule RNA cleavage activity. Thus, ribozyme designs should not be limited to the specific motifs mentioned herein. The information provided through these links is incorporated herein by reference in its entirety for all purposes.

Methods for producing a ribozyme targeting any polynucleotide sequence are known in the art. Ribozymes can be designed as described, for example, in PCT Publications No. WO 93/23569 and WO 94/02595, and synthesized for in vitro and/or in vivo testing as described therein. The information contained in these PCT publications is incorporated herein by reference in its entirety for all purposes.

Ribozyme activity can be optimized by varying the length of the ribozyme binding arms or by chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see, for example, PCT Publications No. WO 92/07065, WO 93/15187, WO 91/03162 and WO 94/13688; EP 92110298.4 and US Patent No. 5334711, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules, the descriptions of which are incorporated herein by reference for all purposes), modifications that increase their effectiveness. in cells, and removal of stem II bases to reduce RNA synthesis time and chemical requirements.

Immunostimulatory oligonucleotides.

The nucleic acids associated with the lipid particles of the present invention may be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single- or double-stranded), capable of eliciting an immune response when administered to a subject, which may be a mammal such as a human. ISS include, for example, certain palindromes leading to secondary structure hairpins (see Yamamoto et al., J. Immunol., 148:4072-6 (1992)) or CpG motifs, as well as other known ISS features (such as multi- G domains; see PCT Publication No. WO 96/11266, the description of which is incorporated herein by reference for all purposes).

Immunostimulatory nucleic acids are considered to be non-sequence specific when they are not required to specifically bind to the target sequence and reduce its expression in order to elicit an immune response. Thus, some immunostimulatory nucleic acids may contain a sequence corresponding to a region of a naturally occurring gene or mRNA, but they may still be considered immunostimulatory nucleic acids with non-specific sequences.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotide contains at least one CpG dinucleotide. The CpG oligonucleotide or dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid includes at least one CpG dinucleotide containing methylated cytosine. In one embodiment, the nucleic acid includes a single CpG dinucleotide, wherein the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid contains at least two CpG dinucleotides, wherein at least one cytosine in the CpG dinucleotides is methylated. In a further embodiment, each cytosine in the CpG dinucleotides present in the sequence is methylated. In another embodiment, the nucleic acid includes a plurality of CpG dinucleotides, wherein at least one of the CpG dinucleotides contains a methylated cytosine. Examples of immunostimulatory oligonucleotides suitable for use in the compositions and methods of this invention are described in

- 26 046751 PCT application No. PCT/US08/88676, filed December 31, 2008, PCT publications No. WO 02/069369 and WO 01/15726, US patent No. 6406705 and Raney et al., J. Pham. Exper. Ther., 298:1185-92 (2001), the descriptions of which are incorporated herein by reference in their entirety for all purposes.

In certain embodiments, the oligonucleotides used in the compositions and methods of this invention have a phosphodiester (PO) backbone or phosphorothioate (PS) backbone and/or at least one methylated cytosine residue in the CpG motif.

mRNA.

In certain embodiments, the invention provides compositions and methods that can be used to express one or more mRNA molecules in a living cell (eg, cells in a human body). mRNA molecules encode one or more polypeptides that are expressed in living cells. In some embodiments, the polypeptides are expressed in a diseased organism (eg, a mammal such as a human), and expression of the polypeptide alleviates one or more symptoms of the disease. The compositions and methods of the invention are particularly useful for the treatment of human diseases caused by the absence or reduced levels of a functional polypeptide in the human body. Accordingly, in some embodiments, the LNP may comprise one or more nucleic acid molecules, such as one or more mRNA molecules (eg, a mixture of mRNA molecules).

In some embodiments, the mRNAs are completely encapsulated in a lipid nucleic acid particle (eg, LNP). For compositions containing a mixture of mRNA, different types of mRNA species present in the mixture (e.g., mRNA having different sequences) may be encapsulated together in the same particle, or each type of mRNA species present in the mixture may be encapsulated in a single particle. The mRNA mixture can be formulated into particles as described herein, using a mixture of two or more individual mRNAs (each having a unique sequence) in identical, similar or different concentrations or molar ratios. In one embodiment, a mixture of mRNA (corresponding to multiple mRNAs with different sequences) is formulated using identical, similar or different concentrations or molar ratios of each type of mRNA, and the different types of mRNA are co-encapsulated in the same particle. In another embodiment, each type of mRNA species present in the mixture is encapsulated in different particles at identical, similar, or different concentrations or molar ratios of mRNA, and the particles thus formed (each containing a different mRNA payload) are administered separately (e.g. , at different times according to the treatment regimen) or they are combined and administered together in a single unit dose (eg, with a pharmaceutically acceptable carrier). The particles described herein are stable in serum, resistant to nuclease degradation, and substantially nontoxic to mammals such as humans.

Modifications of mRNA.

The mRNA used in practice in the present invention may include one, two, or more than two nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in the cell into which the mRNA is introduced compared to the corresponding unmodified mRNA.

In some embodiments, the modified nucleosides include pyridine-4-onribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1 -carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyluridine, 1-methyl-1pseudouridine, 4-thio-1 -methyl-1-pseudouridine, 2-thio-1-methyl-1-pseudouridine, 1-methyl-1-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine, dihydropseuduridine, 2-thiodihydrouridine, 2 -thiodihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine and 4-methoxy-2-thiopseudouridine.

