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WO2024207543A1 - 增加共转染mRNA的蛋白表达水平和表达时长的重组基因及其应用 - Google Patents

增加共转染mRNA的蛋白表达水平和表达时长的重组基因及其应用 Download PDF

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WO2024207543A1
WO2024207543A1 PCT/CN2023/087369 CN2023087369W WO2024207543A1 WO 2024207543 A1 WO2024207543 A1 WO 2024207543A1 CN 2023087369 W CN2023087369 W CN 2023087369W WO 2024207543 A1 WO2024207543 A1 WO 2024207543A1
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mrna
gene
recombinant
expression
protein
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French (fr)
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徐凯
吴秀锦
雷颖雪
陈亮
张耀艺
杨建�
刁泠丹
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成都诺恩基因科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to mRNA gene engineering technology, and in particular to a recombinant gene for protein expression level and expression duration of co-transfected mRNA and its application.
  • Eukaryotic messenger RNA is a single-stranded RNA molecule composed of several different elements, including a 5 ⁇ -m7G cap, a 5 ⁇ -untranslated region (5 ⁇ UTR), a translation start codon, a coding region, a stop codon, a 3 ⁇ UTR, and a polyadenylic acid (poly-A) tail. These elements serve as templates and regulators for the translation of protein sequences.
  • the translation efficiency of mRNA proteins is affected by the components of mRNA and is related to the host cell type.
  • mRNA-mediated gene therapy depends on the protein translation efficiency of the delivered gene and the pharmacodynamic and toxicological characteristics of the protein.
  • the recombinant sequence of medicinal mRNA must be optimized according to the host cell type.
  • the challenge in the preparation of mRNA drugs is to avoid the immunogenicity of mRNA and delivery materials, as well as pyrogenic impurities, while improving the intracellular stability of mRNA, protein translation efficiency, and protein expression kinetics.
  • the immunogenicity of lipid excipients and their degradation products, as well as the autoimmunogenicity of mRNA can stimulate the innate immune mechanism of cells and cause inflammatory and cellular immune responses in the body. This, combined with the intracellular mRNA degradation mechanism, leads to reduced intracellular stability of mRNA. Therefore, in addition to optimizing the design of mRNA sequences, the development of mRNA gene therapy drugs also needs to avoid the influence of host cell innate immune mechanisms and mRNA degradation mechanisms.
  • mRNA degradation mechanisms involve optimization of mRNA recombinant sequences, such as coding optimization, poly-A extension, UTR optimization, and removal of miRNA target sequences, as well as innate immune inhibitors.
  • mRNA recombinant sequences such as coding optimization, poly-A extension, UTR optimization, and removal of miRNA target sequences, as well as innate immune inhibitors.
  • ionizable lipids and auxiliary lipids with low immunogenicity have been developed.
  • nucleoside modification technology mainly pseudouridine modification, is used to reduce the activation of mRNA on the innate immune response of host cells.
  • nucleoside modification technology in the development of mRNA vaccines.
  • Nucleoside-modified mRNA not only improves protein expression levels, but more importantly, avoids the role of activating the innate immune mechanism of cells and produces more specific binding antibodies and neutralizing antibodies.
  • the currently used technical means can only eliminate immunogens as much as possible from the perspective of preparation production, and partially alleviate the impact of innate immune mechanisms and mRNA degradation mechanisms on mRNA stability. Due to the limitations of cognitive level, complexity of nucleoside modification, difficulty of production process, and storage conditions, the improvement effect is greatly restricted.
  • One of the purposes of the present invention is to provide a type of recombinant gene that increases the protein expression level and expression duration of co-transfected mRNA to solve the above problems.
  • the polypeptide is a recombinant ranpirnase or a recombinant derivative of ranpirnase with one or more amino acids deleted, substituted, inserted or added; or a recombinant derivative of its human corresponding gene.
  • the recombinant derivative of ranpirnase is a derivative in which Gln is replaced by amino acids Met and Ser at the N-terminus of ranpirnase, and has an amino acid sequence as shown in SEQ ID NO.1;
  • the recombinant derivative of its human corresponding gene has an amino acid sequence as shown in SEQ ID NO.2 and SEQ ID NO.8, which are rAng (RNase 5) and rRanpK32R, respectively.
  • the polypeptide is a recombinant Amphinase or a recombinant Amphinase derivative with one or more amino acids deleted, substituted, inserted or added.
  • the recombinant Amphinase derivative has an amino acid sequence as shown in SEQ ID NO.3-7, namely: rAmph1 (based on Amph-1 P85072), rAmph2 (based on Amph-2 P85073), rAmph3 (based on Amph-3 P85074), rAmph4 (based on Amph-4 P85075) and rBS-RNase.
  • the second purpose of the present invention is to provide a method for using the above-mentioned recombinant gene exogenous target RNA to increase the expression level in the body, and the technical scheme adopted is: the enhancing gene and the target gene are constructed in series in the same mRNA expression framework to form a fusion protein; or they are isolated by 2A peptide and internal ribosome entry site to express independent recombinant enhancing protein and target protein; or they are independently constructed in the mRNA expression framework, mixed with the target gene mRNA for co-transfection.
  • the nucleotide sequence of the enhancing gene is added to any RNA vector expressed in eukaryotes, and protein expression is performed by independent transfection or co-transfection to increase the expression level of the exogenous target gene in the body and cells.
  • the introduction of the recombinant gene is combined with nucleoside modification.
  • the combination of the recombinant enhancement gene of the present invention and the nucleoside modification technology can have a synergistic effect on enhancing mRNA expression.
  • Another object of the present invention is to provide an mRNA preparation prepared by the above method.
  • the preparation is an injection.
  • Another object of the present invention is to provide the use of the above-mentioned mRNA preparation in the preparation of biological vaccines and gene drugs.
  • the inventors of the present application have discovered through a large number of experiments that by increasing the protein translation efficiency by inhibiting the host cell innate immune response mechanism and mRNA degradation mechanism, the innate immune reactivity of cells to lipids, mRNA and its hydrolysate impurities can be reduced on a larger scale, replacing nucleoside modification, or combining with existing nucleoside modification technology to produce a synergistic effect, thereby greatly improving the protein expression efficiency.
  • Nuclease III is a prokaryotic ribonuclease that degrades double-stranded RNA (dsRNA).
  • Dicer and Drosha are human RN3-type nucleases that cleave miRNA precursors and participate in the maturation of miRNAs, and further participate in the regulation of gene translation levels and mRNA stability.
  • immunogenic substances contained in mRNA vaccine preparations include:
  • LNP lipid nanoparticle
  • PEG lipid glycoprotein
  • ionizable lipids are weakly immunogenic. Impurities and oxidative degradation products of lipid components during storage may stimulate immune responses. These substances stimulate non-specific immune responses and lead to side effects.
  • mRNA The natural mRNA synthesized by eukaryotic cells undergoes post-transcriptional modification and has a large number of modified nucleosides, including pseudouridine and methylated adenosine, and is non-immunogenic.
  • the unmodified mRNA molecule synthesized by in vitro transcription (IVT) is an activating molecule of immune pattern receptors (TLRs, and RIG-1), which can activate intracellular TLR and RIG-I receptors in cells, activate the innate immune response of cells, and induce inflammatory response, leading to translation inhibition and mRNA degradation.
  • TLRs immune pattern receptors
  • RIG-1 immune pattern receptors
  • nucleosides such as N1-methylpseudouridine ( ⁇ ), pseudouridine and N-6-methyladenosine (m6A), which reduces the recognition of the innate immune mechanism, increases the stability of mRNA, and improves the protein translation efficiency by more than 10 times.
  • dsRNA RIG-I and MDA5 are two major intracellular dsRNA receptors. Once they recognize exogenous dsRNA, they activate antiviral signaling pathways and produce anti-inflammatory factors such as interferon.
  • mRNA produced by in vitro transcription (IVT) is accompanied by dsRNA, which stimulates dsRNA receptors and produces a stress response, leading to translation inhibition and mRNA degradation.
  • the uncapped 5 ⁇ -triphosphate motif of the mRNA produced by IVT is immunogenic and activates the cellular innate immune response by binding to RIG-I;
  • Lipids and impurities PEG2000-DMG. Impurities in the PEG group are the source of toxic substances that cause DSPC degradation. Cholesterol is easily oxidized, resulting in a series of cholesterol oxidation products;
  • Impurities generated during storage Studies have shown that naked mRNA molecules can be stably stored for more than 941 days at pH 7.4 in an aqueous phase at 5°C with the addition of nuclease inhibitors (RI). In comparison, the shelf life of the marketed COVID-19 mRNA vaccines stored at ultra-low temperatures (BNT162b2, ⁇ 80°C; mRNA-1273, ⁇ 20°C) is less than one year.
  • the root cause of the shorter shelf life of mRNA-ionizable LNPs is still the internal water content and excipient components in the LNPs formulation. Ionizable LNPs will degrade over time due to aggregation, fusion, mRNA leakage or lipid degradation. During long-term storage of ionizable LNPs, the rate of mRNA decomposition and lipid denaturation is affected by storage temperature, storage time, organic solvents, lipids, and inner core lipids. The most common cause is hydrolysis and oxidation of the nucleic acid phosphodiester backbone in the presence of water or acid/base. The PEG group of PEG2000-DMG and the carboxyl ester bond of DSPC are easily hydrolyzed during storage;
  • lipids present in ionizable LNPs have an indirect effect on the stability and structural integrity of mRNA and is one of the main reasons for their short shelf life.
  • Ranpirnase is an amphibian ribonuclease and a member of the pancreatic ribonuclease (RNase A) protein superfamily. N-pyroglutamyl residues are key components of Ranp's cytotoxic activity and contribute significantly to its intracellular entry and stable conformation.