In some embodiments, the modified nucleosides include 5-azacytidine, pseudoisocytidine, 3-methylcytidine, M'-acetylcytidine, 5-formylcytidine, M'-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolocytidine, pyrrolopseudoisocytidine, 2-thiocytidine, 2-thio -5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methylpseudoisocytidine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebularine, 5-azazebularine, 5-methylzebularine, 5 -aza-2-thiosebularine, 2-thiosebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine and 4-methoxy-1-methylpseudoisocytidine.

In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7 -deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, M ⁶ -methyladenosine, M ⁶ -isopentenyladenosine, M ⁶ -(cis-hydroxyisopentenyl)adenosine, 2-methylthio -M ⁶ -(cishydroxyisopentenyl)adenosine, M ⁶ -glycinylcarbamoyl adenosine, M ⁶ -threonylcarbamoyl adenosine,

2-methylthio-M ⁶ -threonylcarbamoyladenosine, M ⁶ ,M ⁶ -dimethyladenosine, 7-methyladenine, 2-methylthioadenine and

- 27 046751

2-methoxyadenine.

In specific embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)adenosine, 5'-O-(1-thiophosphate)cytidine, 5'-O-(1-thiophosphate)guanosine, 5'-O-( 1-thiophosphate)uridine or 5'-O-(1-thiophosphate)pseudouridine. The α-thio-substituted phosphate moiety is designed to impart stability to RNA polymers through unnatural backbone phosphorothioate bonds. Phosphorothioate RNA has increased resistance to nucleases and, as a result, a longer half-life in the cellular environment. Phosphorothioate-linked nucleic acids are also expected to reduce the innate immune response due to weaker binding/activation of cell innate immune molecules.

In certain embodiments, it is desirable to intracellularly degrade the modified nucleic acid introduced into the cell, for example, if precise timing of protein production is required. Thus, the invention provides a modified nucleic acid containing a degradation domain that can be targeted within a cell.

In other embodiments, modified nucleosides include inosine, 1-methylinosine, viosine, vibutosine, 7-deazaguanosine, 7-deaza-8-azaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8 -azaguanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, U-methylguanosine, U.U-dimethylguanosine. 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, X ² -methyl-6-thioguanosine and H3U-dimethyl-b-thioguanosine.

Optional components of modified nucleic acids.

In additional embodiments, the modified nucleic acids may contain other optional components that may be useful in some embodiments. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intronic nucleotide sequences, internal ribosome entry site (IRES), cap and polyA tails. For example, a 5' untranslated region (UTR) and/or a 3' untranslated region may be provided, where one or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translated region. Nucleic acids containing the Kozak sequence are also provided.

In addition, nucleic acids are provided that contain one or more intronic nucleotide sequences that can be excised from the nucleic acid.

Untranslated regions (UTRs).

Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5'UTR begins at the transcription start site and continues to the start codon, but does not include the start codon; whereas the 3'UTR begins immediately after the stop codon and continues until the transcription termination signal. There is growing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. Regulatory properties of the UTR can be included in the mRNA used in this invention to increase the stability of the molecule. Specific features can also be included to provide controlled suppression of the transcript if it is misdirected to undesired organ sites.

5' capping.

The 5'-cap structure of mRNA is involved in nuclear export, increasing the stability of the mRNA, and binds the cap-binding protein (CBP) of the mRNA, which is responsible for the stability of the mRNA in the cell and the ability to translate due to the association of CBP with the poly(A) binding protein with formation of mature cyclic species of mRNA. The cap also promotes the removal of 5'-proximal introns during mRNA splicing.

Endogenous mRNA molecules may have a cap at the 5' end, forming a 5'-p-5'-triphosphate bond between the terminal guanosine residue of the cap and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to form a Y-methylguanylate residue. The ribose sugars of the terminal and/or pre-terminal 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 can target a nucleic acid molecule, such as an mRNA molecule, for degradation.

IRES sequences.

mRNA containing an internal ribosome entry site (IRES) can also be used in the practice of the present invention. The IRES may act as a single ribosome binding site or may serve as one of multiple ribosome binding sites on mRNA. An mRNA containing more than one functional ribosome binding site can encode more peptides or polypeptides that are independently translated by ribosomes (multicistronic mRNA). If the mRNA is provided with an IRES, a second translation region is optionally provided. Examples of IRES sequences that can be used in accordance with the invention include, but are not limited to, sequences from picornaviruses (eg, foot and mouth disease), pest viruses

- 28 046751 (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 immunodeficiency viruses (S1V) or cricket paralysis virus (CrPV).

Poly(A) tails.

During RNA processing, a long chain of adenine nucleotides (poly(A) tail) can be added to polynucleotides such as mRNA molecules to increase stability. Immediately after transcription, the 3' end of the transcript can be cleaved to release the 3' hydroxyl. Poly(A) polymerase then adds a chain of adenine nucleotides to the RNA. A process called polyadenylation adds a poly-A tail, which can range from 100 to 250 residues in length.

Typically, the length of the poly(A) tail exceeds 30 nucleotides. In another embodiment, 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, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 00, 2500 and 3000 nucleotides ).

In this context, the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer in length than the modified mRNA. The poly(A) tail can also be designed as part of a modified nucleic acid to which it belongs. In this context, the poly(A) tail may comprise 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the modified mRNA or the total length of the modified mRNA minus the polyA tail.

Obtaining mRNA molecules.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, constructing and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al. , Molecular Cloning, A Laboratory Manual ( ^2nd ed. 1989)); as well as PCR methods (see US Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression sequence libraries are also well known to those skilled in the art. Additional basic texts covering general applications of the present invention include Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990) and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The information provided through these links is incorporated herein by reference in its entirety for all purposes.

Encoded polypeptides.

The mRNA component of the nucleic acid-lipid particle described herein can be used to express a polypeptide of interest. Some diseases in humans are caused by the absence or disruption of a functional protein in a particular type of cell where the protein is normally present and active. A functional protein may be completely or partially absent due to, for example, transcriptional inactivity of the coding gene or due to the presence of a mutation in the coding gene that renders the protein completely or partially nonfunctional. Examples of human diseases that are caused by complete or partial inactivation of the protein include X-linked severe combined immunodeficiency (X-SCID) and adrenoleukodystrophy (X-ALD). X-SCID is caused by one or more mutations in the gene encoding the common gamma chain protein, which is a component of receptors for several interleukins that are involved in the development and maturation of B and T cells in the immune system. X-ALD is caused by one or more mutations in the gene for a peroxisomal membrane transport protein called ABCD1. People with X-ALD have very high levels of long-chain fatty acids in tissues throughout the body, causing a variety of symptoms that can lead to mental impairment or death.

Attempts have been made to use gene therapy to treat certain diseases caused by the absence or disruption of a functional protein in a certain type of cell where the protein is normally present and active. Gene therapy typically involves introducing a vector that includes a gene encoding a functional form of the diseased protein into a diseased individual and expressing the functional protein to treat the disease. So far, gene therapy has had limited success. In addition, some aspects of mRNA delivery using LNPs have been described, for example, in international publications WO 2018/006052 and WO 2015/011633.

Thus, there is a continuing need to improve the expression of a functional form of a protein in a human body that suffers from a disease caused by complete or partial absence of a functional protein, and there is a need for improved delivery of nucleic acids (e.g., mRNA) using methods and compositions, for example, that may cause a lesser immune response to therapy. Certain embodiments of the present invention are applicable in this context. Thus, in certain embodiments, expression of the polypeptide alleviates one or more symptoms of a disease or disorder. Certain compositions and methods of the invention may be useful for the treatment of human diseases caused by the absence or reduced levels of a functional polypeptide in the human body. In other embodiments, certain compositions and methods of the invention may be useful for expressing a vaccine antigen for the treatment of cancer.

Self-amplifying RNA.

- 29 046751

In certain embodiments, the nucleic acid is one or more self-amplifying RNA molecules. Self-amplifying RNA (saRNA) may also be called self-replicating RNA, replication competent RNA, replicons, or RepRNA. RepRNA, called self-amplifying mRNA when derived from positive-strand viruses, is generated from a viral genome lacking at least one structural gene; it can be translated and replicated (hence self-amplified) without producing infectious progeny virus. In certain embodiments, RepRNA technology can be used to insert a gene cassette encoding a desired antigen of interest. For example, the alphavirus genome is divided into two open reading frames (ORFs): the first ORF encodes proteins for RNA-dependent RNA polymerase (replicase), and the second ORF encodes structural proteins. In a saRNA vaccine design, the ORF encoding the structural proteins of the virus can be replaced by any antigen of choice, while the viral replicase remains an integral part of the vaccine and drives intracellular RNA amplification after immunization.

PEG-CDMA.

One of ordinary skill in the art will appreciate that the concentration of PEG-C-DMA may vary depending on the rate at which the nucleic acid-lipid particle is to become fusogenic. For example, the rate at which the nucleic acid-lipid particle becomes fusogenic can be changed, for example, by changing the molecular weight of the PEG. In a specific embodiment, PEG-C-DMA has the following structure:

where n is chosen such that the resulting polymer chain has a molecular weight of from about 1000 to about 3000.

In another embodiment, n is selected such that the resulting polymer chain has a molecular weight of about 2000. PEG-C-DMA can be prepared as described by Heyes et al., Synthesis and Characterization of Novel Poly (Ethylene Glycol)-lipid Conjugates Suitable for use in Drug Delivery, Journal of Controlled Release, 2006 and US Patent No. 8936942.

Preparation of lipid particles.

In certain embodiments of the present invention, there is provided an LNP produced by a continuous mixing method, such as a process that includes providing an aqueous solution containing a nucleic acid in a first reservoir, providing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution thereby , so that an organic lipid solution is mixed with an aqueous solution to almost instantly produce liposomes encapsulating a nucleic acid (such as interfering RNA or mRNA). This process and apparatus for carrying it out are described in detail in US Patent Publication No. 20040142025, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Continuous introduction of lipid and buffer solutions into a mixing medium, such as a mixing chamber, causes continuous dilution of the lipid solution with the buffer solution, thereby almost instantly forming liposomes after mixing. As used herein, the expression continuous dilution of a lipid solution with a buffer solution (and variants) generally means that the lipid solution is diluted quickly enough by the process of hydration with sufficient force to cause the formation of vesicles. By mixing an aqueous solution containing a nucleic acid with a solution of an organic lipid, the organic lipid solution undergoes a continuous stepwise dilution in the presence of a buffer solution (ie, an aqueous solution) to obtain a nucleic acid-lipid particle.