  • the C-terminal disulfide bond (87-104) is covalently bound to form an ultrastable conformation of Ranp, which is resistant to endogenous proteases and has a reduced affinity for RI, allowing it to remain active in cells, while most mammalian nucleases are inhibited by RI.
  • Ranp has ribonuclease III (RN3) activity, and its confirmed degradation substrates include: tRNA, dsRNA, miRNA precursors, while mRNA and rRNA are not damaged.
  • RN3 ribonuclease III
  • Ranp's degradation of tRNA leads to the inhibition of protein synthesis; it degrades double-stranded nucleic acids, reducing the stimulation of cellular innate immune responses; it may degrade small RNA precursors, reduce the degradation of RNA by interference mechanisms such as siRNA and miRNA, and produce miRNA and siRNA that affect gene expression.
  • the miRNA-mediated RNA silencing mechanism is one of the main pathways of mRNA degradation. Ranp degrades miRNA precursors and generally downregulates high-abundance miRNAs, especially high-abundance immune regulatory factors such as miR-155 and miR-21, thereby downregulating innate immune function and improving mRNA stability.
  • Ranp has an immunomodulatory mechanism of action by interfering with the nuclear factor kappa light-chain-enhancer of activated B cells (NF ⁇ B) pathway. Ranp inhibits the translocation of NF ⁇ B to the nucleus and regulates the cellular response to stimuli such as stress, free radicals, bacteria and/or viral antigens. In addition, NF ⁇ B plays a key role in regulating the immune response to infection (kappa light chains are an important component of immunoglobulins). By inhibiting the translocation of NF- ⁇ B into the nucleus, the inflammatory process will be suppressed.
  • NF ⁇ B nuclear factor kappa light-chain-enhancer of activated B cells
  • the N-terminus of Ranp is modified with a unique cyclic pyroglutamic acid (Pyr1), which prefers uridine-guanine (UG) substrates and has anti-inflammatory and tumor cell growth inhibitory effects.
  • the polypeptide expressed by the recombinant Ranp (rRanp) gene is headed by Met, which reduces the thermal stability, catalytic activity and antigenicity of rRanp, and is mainly confined to the cytoplasm due to the loss of the secretory signal peptide.
  • the Pyr1 replacement variant maintains a secondary structure similar to that of the wild-type Ranp, but has lower thermal stability and specific catalytic activity for the innate substrate UG.
  • an artificially synthesized recombinant gene encodes a recombinant polypeptide protein, which improves the protein expression efficiency and prolongs the expression time of the target gene by inhibiting the innate immune response and mRNA degradation of the host cells.
  • the protein encoded by rR3GE has the following characteristics: 1. Nuclease III activity, degrading dsRNA; 2. Low immunogenicity, can be soluble or insoluble; 3. No cytotoxicity; 4. No hydrolysis activity on single-stranded RNA; 5. Low affinity with RI; 6. The enhanced function of rR3GE is independent of the target protein type, gene coding sequence, and host cell type.
  • a specific example of the present invention is to co-transfect the target protein mRNA with in vitro synthesized rRanp mRNA.
  • the rR3GE gene is reversely encoded by the polypeptide sequence (4-105) of rRanp with RNA hydrolysis activity.
  • the optimized gene code is recombined and constructed into the plasmid DNA to form an mRNA production template with translation activity.
  • the mRNA is generated by in vitro transcription reaction, and capped and modified by vaccinia capping enzyme to form a mature mRNA with translation activity.
  • the rRanp mRNA and the tracer protein mRNA are encapsulated with traditional LNPs, transfected into in vitro cultured cells or intramuscularly injected into mice. Compared with the control group, it is observed that rRanp promotes a significant increase in the expression of the tracer protein and a prolonged expression time, as shown in Figures 3 and 4. After encapsulation with non-ionized cation-modified LNP (LNP ⁇ ), the same expression enhancement and prolonged expression time were also observed, confirming that the enhancement effect of rRanp may be unrelated to the type of delivery medium and has universality. The in vitro cultured cell colony production experiment confirmed that the rRanp gene had no obvious cytotoxicity, see Table 1.
  • Another specific example of the present invention is to encode and recombinantly encode the rR3GE gene and the target protein gene into the same mRNA segment, with self-cleaving polypeptide (P2A) or IRES interlaced, and simultaneously generate independent rR3GE polypeptide and target protein in the cell.
  • the rRanp gene is reversely encoded by the rRanp polypeptide sequence, and the optimized target gene sequence, P2A gene sequence, and rR3GE gene sequence are arranged in a linear manner and constructed into plasmid DNA under the expression framework of the T7 promoter to form an mRNA production template.
  • the mRNA was encapsulated with LNP and intramuscularly administered to mice. Compared with the control group, it was observed that the rRanp gene promoted a significant increase in the expression of the tracer protein and a prolonged expression time, as shown in Figure 6.
  • Another specific example of the present invention is to use the Amph-1 gene fragment to encode the rR3GE gene, and recombinant it with the target protein gene into the same segment of mRNA, interlaced with a self-cleaving polypeptide (P2A), and simultaneously generate independent rAmph1 protein and target protein.
  • P2A self-cleaving polypeptide
  • the optimized target gene sequence, P2A gene sequence, and rAmph1 gene sequence are arranged in a linear manner and constructed into plasmid DNA to form an mRNA production template with translation activity.
  • the mRNA was encapsulated with LNP and intramuscularly injected into mice. Compared with the control group, it was observed that the rAmph1 gene promoted a significant increase in the expression of the tracer protein and a prolonged expression time, as shown in Table 1.
  • Another specific example of the present invention is to use the polypeptide sequence (4-105) of rRanp with RNA hydrolysis activity, add a translation start codon to the N-terminus of the polypeptide, form a recombinant gene sequence with expression activity in eukaryotic cells, place it under the expression framework of the CMV promoter, and recombinant it in plasmid DNA.
  • Plasmid DNA is used to transfect in vitro cultured cells, and rRanp proteins containing different N-terminal amino acid sequences are translated and expressed under CMV drive, as shown in Figure 1.
  • rR3GE Although the mechanism of action of rR3GE is not very clear, RN3 activity and low RI affinity are two necessary conditions, and there is no degradation effect on linear RNA. Based on these characteristics, ordinary technicians in this field can easily reconstruct recombinant gene fragments that perform similar functions but have different sequences.
  • the mRNA expression enhancing gene of the present invention can be a short artificially synthesized recombinant gene fragment, encoding a recombinant polypeptide with nuclease III activity, which can be co-transfected with the pharmacological mRNA to significantly increase the expression efficiency and duration of the pharmacological protein by inhibiting the innate immune response and mRNA degradation of the host cells.
  • the gene fragment with such function is referred to as rR3GE in this application, i.e. recombinant Ribonuclease III-associated gene expression Enhancer.
  • rR3GE reduces the recognition and degradation of mRNA by the innate immune mechanism of cells, thereby increasing the stability of mRNA and promoting the expression and duration of protein expression.
  • Protein replacement therapy has a wide range of applications.
  • hemophilia in the treatment of hemophilia, hemophilia patients lack coagulation proteins due to gene mutations. Due to the short half-life of proteins, usually only 12 hours, patients need to inject coagulation proteins 3-7 times a week.
  • Preclinical studies on mice have shown that a weekly injection of 0.2-0.5 mg/kg of nucleoside-modified mRNA can maintain effective coagulation factor protein levels.
  • Clinical trials of hemophilia using adenovirus-associated virus (AAV) have shown that protein expression is stable within 2 years after injection.
  • AAV adenovirus-associated virus
  • rR3GE in this application is very low, and there is room for further improvement.
  • Species-derived modification of the rR3GE protein sequence and structure can further reduce the immunogenicity of rR3GE in specific species.
  • humanized recombination of the rR3GE protein can further reduce or eliminate its immunogenicity, which is conducive to long-term repeated medication.
  • Circular RNA is a type of circular RNA with a range of protein-coding and non-coding functions. It does not contain a 5 ⁇ -triphosphate motif, so the RNA-mediated innate immune response is low, and the protein expression of circRNA shows higher stability than that of uridine-modified linear mRNA in adipose tissue. Although circRNA does not contain the triphosphate motif required for typical RIG-I activation, RIG-I may transiently interact with circRNA lacking host nuclear protein protection, leading to a typical RIG-I-mediated inflammatory response. CircRNA may also interact with other RNA sensors, such as endosomal TLR3, 7 and 8, and MDA5, leading to inflammatory responses.
  • rR3GE is compatible with the IRES expression system of circRNA. In the absence of nucleoside modification, rR3GE can be fully utilized to inhibit the host's innate immune response and enhance the expression level and duration of the target gene.
  • the mRNA expression enhancing gene and the method for enhancing gene expression in cells provided by the present invention can increase the protein expression level of the co-transfected gene by 3-4 times and extend the expression time by nearly 1 time by adding a small enhancing gene fragment:
  • rR3GE degrades dsRNA, reducing the difficulty of mRNA production and purification; lowering the threshold for mRNA production;
  • Figure 1 rRanp enhanced gene recombination sequence and co-expressed mRNA molecule sequence analysis
  • Figure 2 rRanp enhanced gene co-expression mRNA recombinant molecule design
  • Figure 3 IVIS imaging results of rRanp enhancing the expression level and expression duration of co-transfected nucleoside-modified mRNA
  • Figure 4 Analysis and comparison of the expression level and expression duration of rRanp-enhanced co-transfected nucleoside-modified mRNA
  • Figure 5 IVIS imaging results of rRanp increasing the expression level and duration of co-transfected unmodified mRNA
  • Figure 6 Analysis and comparison of rRanp enhancing the expression level and expression duration of co-transfected unmodified mRNA
  • Figure 7 IVIS imaging results of rRanp increasing the expression level and expression duration of mRNA with different dsRNA contents co-transfected
  • Figure 8 Analysis of the expression level and expression time of mRNA increased by rRanp in co-transfected cells with different dsRNA contents
  • Figure 9 Comparison of the expression levels of mRNAs increased by rRanp in co-transfected cells with different dsRNA contents
  • Figure 10 Design of rR3GE gene variant mRNA
  • Figure 11 In vivo IVIS imaging results of enhanced expression of the rR3GE gene variant mRNA
  • Figure 12 Colony formation inhibition rate of rRanp recombinant gene in cultured cells
  • Figure 13 rR3GE promotes the stability of LNP formulations stored at 4°C and -20°C.