LNPs formed using the continuous mixing method typically have an average size of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 110 nm. 90 nm. The particles thus formed do not aggregate and are not necessarily sized to achieve uniform particle size.

Another embodiment of the present invention provides LNPs produced by a direct dilution process that involves forming a liposome solution and immediately and directly introducing the liposome solution into a collection vessel containing a controlled amount of dilution buffer. In preferred aspects, the collection vessel includes one or more elements configured to agitate the contents of the collection vessel to facilitate dilution. In one aspect, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of the liposome solution introduced therein. As a non-limiting example, a solution of liposomes in 45% ethanol, when introduced into a collection vessel containing an equal volume of dilution buffer, will preferentially produce smaller particles.

In yet another embodiment of the present invention, LNPs obtained through

- 30 046751 by a direct dilution process in which a third reservoir containing a dilution buffer is fluidly connected to a second mixing zone. In this embodiment, the liposome solution formed in the first mixing zone is immediately and directly mixed with the dilution buffer in the second mixing zone. In preferred aspects, the second mixing zone comprises a T-connector positioned such that the flows of the liposome solution and the dilution buffer solution meet as opposing flows at an angle of 180°; however, connectors that provide smaller angles, such as about 27 to about 180 degrees, can be used. A pumping mechanism supplies a controlled flow of buffer to the second mixing zone. In one aspect, the flow rate of the dilution buffer supplied to the second mixing zone is adjusted to be substantially equal to the flow rate of the liposome solution introduced therefrom from the first mixing zone. This embodiment advantageously allows for better control of the mixing flow of the dilution buffer with the liposome solution in the second mixing zone and therefore also the concentration of the liposome solution in the buffer throughout the second mixing process. This control of the dilution buffer flow rate allows for the preferential formation of small particle sizes at reduced concentrations.

This process and apparatus for carrying it out are described in detail in US Patent Publication No. 20070042031, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

LNPs formed using the direct dilution method typically have an average size of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 110 nm. 90 nm. The particles thus formed do not aggregate and are not necessarily sized to achieve uniform particle size.

If desired, the lipid particles of the invention (eg, LNP) can be measured by any of the methods available for determining the size of liposomes. Size distribution can be carried out to achieve a desired size range and a relatively narrow particle size distribution.

More methods are available to obtain the desired particle size. One method for obtaining the desired sizes, used for liposomes and equally applicable to the present particles, is described in US patent No. 4,737,323, the disclosure of which is incorporated herein by reference for all purposes. Sonication of a particle suspension using a bath or probe results in a gradual reduction in particle size to less than about 50 nm. Homogenization is another method that uses shear energy to break larger particles into smaller ones. In a standard homogenization procedure, particles are recirculated through a standard emulsion homogenizer until the selected particle sizes, typically about 60 to about 80 nm, are met. In both methods, the particle size distribution can be monitored using conventional laser beam particle sizing or QELS.

Extrusion of particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method of reducing particle sizes to a relatively well-defined size distribution. Typically the suspension is passed through the membrane one or more times until the desired particle size distribution is achieved. Particles can be extruded through membranes with progressively smaller pores to achieve gradual size reduction.

In some embodiments, the nucleic acids in the LNP are precondensed, as described, for example, in US patent application No. 09/744103, the disclosure of which is incorporated herein by reference for all purposes.

In other embodiments, the methods further include adding non-lipid polycations that are useful for performing lipofection of cells using the present compositions. Examples of suitable non-lipid polycations include hexadimethrine bromide (sold under the trade name POLYBRENE® from Aldrich Chemical Co., Milwaukee, WI, USA) or other hexadimethrine salts. Other suitable polycations include, for example, the salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-E-lysine, polyallylamine and polyethylenimine. The addition of these salts is preferably after particle formation.

Introduction of lipid particles.

Once formed, the lipid particles of the invention (eg, LNPs) can be used to introduce nucleic acids into cells. Accordingly, the present invention also provides methods for introducing a nucleic acid, such as a nucleic acid (eg, interfering RNA or mRNA), into a cell. The methods are carried out in vitro or in vivo by first forming particles as described above and then contacting the particles with cells for a period of time sufficient to deliver the nucleic acid to the cells.

The lipid particles of the invention (eg LNP) can be adsorbed to virtually any type of cell with which they are mixed or in contact. After adsorption, particles may be subjected to

- 31 046751 endocytosis by part of cells, exchange lipids with cell membranes or fuse with cells. Transfer or incorporation of a portion of a nucleic acid (eg, nucleic acid) can occur through any of these pathways. Specifically, when fusion occurs, the membrane of the particle integrates into the cell membrane, and the contents of the particle combine with the intracellular fluid.

The lipid particles of the invention (eg, LNP) can be administered either alone or in admixture with a pharmaceutically acceptable carrier (eg, saline or phosphate buffer) selected in accordance with the route of administration and standard pharmaceutical practice. Typically, normal buffered saline (eg, 135-150 mM NaCl) is used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for increased stability such as albumin, lipoprotein, globulin, etc. Additional suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa., 17 ^th ed. (1985). As used herein, the term carrier includes any and all solvents, dispersion media, carriers, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retarding agents, buffers, carrier solutions, suspensions, colloids, and the like. The phrase pharmaceutically acceptable refers to molecular substances and compositions that, when administered to the human body, do not cause an allergic or similar adverse reaction.

A pharmaceutically acceptable carrier is typically added after particle formation. Thus, once the particle is formed, it can be diluted with pharmaceutically acceptable carriers such as normal buffered saline.

The concentration of particles in pharmaceutical preparations can vary widely, i.e. from less than about 0.05%, typically from about or at least about 2 to 5%, to about 10 to 90% by weight, and will be selected primarily based on fluid volume, viscosity, etc. according to the specific route of administration chosen. For example, the concentration may be increased to reduce the fluid load associated with treatment. This may be especially desirable in patients suffering from congestive heart failure associated with atherosclerosis or severe hypertension. Alternatively, particles composed of irritating lipids can be diluted to low concentrations to reduce inflammation at the injection site.

The pharmaceutical compositions of this invention can be sterilized by conventional, well known sterilization methods. Aqueous solutions may be packaged for use or aseptically filtered and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution before administration. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH adjusting agents and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride. In addition, the particle suspension may contain lipid-protecting agents that protect lipids from free radicals and lipid peroxidation during storage. Lipophilic free radical scavengers such as alpha-tocopherol and water-soluble iron-specific chelating agents such as ferrioxamine are useful.

In vivo administration.

Systemic delivery for in vivo therapy, such as delivery of a therapeutic nucleic acid to a distal target cell through body systems such as the circulatory system, has been achieved using nucleic acid and lipid based particles such as those described in PCT Publication No. WO 05/ 007196, WO 05/121348, WO 05/120152 and WO 04/002453, the disclosures of which are incorporated herein by reference in their entirety for all purposes. The present invention also provides fully encapsulated lipid particles that protect the nucleic acid from degradation by nuclease in serum, are non-immunogenic, are small in size and are suitable for repeated dosing.

For in vivo administration, administration may be by any route known in the art, for example, injection, oral administration, inhalation (eg, intranasal or intratracheal), transdermal application, or rectal administration. Administration may be in a single or divided dose. The pharmaceutical compositions can be administered parenterally, i.e. intra-articular, intravenous, intraperitoneal, subcutaneous or intramuscular. In some embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally via bolus injection (see, for example, US Pat. No. 5,286,634). Intracellular nucleic acid delivery has also been discussed in Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993). Still other methods of administering lipid-based therapeutics are described, for example, in US Pat. Nos. 3,993,754, 4,145,410, 4,235,871, 4,224,179, 4,522,803, and 4,588,578. Lipid particles can be administered by direct injection into the site of disease or by injection into a site remote from the site of disease ( see, for example, Culver, HUMAN GENE THERAPY, Mary Ann Lie

- 32 046751 bert, Inc., Publishers, New York, p. 70-71 (1994)). The disclosures of the above references are incorporated herein by reference in their entirety for all purposes.

The compositions of this invention, alone or in combination with other suitable components, can be formulated into aerosol formulations (i.e., they can be nebulized) for administration by inhalation (eg, intranasally or intratracheally) (see, Brigham et al., Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed in suitable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

In certain embodiments, pharmaceutical compositions may be delivered via intranasal sprays, inhalations, and/or other aerosol delivery means. Methods for delivering nucleic acid compositions directly to the lungs via nasal aerosols are described, for example, in US Pat. Nos. 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal microparticulate resins and lysophosphatidylglycerol compounds (US Pat. No. 5,725,871) is also well known. in pharmaceuticals. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene matrix is described in US Patent No. 5,780,045. The disclosures of the above patents are incorporated herein by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, for example, intra-articular (into the joints), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatics and solutes. substances that make the composition isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. In the practice of the present invention, the compositions are preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.

Typically, when administered intravenously, the lipid particles are formulated with a suitable pharmaceutical carrier. Many pharmaceutically acceptable carriers can be used in the compositions and methods of this invention. Suitable compositions for use in the present invention can be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, ^17th ed. (1985). Various aqueous vehicles may be used, such as water, buffered water, 0.4% saline, 0.3% glycine, etc., and may contain glycoproteins to improve stability, such as albumin, lipoprotein, globulin, etc. . Typically, normal buffered saline (135-150 mM NaCl) is used as a pharmaceutically acceptable vehicle, but other suitable vehicles will suffice. These compositions can be sterilized by conventional liposomal sterilization methods such as filtration. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH adjusters and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, chloride calcium, sorbitan monolaurate, triethanolamine oleate, etc. These compositions can be sterilized using the methods mentioned above, or alternatively they can be prepared under sterile conditions. The resulting aqueous solutions may be packaged for use or aseptically filtered and lyophilized, the lyophilized formulation being combined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles described herein can be delivered to an individual by oral administration. The particles may be combined with excipients and used in the form of oral tablets, buccal tablets, lozenges, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, wafers, and the like. (See, for example, US Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosures of which are incorporated herein by reference in their entirety for all purposes). These oral dosage forms may also contain the following: binders, gelatin; excipients, lubricants and/or flavorings. When the unit dosage form is a capsule, it may contain, in addition to the materials described above, a liquid carrier. A variety of other materials may be present in the form of coatings or may otherwise modify the physical form of the dosage unit. Of course, any material used in the preparation of any unit dosage form must be pharmaceutically pure and substantially non-toxic in the quantities used.