  • Ionizable lipids such as ALC-0315, MC3, DHA-1, L319, SM-102, etc.
  • the ionizable lipid used in the embodiment of the present invention is ALC-0315;
  • Non-ionized cationic lipid an amphiphilic molecule with a hydrophilic group and a hydrophobic group, consisting of a polar head (hydrophilic group), a connecting bond, and a hydrophobic tail.
  • the hydrophilic head is a quaternary ammonium salt, which is a permanent cation and does not have an ionizable characteristic.
  • the non-ionized cationic lipid used in the embodiment of the present invention is DOTAP;
  • Nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, including DNA and RNA.
  • RNA can be in the form of siRNA, microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, circular RNA and combinations thereof.
  • Nucleic acids can be synthetic, naturally occurring and non-naturally occurring. Including but not limited to phosphorothioates, phosphoramidates and peptide nucleic acids (PNA), and including nucleic acids containing known natural nucleotide analogs and artificially modified nucleotides, such as pseudouracil, methylated, methyl pseudouracil modified.
  • DNA can be double-stranded DNA, single-stranded DNA, plasmid DNA, etc.
  • Example 1 Raw materials and preparation production
  • the mRNA used in the examples of the present invention was obtained by IVT reaction production.
  • the general process is enzyme digestion of plasmid DNA template; column purification to obtain linearized plasmid DNA; IVT transcription production of RNA (Thermo Fisher, MEGAscript® Kit); after transcription, RNA was purified using oligo-dT column (Sartorius). Unless otherwise specified, the transcription reaction substrate UTP was replaced by N1-methyl pseudouridine ( ⁇ ).
  • the capping reaction of mRNA was completed with Vaccinia Capping Enzyme of near-shore protein.
  • the mRNA capping reaction was set up according to the reaction system recommended by the kit, and the reaction conditions were 37°C for 1 hour.
  • the capping product was purified using an oligo-dT affinity column. The purified mRNA was dissolved in sterile water for injection, analyzed by RNA gel electrophoresis and identified by Qubit concentration.
  • LNP and LNP ⁇ preparations are composed of ionizable lipids, non-ionized cationic lipids, DSPC, cholesterol (Chol) and PEG2000-DMG in a certain molar ratio.
  • the specific composition is as follows:
  • Lipid materials were dissolved in anhydrous ethanol, and nucleic acids were dissolved in citric acid aqueous solution (10 mM, pH 4.0). The aqueous solution and organic solution were mixed at a volume ratio of 3:1 through a microfluidic chip (Shanghai Pengzan Bio, Luer connector microfluidic chip) with a total flow rate of 12 ml/min.
  • the LNP preparation was dialyzed against 1 ⁇ PBS solution overnight, and then transferred to a glass bottle and stored at 4°C or -20°C. Final mRNA concentration: 0.1–0.375 ⁇ g/ ⁇ l.
  • Example 2 P2A self-cleavage of rRanp gene enhances nucleoside-modified mRNA expression level and expression time
  • This example compares the effect of rR3GE on the expression level of nucleoside-modified mRNA delivered by LNP and LNP ⁇ 46 lipids.
  • N1-methyl pseudouracil nucleotides were used instead of UTP as substrates to prepare ⁇ Fluc and ⁇ Fluc-rRanp mRNA.
  • the mRNA was encapsulated with LNP and LNP ⁇ 46 lipids to prepare a series of LNPs containing different mRNAs.
  • the protein sequence design of rRanp is shown in Figure 1, and the composition of mRNA is shown in Figure 2.
  • rRanp uses the polypeptide sequence of mature Ranp, removing Gln (Q) at the N-terminus and replacing it with Met and Ser.
  • the amino acid sequence of "rRanp" in the following example is shown in SEQ ID NO. 1.
  • the co-expressed mRNA of Fluc (firefly luciferase gene) and rRanp is connected by the self-cleaving polypeptide P2A sequence to form Fluc-rRanp single-stranded mRNA.
  • the polypeptide is cleaved from the 21st position of P2A, dividing the transcript into two polypeptide segments: Fluc and rRanp.
  • mice Seven-week-old female Balb/c mice were divided into six groups, with three mice in each group.
  • the drug was administered by intramuscular injection (IM) of the right lower limb at a dose of 5.0 ⁇ g/30 ⁇ l to test the expression kinetics of four lipid particle preparations, namely ⁇ Fluc-LNP, ⁇ Fluc-rRanp-LNP, ⁇ Fluc-LNP ⁇ 46, and ⁇ Fluc-rRanp-LNP ⁇ 46.
  • IM intramuscular injection
  • IVIS imaging analysis was performed at different time points. The results of in vivo IVIS imaging of mice are shown in Figure 3.
  • the experimental results showed that the tracer gene in the LNP preparation group mice was highly expressed at the intramuscular injection site and liver tissue, as shown in Figure 4A.
  • the tracer protein expression of the mice in the ⁇ Fluc-rRanp-LNP experimental group with the addition of rRanp recombinant fragments was significantly increased, calculated by the area under the curve (AUC), which was 6.14, 1.60, and 3.88 times the protein expression in the liver, muscle, and whole body of the mice in the ⁇ Fluc-LNP control group, respectively, as shown in Figure 4C.
  • AUC area under the curve
  • the expression duration of the tracer protein in the ⁇ Fluc-rRanp-LNP experimental group mice was also significantly increased, with the background fluorescence intensity as the lower limit, the expression of the tracer protein in the liver was extended by 48 hours, and the expression time of the protein in the intramuscular injection site was extended by 96 hours.
  • the half-life of the tracer protein in the muscle site was increased from 20.2 hours in the control group to 23.2 hours.
  • the tracer signal at the intramuscular injection site of mice in the rRanp group decreased sharply at the beginning of transfection, but a turning point occurred 24 hours later, the decay rate of the tracer protein signal slowed down, and the curvature of the tracer protein expression kinetics changed significantly thereafter, as shown in Figure 4A.
  • mice in the intramuscular injection-LNP ⁇ 46 group expressed high levels of tracer protein only at the intramuscular injection site.
  • the expression of tracer protein at the intramuscular injection site of the mice in the ⁇ Fluc-rRanp-LNP ⁇ 46 experimental group was significantly increased.
  • the protein expression level of the mice in the ⁇ Fluc-rRanp-LNP ⁇ 46 experimental group was 1.77 times that of the mice in the ⁇ Fluc-LNP ⁇ 46 control group, as shown in Figure 4D.
  • the expression duration of tracer protein in the mice in the ⁇ Fluc-rRanp-LNP ⁇ 46 experimental group was significantly increased, and the protein expression at the intramuscular injection site was extended to 7 days, which was 1.87 times that of the ⁇ Fluc-LNP ⁇ 46 control group.
  • the half-life of the tracer protein in the muscle site increased from 27.8 hours in the control group to 37.2 hours; the tracer signal at the intramuscular injection site of the LNP ⁇ 46 group of mice decreased sharply at the beginning of transfection, and slowly recovered at 24 to 48 hours. Thereafter, the kinetic curvature of the tracer protein expression changed significantly, as shown in Figure 4B.
  • rRanp significantly enhanced the expression kinetics of nucleoside-modified mRNA delivered by LNP and LNP ⁇ 46 lipids.
  • the protein expression increased by 1.80 to 6.24 times and the expression time was prolonged by about 1 time depending on the expression site.
  • the separation of the tracer protein and rRanp protein by the P2A self-cleavage peptide had no effect on each other's functions.
  • the expression kinetics of the tracer protein at the intramuscular injection site was superimposed by multiple modification effects. Pseudouridine modification increased the half-life of the tracer protein from 20.2 hours to 23.2 hours; LNP ⁇ 46 increased it to 27.8 hours; and the addition of rRanp increased the half-life to 37.2 hours.
  • This example compares the effect of rRanp on the expression level of mRNA without nucleoside modification delivered by LNP lipids.
  • UTP or N1-methyl pseudouracil nucleotides were used instead of UTP as substrates to prepare Fluc-rRanp mRNA and ⁇ Fluc mRNA, respectively.
  • the mRNA was encapsulated with LNP lipids to prepare mRNA-LNPs.
  • mice Seven-week-old female Balb/c mice were divided into three groups, each with 3 mice, and administered by intramuscular injection into the right lower limb.
  • the nucleic acid-modified ⁇ Fluc-LNP hereinafter referred to as the "control group”
  • the unmodified mRNA liposome Fluc-rRanp-LNP hereinafter referred to as the "experimental group”
  • 5 ⁇ g and 10 ⁇ g lipid nanoparticle preparations were tested respectively.
  • intramuscular administration in vivo IVIS imaging analysis was performed at different time points. The results of in vivo IVIS imaging of mice are shown in Figure 5.
  • the experimental results show that under intramuscular administration, the tracer gene in the LNP preparation group mice has a high level of expression in the intramuscular injection site and liver tissue.
  • the total amount of tracer protein expression in the experimental group mice with a dose of 5 ⁇ g mRNA was significantly lower than that in the control group, and the protein expression in the liver, muscle, and whole body was 49%, 15%, and 16%, respectively, Figure 6A, Figure 6C.
  • the tracer protein of the control group of this embodiment was mainly expressed on the first day after administration, and the expression level was far higher than that of the same dose group, but the tracer protein expression of the experimental group mice exceeded that of the 3rd to 13th day, which was 1.47 times that of the experimental group mice, and the half-life of the tracer protein was increased to 32.9 hours, Figure 6A.
  • the total amount of tracer protein expression in the 10 ⁇ g mRNA dose experimental group was higher than that in the low dose (5 ⁇ g mRNA) experimental group, which was 1.73, 1.47, and 3.75 times higher than that in the liver, muscle, and whole body (Figure 6D), respectively, and the half-life of the tracer protein was increased to 31.6 hours ( Figure 6B).
  • the kinetic spectra of tracer protein expression in the muscle of the experimental groups of mice at different doses were similar, and the protein expression levels exceeded that of the control group from the 3rd to the 13th day, which was 2.21 times higher than that of the control group.
  • the tracer signal at the intramuscular injection site slowly recovered from 24 to 48 hours, which was particularly obvious in the experimental group.
  • the recovery intensity was proportional to the mRNA injection dose. Thereafter, the kinetic curvature of tracer protein expression changed significantly, Figure 6A, Figure 6B.
  • the corresponding protein expression amount of 10 ⁇ g rRanp mRNA is 1.47 to 3.75 times that of 5 ⁇ g rRanp mRNA, but the rRanp mRNA dose has no effect on the expression kinetic spectrum, and increasing the mRNA dose does not prolong the protein expression time.
  • the expression signal of the tracer gene delivered by LNP ⁇ 46 in Figure 4B was extended on the second day after intramuscular injection, the recovery of the tracer protein signal at the injection site one day after intramuscular injection was caused by the rRanp gene, and the recovery intensity was proportional to the mRNA dose, which was particularly obvious in unmodified mRNA.
  • the period of signal recovery in the early stage of transfection is exactly the time when the rRanp gene is expressed and takes effect, which also confirms that the expression enhancement effect of nucleoside-modified mRNA is independent of the expression enhancement effect of rR3GE, that is, mRNA without nucleoside modification and only integrating rR3GE also shows obvious expression enhancement effect, and the two have synergistic effect when used simultaneously.
  • Fluc-LNP and Fluc-rRanp-LNP were prepared using Fluc mRNA and Fluc-rRanp mRNA containing different concentrations of dsRNA.
  • Seven-week-old female Balb/c mice were divided into four groups, with 3 mice in each group, and intramuscular administration was performed to test LNPs encapsulating H-Fluc and H-Fluc-rRanp mRNA containing high concentrations of dsRNA (1.26%, hereinafter referred to as "H-Fluc-rRanp group”), and L-Fluc-rRanp mRNA containing low concentrations of dsRNA (0.14%, hereinafter referred to as "L-Fluc-rRanp group”).
  • intramuscular administration 5 ⁇ g or 10 ⁇ g mRNA
  • in vivo IVIS imaging analysis was performed at different time points. The results of in vivo IVIS imaging of mice are shown in Figure 7.
  • mice in the group of mRNA-LNP preparations containing high concentrations of dsRNA showed high expression of tracer proteins in the intramuscular injection site and liver tissue.
  • the total amount of protein expressed in the liver and intramuscular injection site of mice in the rRanp-mRNA group was 2.50 times and 2.90 times higher than that in the H-Fluc-rRanp group, respectively, and the expression time was extended by 48 hours and 72 hours, respectively.
  • the total amount of protein expressed in the liver of mice in the L-Fluc-rRanp group was 4.16 times higher than that in the H-Fluc-rRanp group, and the expression time was extended by 24 hours.
  • the total amount of protein expressed in the intramuscular injection site was 1.72 times higher, but the expression time was not extended, as shown in Figures 8 and 9.
  • the present invention analyzes the protein amino acid sequence, structure and functional groups of rRanp based on performance indicators such as RN3 activity and low RI binding that may be involved in enhancing gene expression, and uses the rRanp molecule as a template to search for genes with similar structures in the NIH-NCBI gene library. It is understandable to those skilled in the art that it is possible to edit and recombinantly use these sequences as templates to construct a new rR3GE with similar functions to rRanp.
  • rRanp molecular sequence According to the inventor's analysis and testing of rRanp molecular sequence, structural features, and functional groups, the common characteristics of rR3GE are summarized.
  • the present invention further explores and tests the rR3GE molecular sequence, structure, and functional groups that may have gene expression enhancement functions, verifying the inventor's summary of the sequence, structure, and function of rR3GE.
  • RN3 activity and low RI affinity affect the gene expression enhancement of rR3GE, while the degradation activity of mRNA (single-stranded RNA) needs to be avoided.
  • the catalytic triad of RNase A (His12, Lys41 and His119) is a key site for the high nuclease activity of RNase A. It is strictly evolutionarily conserved in all variants of Ranp (His10, Lys31 and His97) and Amph (His15, Lys42 and His107). The nucleolytic activity of Ranp and Amph-1 is significantly lower than that of RNase A and most homologs. When Lys41 of RNase A was replaced by an arginine residue, it was shown that the variant has about 2% of the enzymatic activity of wild-type RNase A. The molecular basis of this difference has not been fully elucidated.
  • the inventors constructed a group of recombinant gene-enhanced mRNAs with different RNase A hydrolysis activities and RI affinities based on the characteristics of rR3GE.
  • the constructs are shown in FIG10 .
  • the mRNA-LNP was prepared. Seven-week-old female Balb/c mice were divided into six groups, with three mice in each group. 5 ⁇ g was intramuscularly injected, and in vivo IVIS imaging analysis was performed on days 0.25, 1 to 9 of administration. The results are shown in Figure 11.
  • the K32R mutation reduced the RNase A hydrolase activity (RN1) of rRanpK32R, and further increased the expression level of the co-transfected gene on the basis of rRanp, indicating that the RNase A hydrolysis activity has a limited promoting effect on the protein expression level of the co-transfected mRNA in a short period of time, and has no obvious help in increasing the expression duration of the co-transfected gene.
  • the K d value of dimeric BS-RNase is 2E-06; the K d value of monomeric BS-RNase is 1E-12;
  • RN3 activity and low RI affinity are required for the gene expression enhancement of rR3GE.
  • excessive RNase I was expressed in a short period of time, degrading various RNAs in the cytoplasm, lowering the normal metabolic level of cells, and assisting in enhancing the protein expression of co-transfected mRNA, but it did not increase the expression duration of the target gene.
  • Reducing the RN1 of rR3GE by mutation can further improve the expression level of the co-transfected gene.
  • A549 cells are p53 wild-type, H1299 cells are p53-deficient, and H322 cells are p53 mutant heterozygous.
  • A549, H322, and H1299 cells were seeded into 6-well plates at 2 ⁇ 10 5 cells/mL and incubated overnight at 37°C in 5% CO2.
  • pcDNA3.1 was mixed with pCMV-eGFP, pCMV-BP-RNase, pCMV-Bax, pCMV-p53, pCMV-rMQD-Ranp, and pCMV-rMSD-Ranp plasmid DNA at a weight ratio of 1:9, encapsulated with Dotap:Lecithin (molar ratio 20:9) liposomes, and then diluted with culture medium containing 10% FBS, with 1 ⁇ g or 5 ⁇ g DNA per ml of culture medium. The cell culture medium was replaced with the culture medium containing DNA liposomes and continued to be cultured in the incubator.
  • the colony formation inhibition rate of the rRanp recombinant gene in cultured cells is shown in Figure 12.
  • the experiment shows that the Bax and p53 genes have different cell colony formation inhibition rates on these three cultured cell lines.
  • the Bax gene significantly inhibits the formation of colonies in A549 cells and H322 cells; the p53 gene inhibits the formation of colonies in H1299 cells.
  • rMQD-Ranp and rMSD-Ranp have no inhibitory effect on the formation of colonies in these three cells, Figure 12A-F.
  • the BP-RNase recombinant gene did not show obvious cell colony growth inhibition ability.
  • the inventors conducted a comparative experiment on the expression intensity of the tracer gene in mice under the storage conditions of 4°C and -20°C for LNP preparations.
  • the experiment used ⁇ Fluc mRNA and ⁇ Fluc-rRanp mRNA purified by oligo dT column. Microfluidic chips were prepared with LNP respectively.
  • the prepared mRNA-nLNP was stored in a 4°C refrigerator and a -20°C refrigerator respectively.
  • mice After storage, 0W, 1W, 2W, 4W, and 8W were selected from 4°C and -20°C respectively, and 400 uL 1XPBS was added for dilution (1:2 dilution, mRNA concentration 0.1667ug/uL) for particle size measurement and mRNA encapsulation rate measurement.
  • the tracer protein expression experiment of mice intramuscular administration was carried out, and IVIS live imaging detection was performed 0.25, 1, and 2 days after administration.
  • the rRanp gene fragment significantly prolonged the storage time of mRNA-rR3GE-LNP preparations at 4°C or -20°C, extending the storage time by at least twice.
  • the present invention is a synthetic recombinant gene fragment, rR3GE, which encodes a recombinant polypeptide with nuclease III activity, and is co-transfected with the pharmacological mRNA to improve the expression efficiency and expression time of the pharmacological protein by inhibiting the innate immune response of the host cell and mRNA degradation.
  • rR3GE can be integrated into mRNA without changing the physicochemical properties of mRNA, and is therefore suitable for all delivery methods and administration methods of delivering mRNA without changing the biological function and pharmacological efficacy of mRNA.