Typically, these oral formulations may contain at least about 0.1% lipid particles or more, although the percentage of particles can, of course, vary and typically range from about 1 or 2% to about 60 or 70% or more by weight or the volume of the entire composition. Naturally, the number of particles in each therapeutically useful composition can be obtained such that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological features will be taken into account by specialists

- 33 046751 in the art when preparing such pharmaceutical compositions, and therefore different dosages and treatment regimens may be desirable.

Dosage forms suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of a packaged therapeutic agent, such as a nucleic acid (eg, interfering RNA or mRNA), suspended in diluents such as water, saline, or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of a therapeutic agent, such as a nucleic acid (eg, interfering RNA or mRNA), in the form of liquids, solids, granules or gelatin; (c) suspensions in a suitable liquid; and (d) suitable emulsions. Tablet forms may contain one or more of the following components: lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, dyes , fillers, binders, diluents, buffering agents, wetting agents, preservatives, flavorings, coloring agents, disintegrants and pharmaceutically compatible carriers. Lozenges may contain a therapeutic agent such as a nucleic acid (e.g., interfering RNA or mRNA) in a flavoring agent such as sucrose, as well as lozenges containing the therapeutic agent in an inert base such as gelatin and glycerin or sucrose and gum emulsions, gels, etc. etc., containing, in addition to the therapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be included in a wide range of topical dosage forms. For example, a suspension containing nucleic acid and lipid based particles, such as LNPs, can be formulated and administered as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical formulations of the lipid particles of the invention, it is preferable to use a number of particles that have been purified to reduce or remove empty particles or particles with therapeutic agents, such as nucleic acid, associated with the outer surface.

The methods of this invention can be used in a variety of hosts. Preferred hosts include mammalian species such as primates (eg, humans and chimpanzees, as well as other non-human primates), dogs, cats, horses, cows, sheep, goats, rodents (eg, rats and mice), lagomorphs, and pigs.

The number of particles administered will depend on the ratio of therapeutic agent (eg, nucleic acid) to lipid, the specific therapeutic agent (eg, nucleic acid) used, the disease or disorder being treated, the age, weight and condition of the patient, the patient and the physician's judgment, but generally is from about 0.01 to about 50 mg per kg of body weight, preferably from about 0.1 to about 5 mg/kg of body weight or about 10-10 particles per administration (eg, injection).

In vitro administration.

For in vitro applications, delivery of therapeutics such as nucleic acids (eg, interfering RNA or mRNA) can be achieved into any cell grown in culture, whether it is from plants or animals, vertebrates or invertebrates, or any fabric or type. In preferred embodiments, the cells are animal cells, more preferably mammalian cells, and most preferably human cells.

Contact between cells and lipid particles, when carried out in vitro, occurs in a biologically compatible environment. The particle concentration varies widely depending on the specific application, but is typically from about 1 µmol to about 10 mmol. Treatment of cells with lipid particles is typically carried out at physiological temperatures (about 37°C) for periods of about 1 to 48 hours, preferably from about 2 to 4 hours.

In one set of preferred embodiments, a suspension of lipid particles is added to 60-80% confluent cells on the plate having a cell density of about 10 ³ to about 10 ⁵ cells/ml, more preferably about 2x10 ⁴ cells/ml. The concentration of the suspension added to the cells is preferably about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

Using endosome release parameter (ERP) analysis, the delivery efficiency of the LNP or other lipid particle of the present invention can be optimized. ERP analysis is described in detail in US Patent Publication No. 20030077829, the disclosure of which is incorporated herein by reference in its entirety for all purposes. More specifically, the goal of the ERP assay is to distinguish the effects of different cationic lipids and accessory lipid components of LNP based on their relative effects on binding/uptake or fusion with/destabilization of the endosomal membrane. This assay quantifies how each LNP component or other

- 34 046751 lipid particle affects delivery efficiency, thereby optimizing the LNP or other lipid particle. Typically, an ERP assay measures the expression of a reporter protein (e.g., luciferase, β-galactosidase, green fluorescent protein (GFP), etc.), and in some cases an LNP formulation optimized for an expression plasmid will also be suitable for encapsidating interfering RNA or mRNA. In other cases, the ERP assay can be adapted to measure transcriptional or translational inhibition of a target sequence in the presence or absence of interfering RNA (eg, siRNA). In other cases, the ERP assay can be adapted to measure the expression of a target protein in the presence or absence of mRNA. By comparing the ERPs for each of the different LNPs or other lipid particles, one can easily identify an optimized system, such as the LNP or other lipid particle, that has the highest cellular uptake.

Cells for delivery of lipid particles.

The compositions and methods of this invention are used to treat a wide variety of cell types in vivo and in vitro. Suitable cells include, for example, hematopoietic progenitor cells (stem cells), fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, slow or non-cycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, etc. In one embodiment, a nucleic acid, such as one or more nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) or mRNA), is delivered to cancer cells, such as, for example, lung cancer cells, colon cancer cells, cancer cells rectum, anal cancer cells, bile duct cancer cells, small intestine cancer cells, stomach cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, cancerous ovarian cells, cervical cancer cells, prostate cancer cells, kidney cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells and cancer cells blood.