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Abstract

提供了增加共转染mRNA的蛋白表达水平和表达时长的重组基因，该重组基因编码的多肽为具有核糖核酸酶Ⅲ活性、不受核糖核酸酶抑制剂抑制并且不降解单链RNA的多肽；该重组基因和目标基因可以串联或平行构建，形成融合蛋白或独立表达的重组蛋白；或以2A肽、内部核糖体进入位点等进行隔离，表达出独立的重组增强蛋白及目标蛋白；重组增强蛋白通过降解dsRNA、tRNA、miRNA及其前体分子，抑制宿主细胞先天免疫机制和mRNA降解机制，使共转染基因的蛋白表达水平提高3-4倍，表达时间延长近1倍。

Description

增加共转染mRNA的蛋白表达水平和表达时长的重组基因及其应用 技术领域
本发明涉及mRNA基因工程技术，尤其涉及一类加共转染mRNA的蛋白表达水平和表达时长的重组基因及其应用。
 背景技术
真核信使RNA(mRNA)是由几个不同元素组成的单链核糖核酸分子，包括一个5¢-m7G 帽、一个5¢-非翻译区(5¢UTR)、一个翻译起始密码子、一个编码区、终止密码子、一个3¢UTR和聚腺苷酸(poly-A)尾巴。这些元件用作蛋白质序列翻译的模板和调控。mRNA蛋白的翻译效率受mRNA各成分的影响，并与宿主细胞类型有关。mRNA介导的基因治疗取决于递送基因的蛋白质翻译效率以及蛋白质的药效学和毒理学特征。药用mRNA的重组序列必须根据宿主细胞类型进行优化。而mRNA药物制备中面临的挑战是避免mRNA和递送材料的免疫原性，以及热源杂质，同时提高mRNA细胞内稳定性、蛋白质翻译效率和蛋白质表达动力学特性。脂质原辅料及其降解产物的免疫原性，以及mRNA的自身免疫原性，可以激发细胞先天免疫机制，引起机体的炎症反应和细胞免疫反应。这与细胞内mRNA降解机制相结合，导致mRNA胞内稳定性降低。因此，mRNA基因治疗药物开发，除了需要优化设计mRNA序列外，也需要规避宿主细胞先天免疫机制和mRNA降解机制的影响。
目前针对细胞内mRNA降解机制的技术解决方案涉及mRNA重组序列优化，例如编码优化、poly-A延伸、UTR优化和miRNA靶序列去除等方法，以及先天免疫抑制剂。对于递送脂质，开发出来了具有低免疫原性的可电离脂质和辅助脂质。为了解决mRNA的自身免疫原性，核苷修饰技术，主要是假尿嘧啶修饰，被用来减少mRNA对宿主细胞先天免疫反应的激活。这些方法是目前提高基于mRNA疗法的稳定性和功效的主流技术途径。尤其是辉瑞/BioNTech、和Moderna使用甲基假尿嘧啶核苷修饰技术成功开发新冠病毒mRNA疫苗的经验，和CureVac等利用天然非修饰mRNA开发疫苗的失败教训，确立了核苷修饰技术在mRNA疫苗研发中的关键作用。经核苷修饰的mRNA不仅仅提升蛋白表达水平，更重要的是避免激活细胞先天免疫机制的作用，产生更为专一的结合抗体和中和抗体。
 技术问题
但是，目前应用的技术手段只能从制剂生产的角度，尽可能的消除免疫原，部分缓解先天免疫机制和mRNA降解机制对mRNA稳定性造成的影响。受到认知水平、核苷修饰复杂性、生产工艺难度、及储存条件等因素的限制，改进效果受到很大制约。
 技术解决方案
本发明的目的之一，就在于提供一类增加共转染mRNA的蛋白表达水平和表达时长的重组基因，以解决上述问题。  
为了实现上述目的，本发明采用的技术方案是这样的：
增加共转染mRNA的蛋白表达水平和表达时长的重组基因，所述重组基因编码的多肽为具有核糖核酸酶Ⅲ活性、不受核糖核酸酶抑制剂抑制并且不降解单链RNA的多肽。
作为优选的：所述多肽为重组豹蛙酶或缺失、取代、插入或增加一个或多个氨基酸的豹蛙酶重组衍生物；或其人类对应基因的重组衍生物。
作为进一步优选的：所述豹蛙酶重组衍生物为在豹蛙酶的N-端用氨基酸Met和Ser取代Gln，其具有如SEQ ID NO.1所示的氨基酸序列；所述其人类对应基因的重组衍生物具有如SEQ ID NO.2和SEQ ID NO.8所示的氨基酸序列，分别为rAng (RNase 5)和rRanpK32R。
作为优选的：所述多肽为重组Amphinase或缺失、取代、插入或增加一个或多个氨基酸的Amphinase重组衍生物。
作为进一步优选的：所述Amphinase重组衍生物为具有如SEQ ID NO.3-7所示的氨基酸序列，依次为：rAmph1 (基于Amph-1 P85072)、rAmph2 (基于Amph-2 P85073)、rAmph3 (基于Amph-3 P85074)、rAmph4 (基于Amph-4 P85075)和rBS-RNase。
本发明的目的之二，在于提供一种利用上述的重组基因外源目标RNA在机体中表达水平的方法，采用的技术方案为：所述增强基因和目标基因以串联方式构建于同一mRNA表达框架下，形成融合蛋白；或以2A肽、内部核糖体进入位点进行隔离，表达出独立的重组增强蛋白及目标蛋白；或独立构建于mRNA表达框架，与目标基因mRNA混合制备，实施共转染。
作为优选的：所述增强基因的核苷酸序列添加到任意在真核表达的RNA载体中，以独立转染或共转染的方式进行蛋白表达，提高外源目的基因在机体和细胞中的表达水平。
作为优选的：所述重组基因的引入与核苷修饰相结合。本发明的重组增强基因与核苷修饰技术相结合，能够对于mRNA表达增强有协同增效的效果。
本发明的又一个目的，在于提供上述方法制得的mRNA制剂。
作为优选的：所述制剂为注射剂。
本发明的再一个目的，在于提供上述的mRNA制剂在制备生物疫苗及基因药物中的应用。
本申请的发明人通过大量试验发现，通过抑制宿主细胞先天免疫反应机制和mRNA降解机制的方法来增加蛋白翻译效率，可以在更大维度上降低细胞对脂质、mRNA及其水解物杂质的先天免疫反应性，取代核苷修饰，或与现有的核苷修饰技术结合以产生协同效应，从而更大地提高蛋白表达效率。
核酸酶Ⅲ(RN3, RNase Ⅲ)是一种原核生物的核糖核酸酶，降解双链RNA（dsRNA）。Dicer和Drosha是人RN3型核酸酶，切割miRNA前体而参与miRNA的成熟转化，并进一步参与基因转译水平和mRNA稳定性的调控。
具体而言：mRNA疫苗制剂中含有的免疫原性物质，包括：
LNP（脂质纳米颗粒）是一种复合物制剂，其组成成分被认为是无药效活性并且毒性较小，但所用组份，如PEG、可电离脂质具有弱免疫原性。杂质及保存过程中脂质成分的氧化降解产物均有可能刺激免疫反应。这些物质刺激产生非特异免疫反应，导致副反应。下面是对主要免疫原成份及其对mRNA表达效率影响分析：
mRNA：真核细胞合成的天然mRNA经过转录后修饰，拥有大量修饰核苷，包括假脲嘧啶和甲基化腺苷，没有免疫原性。体外转录（IVT）合成的未修饰mRNA分子是一种免疫模式受体 (TLRs，及RIG-1) 的激活分子，在细胞中能够激活细胞内TLR和RIG-I受体，激活细胞先天免疫反应，并诱发炎症反应，导致翻译抑制和mRNA降解。体外合成中用N1-甲基假尿苷(Ψ)、假尿苷和N-6-甲基腺苷（m6A）等核苷修饰，降低先天免疫机制的识别，mRNA稳定性增加，蛋白翻译效率提升10倍以上。
dsRNA：RIG-I和MDA5是两个主要的细胞内dsRNA受体，一旦识别到外源dsRNA，就会激活抗病毒信号途径，产生抗炎症因子，如干扰素。体外转录反应(IVT)生产的mRNA，伴生dsRNA，刺激dsRNA感受器，产生应激反应，导致翻译抑制和mRNA降解。
5¢-三磷酸基序（Triphosphate Motif）：IVT生产的mRNA，经过加帽修饰步骤后，未加帽的5¢-三磷酸基序具有免疫原性，通过结合RIG-I而激活细胞先天免疫反应；
脂质及杂质：PEG2000-DMG。PEG基团中存在的杂质是导致DSPC降解的有毒物质的来源。胆固醇容易被氧化，导致一系列的胆固醇氧化产物；
储存过程产生杂质：有研究表明，在加有核酸酶抑制剂（RI）的5℃的水相中，在pH7.4条件下，裸mRNA分子可稳定保存超过941天，相比而言，已上市的COVID-19 mRNA疫苗在超低温下储存（BNT162b2，−80℃；mRNA-1273，−20℃），其保质期还是只有不到一年。通过对现有mRNA-可电离脂质LNPs和商业化mRNA-可电离脂质LNPs不同的储存条件和保质期的总结发现，mRNA-可电离化脂质LNPs的辅料和制造方法对期存储稳定性有显著影响。mRNA-可电离LNP的保质期较短的根本原因仍然是LNPs配方中内部的水含量和辅料成分。可电离LNPs会由于聚集、融合、mRNA泄漏或脂质降解，随着时间的推移而降解。在可电离LNP长期储存过程中，mRNA的分解和脂质变性速率受储存温度、储存时间、有机溶剂、脂质和内核脂质的影响。最常见的原因是核酸磷酸二酯主链在水或酸/碱存在下，发生水解和氧化。PEG2000-DMG的PEG基团及DSPC的羧基酯键在储存中过程中易于水解；