In vivo delivery of lipid particles, such as LNPs, encapsulating one or more nucleic acid molecules (eg, interfering RNA (eg, siRNA) or mRNA) is suitable for targeting any cell type. The methods and compositions can be used with cells from a wide variety of vertebrates, including mammals, such as, for example, canines, felines, horses, cattle, sheep, goats, rodents (for example, mice, rats and guinea pigs), lagomorphs, pigs and primates (eg monkeys, chimpanzees and humans).

The extent to which tissue culture of cells may be required is well known in the art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, ^3rd Ed., Wiley-Liss, New York (1994), Kuchler et al., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. . (1977), and the references cited therein constitute a general guide to cell culture. Cultured cell systems are often monolayers of cells, although cell suspensions are also used.

Detection of lipid particles.

In some embodiments, the lipid particles of this invention (eg, LNP) are detectable in a subject after about 1, 2, 3, 4, 5, 6, 7, 8 hours or more. In other embodiments, the lipid particles of the invention (e.g., LNP) are detectable in the subject at about 8, 12, 24, 48, 60, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16, 18. 19, 22, 24, 25 or 28 days after particle administration. The presence of particles may be detected in the cells, tissues, or other biological samples of the subject. The particles can be detected, for example, by direct particle detection, detection of a therapeutic nucleic acid such as an interfering RNA sequence (eg, siRNA) or mRNA sequence, detection of a target sequence of interest (eg, by detecting a change in expression of the sequence of interest), or their combination.

Particle detection.

The lipid particles of the invention, such as LNP, can be detected using any method known in the art. For example, the label can be directly or indirectly associated with a component of the lipid particle using methods well known in the art. A variety of labels can be used, with the choice of label depending on the sensitivity required, ease of conjugation to the lipid particle component, stability requirements, and available tools and disposal provisions. Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green™; rhodamine and derivatives such as Texas red, tetrarhodimine isothiocyanate (TRITC), etc. ., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, etc.; radioactive labels such as ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P, ³³ P, etc.; ne

- 35 046751 horseradish roxidase, alkaline phosphatase, etc.; spectral colorimetric tags such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc. The mark can be detected using any means known in the art.

Detection of nucleic acids.

Nucleic acids (eg, interfering RNA or mRNA) are detected and quantified herein by any of a number of methods well known to those skilled in the art. Detection of nucleic acids can be carried out by well-known methods such as Southern blotting, Northern blotting, gel electrophoresis, PCR, radiolabeling, scintillation counting and affinity chromatography. Additional analytical biochemical techniques such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and hyperdiffusion chromatography may also be used.

The choice of the nature of nucleic acid hybridization is not critical. Various hybridization patterns of nucleic acids are known to those skilled in the art. For example, common characters include sandwich analyzes and competition or displacement analyses. Hybridization methods are generally described, for example, in Nucleic Acid Hybridization, A Practical Approach, Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of hybridization assays can be increased by using a nucleic acid amplification system that multiplies the target nucleic acid to be detected. In vitro amplification methods are known that are suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning. Examples of methods sufficient to guide those skilled in the art in such in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), QP replicase amplification, and other RNA polymerase-mediated methods (eg, NASBA™) can be found in Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002); as well as US patent No. 4683202; PCR Protocols, A Guide to Methods and Applications (Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990); Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research 3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell et al., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077 (1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene, 4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan and Malek, Biotechnology, 13:563 (1995). Improved methods for cloning in vitro amplified nucleic acids are described in US Pat. No. 5,426,039. Other methods described in the art are nucleic acid sequence-based amplification systems (NASBA™, Cangene, Mississauga, Ontario) and QP replicase systems. These systems can be used to directly identify mutants in which PCR or LCR primers are designed to extend or ligate only when a selected sequence is present. Alternatively, the selected sequences can be routinely amplified using, for example, non-specific PCR primers and the amplified target region later probed for a specific mutation-indicative sequence. The disclosures of the above references are incorporated herein by reference in their entirety for all purposes.

Nucleic acids for use as probes, for example in in vitro amplification methods, for use as gene probes or as inhibitor components, are typically synthesized chemically according to the solid phase triester phosphoramidite method described in Beaucage et al., Tetrahedron Letts., 22: 1859 1862 (1981), e.g., using an automated synthesizer, as described in Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984). If necessary, purification of polynucleotides is usually carried out either by native acrylamide gel electrophoresis or by anion exchange HPLC, as described in Pearson et al., J. Chrom., 255:137 149 (1983). The sequence of synthetic polynucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980), described in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.

An alternative way to determine the level of transcription is in situ hybridization. In situ hybridization assays are well known and generally described in Angerer et al., Methods Enzymol., 152:649 (1987). In an in situ hybridization assay, cells are fixed to a solid support, usually a glass slide. If DNA needs to be probed, the cells are denatured by heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to allow the specific labeled probes to anneal. The probes are preferably labeled with radioisotopes or fluorescent labels.

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Examples

The present invention will be described in more detail with the help of specific examples. The following examples are offered for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize a number of non-critical parameters that can be changed or modified to produce substantially identical results.

Example 1.