可电离LNPs中存在的脂质对mRNA的稳定性和结构完整性有间接影响，也是其保质期较短的主要原因之一。
豹蛙酶详细说明
豹蛙酶（Ranpirnase，Ranp）是一种两栖类核糖核酸酶，属胰腺核糖核酸酶（RNase A）蛋白超级家族成员。N-焦谷氨酰残基是Ranp产生细胞毒活性的关键组成部分，对其入胞、和稳定构象有重要贡献。C-端二硫键（87-104）共价结合，形成Ranp超稳构象，对内源性蛋白酶产生抗性，并降低对RI的亲和力，使其在细胞内保持活性，而大多数哺乳动物核酸酶受到RI抑制。
Ranp具有核糖核酸酶Ⅲ（RN3）活性，已经证实的降解底物有：tRNA, dsRNA, miRNA前体，而mRNA和rRNA不受损害。
Ranp对tRNA的降解导致蛋白质合成抑制；降解双链核酸，降低刺激细胞先天免疫反应；可能降解小RNA前体，减少siRNA和miRNA等干扰机制对RNA的降解，并产生影响基因表达的miRNA和siRNA。
miRNA介导的RNA沉默机制是mRNA降解的主要途经之一。Ranp降解miRNA前体，普遍下调高丰度miRNA，尤其是miR-155，miR-21等高丰度免疫调节因子，以此下调先天免疫功能并提升mRNA稳定性。
Ranp通过干扰活化B细胞的核因子κ轻链增强子（NFκB）途径而具有免疫调节作用机制。Ranp抑制NFκB向细胞核的易位，并调节细胞对刺激的反应，如应激、自由基、细菌和/或病毒抗原。此外，NFκB在调节感染的免疫反应中起着关键作用（κ轻链是免疫球蛋白的重要组成部分）。通过抑制NF-κB向细胞核内的移位，炎症过程将被抑制。
Ranp的N端为特有的环焦谷氨酸(Pyr1)修饰，偏好尿苷-鸟嘌呤(UG)底物，具有抗炎和抑制肿瘤细胞生长的作用。作为其中一个实施例，重组Ranp（rRanp）基因表达的多肽以Met为首，减少了rRanp的热稳定性、催化活性和抗原性，而且因失去分泌信号肽而主要被局限于细胞质内。Pyr1替换变体保持与野生型Ranp相似的二级结构，但对先天底物UG的热稳定性和特异性催化活性较低。
rR3GE的设计和功能验证
作为其中一个实施例，作为一段人工合成的重组基因（rR3GE），其编码的重组多肽蛋白，通过抑制宿主细胞的先天免疫反应和mRNA降解，提高目标基因的蛋白表达效率和延长表达时间。
rR3GE编码的蛋白具有以下特征：1.核酸酶Ⅲ活性，降解dsRNA；2.低免疫原性，可以为溶解型或非溶型；3.无细胞毒性；4.对单链RNA没有水解活性；5.与RI亲和力低；6.rR3GE的增强功能独立于目标蛋白质类型，基因编码序列，以及宿主细胞类型。
本发明一个具体实例，是利用体外合成的rRanp mRNA同目标蛋白mRNA进行共转染。利用rRanp具有RNA水解活性的多肽序列（4-105）逆向编码rR3GE基因，在T7启动子的表达框架下，将优化的基因密码重新组合构建到质粒DNA中，形成具有翻译活性的mRNA生产模板。利用体外转录反应生成mRNA，经牛痘加帽酶进行加帽修饰，形成具有翻译活性的成熟mRNA。将rRanp mRNA同示踪蛋白mRNA用传统LNP进行包裹，转染体外培养细胞或向小鼠进行肌注给药，同对照组比，观察到rRanp促进示踪蛋白表达量显著增加和表达时间延长，见图3,4。经过以非电离阳离子修饰LNP（LNP Å）进行包裹，也观察到同样的表达增强和表达时长延长，证实rRanp的增强作用与递送介质类型可能无关，具有通用性。rRanp通过体外培养细胞集落生产实验证实，rRanp基因没有明显细胞毒性，见表1。
本发明另一个具体实例，是将rR3GE基因同目标蛋白基因，编码重组到同一段mRNA中，以自切割多肽（P2A）或IRES相间，在细胞中同时生成独立的rR3GE多肽和目标蛋白。利用rRanp多肽序列逆向编码rRanp基因，T7启动子的表达框架下，将优化后的目标基因序列、P2A基因序列、rR3GE基因序列，以线性方式排列并构建到质粒DNA中，形成mRNA生产模板。用LNP对该mRNA进行包裹，对小鼠进行肌注给药，同对照组比，观察到rRanp基因促进示踪蛋白表达量显著增加和表达时间延长，见图6。
本发明再一个具体实例，是利用Amph-1基因片段编码rR3GE基因，同目标蛋白基因重组到同一段mRNA中，以自切割多肽（P2A）相间，同时生成独立的rAmph1蛋白和目标蛋白。利用Amph1具有RNA水解活性的多肽序列（4-105），T7启动子的表达框架下，将优化后的目标基因序列、P2A基因序列、rAmph1基因序列，以线性方式排列并构建到质粒DNA中，形成具有翻译活性的mRNA生产模板。用LNP对该mRNA进行包裹，对小鼠进行肌注给药，同对照组比，观察到rAmph1基因促进示踪蛋白表达量显著增加和表达时间延长，见表1。
本发明另一个具体实例，是利用rRanp具有RNA水解活性的多肽序列（4-105），多肽N端加上翻译起始密码子，形成具有真核细胞中表达活性的重组基因序列，置于CMV启动子的表达框架下，重组于质粒DNA中。利用质粒DNA转染体外培养细胞，在CMV驱动下翻译表达出含不同N-端氨基酸序列的rRanp蛋白，见图1。尽管对于rR3GE作用机理还不是很清楚，但RN3活性和低RI亲和力是两个必要的条件，而且对线性RNA也没有降解作用。基于这些特性，本领域普通技术人员可以方便地重组构建出行使类似功能但序列相异的重组基因片段。
rR3GE潜在作用机制：
（1）降解tRNA，降低细胞翻译效率，维持低水平的蛋白表达；减少对外源mRNA降解及蛋白质的降解；
（2）特异性切割miRNA及其前体，介入miRNA/siRNA的成熟转化，降低细胞先天免疫反应性，削弱细胞RNA降解机制；
（3）敲除dsRNA，避免刺激细胞先天免疫反应；
（4）介入NF-κB通路和MMP9活性，降低细胞先天免疫反应性
作为优选的，本发明的mRNA表达增强基因，可以为一段人工合成重组基因短片段，编码具有核酸酶Ⅲ活性的重组多肽，与药效mRNA共转染，通过抑制宿主细胞的先天免疫反应和mRNA降解，显著增加药效蛋白表达效率和表达时长，具有这样功能的基因片段，本申请简称为rR3GE ，即recombinant Ribonuclease Ⅲ-associated gene expression Enhancer。
本发明从机制上讲，rR3GE减少细胞先天免疫机制对mRNA的识别和降解，从而增加mRNA的稳定性，促进蛋白质的表达和表达时长。这些发现表明rR3GE有潜力用作mRNA疫苗（药物）的通用助推器，这对于生物制品、药物和疫苗的生物医学研究和开发具有重要意义。这种增强的程度不因蛋白质类型、细胞密度/功能、转染效率、递送机制、报告剂量、分泌信号和2A介导的自动切割效率而异，也不改变基因表达的组织分布。
蛋白质替代疗法有很大的应用范围。如在血友病的治疗中，血友病患者由于基因突变缺少凝血蛋白，由于蛋白质半衰期短，通常只有12小时，因此患者需要每周注射3 - 7次凝血蛋白。而针对小鼠的临床前研究显示，每周注射一次0.2-0.5mg/kg剂量的核苷修饰mRNA就能保持有效的凝血因子蛋白水平。使用腺病毒相关病毒（AAV）的血友病临床试验显示，在注射后2年内，蛋白质表达稳定。但一些最近的研究显示，由于免疫系统对病毒载体的排斥，在注射5-7年后可能需要再次注射，而病毒载体有其自身的安全性问题，特别是在儿科疾病治疗中。另外，具有基础免疫力的病人也无法使用AAV基因疗法。基于本申请的rR3GE增强型凝血因子VⅢ mRNA疗法，可能替代传统疗法治疗血友病。
目前，在mRNA新冠疫苗接种中，小剂量mRNA局部递送已观察到炎症并发症，而mRNA疗法是通过高剂量长期给药，这将放大炎症并发症及其它副作用。而本申请rR3GE的免疫原性很低，而且还有进一步改善空间。对rR3GE蛋白序列和结构进行种属源修饰，可以进一步降低rR3GE在特定种属的免疫原性。如，对rR3GE蛋白进行人源化重组，可以进一步降低或消除其免疫原性，有利于长期重复用药。
环状RNA（circRNA）是一类具有一系列蛋白质编码和非编码功能的环状RNA，不含5¢-三磷酸基序，因此RNA介导的先天免疫反应较低，circRNA的蛋白质表达表现出比脂肪组织中尿苷修饰的线性mRNA更高的稳定性。虽然circRNA不包含典型的RIG-I激活所需的三磷酸基序，RIG-I可能与缺乏宿主核蛋白保护的circRNA瞬时相互作用，导致典型的RIG-I介导的炎症反应。circRNA也可能与其他RNA传感器相互作用，例如内涵体TLR3、7和8，以及MDA5等，导致炎症反应。而circRNA的蛋白质表达受IRES启动，核苷修饰影响其蛋白翻译水平，因此无法借助核苷修饰降低宿主对circRNA的先天免疫反应。rR3GE与circRNA的IRES表达系统兼容，在没有核苷修饰的情形下，可以充分利用rR3GE抑制宿主先天免疫反应，增强目的基因表达水平和表达时长。
 有益效果
本发明的优点在于：本发明提供的mRNA表达增强基因及用于细胞内基因表达增强的方法，通过加入增强基因小片段，将共转染基因蛋白表达水平提升3 - 4倍，表达时间延长近1倍：
（1）增强共转染基因蛋白表达量3 - 4倍，减少mRNA制剂用量；
（2）降低细胞蛋白整体水平，减少对外源mRNA降解，延长基因表达时长；
（3）抑制细胞先天免疫机制，降低细胞对制剂热源（包括杂质及降解物）的反应性；在特定应用中，可以代替核苷修饰，规避宿主先天免疫机制对mRNA的破坏；
（4）rR3GE降解dsRNA，降低mRNA生产及纯化难度；降低mRNA生产门槛；
（5）简化mRNA-LNP生产工艺；延长mRNA-LNP保存持时间；
（6）降低肌肉细胞中先天免疫反应，使特异蛋白呈递更具专一性。
 附图说明
图1：rRanp增强基因重组序列及共表达mRNA分子序列分析；
图2：rRanp增强基因共表达mRNA重组分子设计；
图3：rRanp增强共转染核苷修饰mRNA表达水平和表达时长的IVIS成像结果；
图4：rRanp增强共转染核苷修饰mRNA的表达水平和表达时长分析比较；
图5：rRanp提升共转染未修饰mRNA表达水平和表达时长的IVIS成像结果；
图6：rRanp提升共转染未修饰mRNA表达水平和表达时长分析比较；
图7：rRanp增加共转染不同dsRNA含量mRNA的表达水平和表达时长的IVIS成像结果；
图8：rRanp增加共转染不同dsRNA含量mRNA的表达水平和表达时间分析；
图9：rRanp增加共转染不同dsRNA含量mRNA的表达水平差异比较；
图10：rR3GE基因变异体mRNA设计；
图11：rR3GE基因变异体mRNA的表达增强活体IVIS成像结果；
图12：培养细胞中rRanp重组基因的集落形成抑制率；
图13：rR3GE促进LNP制剂在4℃及-20℃保存条件下的稳定性。
 本发明的最佳实施方式
下面将结合附图对本发明作进一步说明。
名词解释：可电离脂质：比如ALC-0315、MC3、DHA-1、L319、SM-102等，本发明实施例所使用的可电离脂质为ALC-0315；
非电离阳离子脂质：具有亲水基团和疏水基团的双性分子，由极性头部(亲水基团)、连接键、疏水尾部组成。亲水头部为季铵盐，为永久性阳离子，不具可电离特征。本发明实施例所使用的非电离阳离子脂质为DOTAP；
核酸：是指含有至少两个单链或双链形式的脱氧核糖核苷酸或核糖核苷酸的聚合物，包括DNA和RNA。RNA可以是siRNA、微小RNA(miRNA)、mRNA、tRNA、rRNA、tRNA、环状RNA及其组合的形式。核酸可以是合成的、天然存在的和非天然存在的。包括但不限于硫代磷酸酯、氨基磷酸酯和肽核酸(PNA)，并包括含有已知的天然核苷酸类似物以及人工修饰核苷酸，如假尿嘧啶、甲基化、甲基假脲嘧啶修饰的核酸。DNA可以是双链 DNA、单链DNA、及质粒DNA等。
实施例1：原材料及制剂生产
1.1 RNA制备
本发明实施例中所使用的mRNA均由IVT反应生产获得。大致过程为质粒DNA模板的酶切处理；柱纯化得到线性化质粒DNA；RNA的IVT转录生产（赛默飞，MEGAscript® Kit）；转录完成后，用oligo-dT柱（Sartorius）纯化RNA。除非特殊说明，转录反应底物UTP用N1-甲基假尿苷酸（ѱ）代替。
mRNA的加帽修饰反应用近岸蛋白的痘苗加帽酶（Vaccinia Capping Enzyme）完成。mRNA加帽修饰反应遵照试剂盒推荐的反应体系设置，反应条件为37℃ 1小时。反应完成后，加帽产物用oligo-dT亲和柱纯化。纯化的mRNA溶解于无菌注射用水，经RNA凝胶电泳分析及Qubit浓度鉴定。
1.2 LNP、LNP Å制剂的制备
LNP、LNP Å制剂由可电离脂质、非电离阳离子脂质、DSPC、胆固醇（Chol）及PEG2000-DMG以一定的摩尔比组成，具体组成如下：
LNP脂质摩尔数量比为：ALC-0315:DSPC:Chol:PEG = 46.29:9.4:42.67:1.64；N/P比为6.09；
LNP Å46脂质摩尔数量比为：Dotap:ALC-0315:DSPC:Chol:PEG = 23.01:23.01:9.35:42.43:2.2；N/P比为12.1；
脂质物料溶解于无水乙醇，核酸溶解于柠檬酸水溶液(10mM，pH 4.0)。水溶液同有机溶液以3:1体积比通过微流控芯片（上海澎赞生物，鲁尔接头微流控芯片）进行混合，总流速为12 ml/min。LNP制剂经1×PBS溶液透析过夜，完毕后转移至玻璃瓶，4℃或-20℃保存。mRNA终浓度：0.1–0.375 µg/µl。
实施例2  P2A自切割rRanp基因增强核苷修饰mRNA表达水平及表达时间
本实施例比较了LNP及LNP Å46脂质递送的核苷修饰mRNA表达水平受rR3GE的影响。在IVT反应中，用N1-甲基假尿嘧啶核苷酸代替UTP为底物，制备ѱFluc、及ѱFluc-rRanp mRNA。用LNP及LNP Å46脂质分别对mRNA进行包裹，制成一系列含不同mRNA的LNPs。rRanp的蛋白序列设计见图1，mRNA的构成见图2。
从图1中可以看出，rRanp采用成熟Ranp的多肽序列，在N-端去掉Gln（Q）、代之以Met和Ser而成，下述实施例的“rRanp”的氨基酸序列如SEQ ID NO.1所示。Fluc（萤火虫荧光素酶基因）和rRanp的共表达mRNA由自切割多肽P2A序列连接，形成Fluc-rRanp单链mRNA。基因转录时，多肽从P2A第21位切割，将转录子分割为两段多肽：Fluc和rRanp。
将7周龄雌性Balb/c小鼠分为六组，每组3只，通过右下肢肌肉注射（IM）的方式给药，给药剂量为5.0 µg/30 µl，测试ѱFluc-LNP、ѱFluc-rRanp-LNP、ѱFluc-LNP Å46、ѱFluc-rRanp-LNP Å46等四种脂粒制剂的表达动力学。肌注给药后，在不同时间点进行活体IVIS成像分析，小鼠活体IVIS成像结果见图3。
实验结果显示，LNP制剂组小鼠中示踪基因在肌注给药部位及肝脏组织中具有高水平表达，如图4A所示。加入rRanp重组片段的ѱFluc-rRanp-LNP实验组小鼠的示踪蛋白表达量显著升高，以曲线下面积（AUC）计算，分别为ѱFluc-LNP对照组小鼠在肝脏、肌肉、及全身蛋白表达量的6.14、1.60、及3.88倍，如图4C所示。ѱFluc-rRanp-LNP实验组小鼠的示踪蛋白表达时长也显著增大，以达到背景荧光强度为下限，肝脏中示踪蛋白表达延长48小时，肌注部位蛋白表达时间延长96小时。同时，肌肉部位示踪蛋白半衰期从对照组的20.2小时提升到23.2小时。rRanp组小鼠肌注部位示踪信号在转染初期急剧减少，但在24小时后发生转折，示踪蛋白信号衰减速度减缓，此后的示踪蛋白表达动力学曲率发生明显改变，如图4A所示。
肌注型-LNP Å46制剂组小鼠仅在肌注给药部位高水平表达示踪蛋白。ѱFluc-rRanp-LNP Å46实验组小鼠肌注部位示踪蛋白表达量显著升高，以曲线下面积（AUC）计算，ѱFluc-rRanp-LNP Å46实验组小鼠蛋白表达量是ѱFluc-LNP Å46对照组小鼠蛋白表达量的1.77倍，如图4D所示。ѱFluc-rRanp-LNP Å46实验组小鼠示踪蛋白表达时长显著增大，以达到背景荧光强度为限，肌注部位蛋白表达延长7天，为ѱFluc-LNP Å46对照组的1.87倍。同时，肌肉部位示踪蛋白半衰期从对照组的27.8小时提升到37.2小时；LNP Å46组小鼠肌注部位示踪信号在转染初期急剧减少，在24至48小时时缓慢回升，此后，示踪蛋白表达动力学曲率发生明显改变，如图4B。
结论：rRanp对LNP及LNP Å46脂质递送的核苷修饰mRNA表达动力学均有显著增强效果，依据不同表达部位，蛋白表达量增加1.80至6.24倍，表达时间延长约1倍。由P2A自切割多肽分离示踪蛋白和rRanp蛋白，对彼此功能作用没有影响。在肌注部位示踪蛋白表达动力学特性由多重修饰效果叠加而成，假尿嘧啶核苷修饰将示踪蛋白半衰期从20.2小时提升到23.2小时；LNP Å46将其提升到27.8小时；而rRanp的加入，使得半衰期增加到37.2小时。相比下，文献报道中，同剂量核苷修饰mRNA肌肉注射及皮内注射表达的示踪蛋白，其半衰期分别为20.6小时和29.6小时(Pardi, Tuyishime et al. 2015)。因此，假尿嘧啶修饰、LNP Å46制剂、及rRanp的表达增强效果具有协同增效效应，当同时应用时，即便采用肌肉注射方式给药，给药15天后，仍能用IVIS显影仪检测到明确示踪蛋白信号。与立刻起效的核苷修饰不同的是，rRanp需要约24小时进行表达并起效，在24至48小时之间产生特征性示踪蛋白表达水平回升转折，并改变随后的蛋白表达动力学曲率。