An exemplary lipid composition according to the invention is shown in table. 1, in which CL1 is

or its salt.

Table 1

Exemplary Lipid Compositions

Example Composition Molar ratios 1 PEG-S-OMA:(X1 :CHOL:DSPC 1.5: 50.0: 38.5: 10.0

The composition was prepared using the following procedure.

Stock lipids (total lipid content about 7 mg/mL) were prepared in 100% ethanol using the described lipid characteristics and molar ratios. The mRNA was diluted with pH 5 acetate and nuclease-free water to achieve an mRNA concentration of 0.366 mg/mL in 100 mM pH 5 acetate. Equal volumes of each solution were mixed at 400 mL/min in a T-connector and diluted with approximately 4 volumes of PBS, pH 7.4 using the direct dilution method described in US Patent No. 9404127. The formulations were then placed in Slide-A-Lyzer dialysis units (MWCO 10000) and dialyzed overnight with 10 mM Tris, 500 mM NaCl pH 8 buffer Tris/NaCl). After dialysis, the formulations were evaporated to approximately 0.6 mg/mL using VivaSpin concentrators (MWCO 100,000) and then filtered through a 0.2 μm syringe filter.

Example 2: In Vivo Assay.

Typically, LNP was administered intravenously at a dose of 0.5 mg/kg to female Balb/C mice aged 5–8 weeks, and blood was collected 4–6 h after dosing; blood was collected in K2EDTA and plasma isolated, then stored frozen at -80°C until analysis. Activity was assessed by testing plasma for human EPO expression using a human EPO ELISA kit from either StemCell (cat. no. 01630) or R&D Systems (cat. no. DEP00) according to the manufacturer's instructions. The data is presented in table. 2.

As can be seen from the data in table. 2, the composition of the present invention is significantly more effective than the MC3 composition used in patisiran (Onpattro), an approved LNP product for the treatment of TTR amyloidosis.

table 2

Efficacy of lipid composition 1 0.5 mg/kg and patisiran-based composition containing human EPO mRNA 4 hours after intravenous administration to Balb/C mice (n=4)

Lipid composition EPO (mU/ml) Std. off (mU/ml) Lipid composition 1 with CL1 (1.5: 50.0: 38.5: 10.0) 136439 17373 Composition based on patisiran with MSZ (1.5: 50.0: 38.5: 10.0) 42230 2697

It should be understood that the above description is intended to be illustrative and not limiting. Many embodiments will become apparent to those skilled in the art after reading the above description. Therefore, the scope of the invention should not be determined by reference to the foregoing description, but instead should be determined by reference to the accompanying claims, together with the full scope of equivalents to which such claims are entitled. Disclosures of all articles and references, including patent applications, patents, PCT publications, and Genbank accession numbers, are incorporated herein by reference for all purposes.

Claims (14)

1. Lipid nanoparticle for delivering nucleic acid to a patient or into a cell, containing: (a) one or more nucleic acid molecules; (b) cholesterol; (c) DSPC; (d) PEG-C-DMA; (e) cationic lipid of formula or a salt thereof, where the mole percentage of total lipid for PEG-C-DMA, cationic lipid, cholesterol and DSPC is as follows: PEG-C-DMA: about 1.5; cationic lipid: about 50.0; cholesterol: about 38.5; DSPC: about 10.0. 2. Lipid nanoparticle according to claim 1, in which one or more nucleic acid molecules contain siRNA. 3. Lipid nanoparticle according to claim 1, in which one or more nucleic acid molecules contain mRNA. 4. A lipid nanoparticle according to any one of claims 1 to 3, having a mass ratio (total lipid):(nucleic acid) of more than about 17. 5. A lipid nanoparticle according to any one of claims 1 to 3, having a mass ratio (total lipid):(nucleic acid) of more than about 18. 6. A lipid nanoparticle according to any one of claims 1 to 3, having a mass ratio (total lipid):(nucleic acid) of more than about 19. 7. A lipid nanoparticle according to any one of claims 1 to 3, having a weight ratio (total lipid):(nucleic acid) of about 22 to about 25. 8. A pharmaceutical composition for the therapeutic or prophylactic treatment of a disease, containing a lipid nanoparticle according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 9. Pharmaceutical composition according to claim 8, formulated for subcutaneous administration. 10. Pharmaceutical composition according to claim 8 or 9 for the therapeutic or prophylactic treatment of a disease characterized by a genetic disorder that leads to a deficiency of a functional protein. 11. Pharmaceutical composition according to claim 8 or 9 for therapeutic or prophylactic treatment of a disease characterized by overexpression of a polypeptide. 12. A method for delivering a nucleic acid into a cell, including bringing the cell into contact, in vitro, with a lipid nanoparticle according to any one of claims 1-7. 13. A method of treating a disease characterized by a genetic disorder that results in a deficiency of a functional protein, comprising administering to a subject with the disease a lipid nanoparticle according to any one of claims 1 to 7, wherein the nucleic acid molecule is an mRNA encoding a functional protein or a protein having such the same biological activity as the functional protein. 14. A method of treating a disease characterized by overexpression of a polypeptide, comprising administering to a subject with the disease a lipid nanoparticle according to any one of claims 1 to 7, wherein the nucleic acid molecule is an siRNA that targets expression of the overexpressed polypeptide.