实施例
本实施例比较了rRanp对LNP脂质递送未经核苷修饰mRNA表达水平的影响。在IVT反应中，用UTP或N1-甲基假尿嘧啶核苷酸代替UTP为底物，分别制备Fluc-rRanp mRNA、及ѱFluc mRNA。用LNP脂质对mRNA进行包裹，制成mRNA-LNPs。
将7周龄雌性Balb/c小鼠分为三组，每组3只，通过右下肢肌肉注射的方式给药，分别测试核酸修饰ѱFluc-LNP（后称“对照组”）、及未修饰mRNA脂粒Fluc-rRanp-LNP（后称“实验组”）（5 µg和10 µg）脂质纳米颗粒制剂。肌注给药后，在不同时间点进行活体IVIS成像分析，小鼠活体IVIS成像结果见图5。
实验结果显示，肌注给药下，LNP制剂组小鼠中示踪基因在肌注给药部位及肝脏组织中具有高水平表达。5 µg mRNA剂量的实验组小鼠的示踪蛋白表达总量显著低于对照组，在肝脏、肌肉、及全身蛋白表达量分别为其49%、15%、及16%，图6A、图6C。但是，实验组小鼠的示踪蛋白表达时长显著增加，以达到背景荧光强度为低限，肝脏中示踪蛋白表达延长48小时，而肌注部位蛋白表达延长96小时，同实施例2中核苷修饰ѱFluc-rRanp-LNP实验组一致（图4A）。肌肉注射部位示踪蛋白表达动力学谱发生明显改变，本实施例的对照组的示踪蛋白主要在给药后第一天表达，表达量远超同剂量组，但实验组小鼠在第3至13天示踪蛋白表达量反超，为其1.47倍，且示踪蛋白半衰期提升到32.9小时，图6A。
10 µg mRNA剂量实验组小鼠的示踪蛋白表达总量比低剂量（5 µg mRNA）实验组有所提升，分别为其在肝脏、肌肉、及全身示踪蛋白表达量的1.73、1.47、及3.75倍（图6D），示踪蛋白半衰期提升到31.6小时（图6B）。不同剂量下实验组小鼠肌肉部位示踪蛋白表达动力学谱相近，蛋白表达量均在第3至13天反超对比组，为其2.21倍。肌注部位示踪信号在经历了第一个24小时的急剧减少后，在24至48小时有缓慢回升，实验组尤为明显，回升强度与mRNA注射剂量有正比关系。此后，示踪蛋白表达的动力学曲率明显改变，图6A、图6B。
结论：相同mRNA剂量水平下，未修饰rRanp增强mRNA的示踪蛋白表达总量低于核苷修饰mRNA，但rRanp多肽影响示踪蛋白表达动力学特性，延长蛋白表达时间一倍以上。rRanp对未修饰mRNA的示踪蛋白表达增量存在正向的剂量关系。依据其表达部位不同，10 µg rRanp mRNA相应的蛋白表达量是5 µg rRanp mRNA的1.47至3.75倍，但rRanp mRNA剂量对表达动力学谱没有影响，提高mRNA剂量没有延长蛋白表达时间。尽管图4B中LNP Å46递送的示踪基因在肌注后第二天表达信号有所延拓，肌注1天后肌注部位示踪蛋白信号回升为rRanp基因所致，回升强度与mRNA剂量成正比，在未修饰mRNA中表现尤其明显。转染初期信号回升时段，正好是rRanp基因表达并发挥作用的时间，也证实核苷修饰mRNA的表达增强效果与rR3GE的表达增强效果是相互独立的，即不进行核苷修饰、仅整合rR3GE的mRNA也表现出明显的表达增强效果，而且二者同时使用时具有协同增效性。
实施例
用含不同浓度dsRNA的Fluc mRNA、Fluc-rRanp mRNA分别制备Fluc-LNP、Fluc-rRanp-LNP。将7周龄雌性Balb/c小鼠分为四组，每组3只，肌肉给药，分别测试包裹含高浓度dsRNA（1.26%，后称“H-Fluc-rRanp组”）的H-Fluc、H-Fluc-rRanp mRNA、及低浓度dsRNA(0.14%，后称“L-Fluc-rRanp组”)的L-Fluc-rRanp mRNA的LNPs。肌注给药（5 µg 或 10 µg mRNA）后，在不同时间点进行活体IVIS成像分析，小鼠活体IVIS成像结果见图7。
实验结果显示，肌注给药下，包裹含高浓度dsRNA mRNA-LNP制剂组小鼠均在肌注部位和肝脏组织中显示出示踪蛋白高表达，rRanp-mRNA组小鼠在肝脏及肌注部位表达的蛋白总量分别比H-Fluc-rRanp组高2.50倍和2.90倍，表达时间分别延长48小时和72小时。L-Fluc-rRanp组小鼠在肝脏表达的蛋白总量比H-Fluc-rRanp组高4.16倍，表达时间延长24小时，肌注部位表达的蛋白总量高1.72倍，但表达时间没有延长，如图8和图9所示。
本实施例证实：高浓度dsRNA及假尿嘧啶核苷修饰对rRanp-mRNA基因表达时长没有显著影响，但影响转染初期蛋白表达量。
实施例5  rR3GE蛋白序列分析
本发明根据可能参与基因表达增强的RN3活性、低RI结合性等性能指标，对rRanp的蛋白质氨基酸序列、结构及功能基团进行了分析，并以rRanp分子为模板，搜寻了NIH-NCBI基因库中具有相似结构的基因，本领域普通技术人员能够理解的，以这些序列为模板进行编辑、重组，构建出具有类似rRanp功能的新型rR3GE是可能的。
。
实施例6
根据发明人对rRanp分子序列、结构特征、功能基团的分析和测试，总结出了rR3GE的共同特性。本发明进一步对可能具有基因表达增强功能的rR3GE分子序列、结构和功能基团进行了探讨和测试，验证了发明人对rR3GE的序列、结构和功能的归纳。RN3活性和低RI亲和性对rR3GE基因表达增强作用的影响，而对mRNA（单链RNA）的降解活性则是需要避免的。
RNase A的催化三联体（His12、Lys41 和 His119）是RNase A核酸酶高活性的关键位点。在 Ranp中（His10、Lys31 和 His97）和Amph（His15、Lys42 和His107）的所有变体中严格进化保守的。Ranp和Amph-1的核酸水解活性明显低于RNase A和大多数同系物。当RNase A的Lys41被精氨酸残基取代的变体被证明具有大约 2% 的野生型RNase A酶水解活性。这种差异的分子基础尚未完全阐明。最明显的因素是催化残基的非典型构象，Ranp中的Lys31和His97以及Amph-1中的等效Lys42和His107。这些残基，与RNase A超家族的高活性成员相比，与相邻残基的氢键结合较少，两种酶的底物结合受到损害。
本实施例中，发明人依据rR3GE的特征，构建了一组含不同RNase A水解活性和RI亲和性的重组基因增强mRNA，构建体见图10。
本实施例制备上述mRNA-LNP。将7周龄雌性Balb/c小鼠分为六组，每组3只。肌注给药5 µg，在给药0.25、1 至 9天，进行活体IVIS成像分析，结果见图11。
实验结果如表1所示，肌注给药下，rRanp、rRanpK32R、rAmph1、rBS-RNase等具有RN3水解活性和低RI亲和性的重组基因具有类似rR3GE的基因表达增强功能，而rRNaseI在给药1天内肝脏中表达的蛋白质表达水平略有增加，但没有增加基因表达时长。K32R突变降低rRanpK32R的RNase A水解酶活性（RN1），在rRanp的基础上进一步提高了共转染基因的表达水平，说明RNase A水解活性对共转染mRNA的蛋白质表达水平短时间内具有有限的促进作用，对提升共转染基因的表达时长没有明显帮助。
表1：不同rR3GE基因的活性特征及其基因表达增强作用比较
*: 二聚体 BS-RNase 的 K d 值为2E-06；单体BS-RNase的 K d 值为1E-12；
**:  基于rRanp 的RNaseⅢ活性
***：Rutkoski, T. J. and R. T. Raines (2008). "Evasion of ribonuclease inhibitor as a determinant of ribonuclease cytotoxicity." Curr Pharm Biotechnol 9(3): 185-189.
结论： RN3活性和低RI亲和性，是rR3GE基因表达增强作用所需要的。RNase I共转染细胞后，短时间内表达过量RNase I，降解细胞质中各种RNA，调低细胞正常代谢水平，对共转染的mRNA的蛋白质表达有辅助增强作用，但并不能增加目的基因的表达时长。通过突变降低rR3GE的RN1，可以进一步提高共转染基因的表达水平。
实施例7
A549细胞为p53野生型，H1299细胞是p53缺失型，而H322细胞是p53突变杂合型。为了评估体外细胞中rRanp对示踪基因的影响，以2×10 5细胞/mL分别接种A549、H322、及H1299细胞到6孔板，在5%的CO2中37℃孵育过夜。将pcDNA3.1分别与pCMV-eGFP、pCMV-BP-RNase、pCMV-Bax、pCMV-p53、pCMV-rMQD-Ranp、pCMV-rMSD-Ranp质粒DNA以1：9重量比混合，以Dotap:Lecithin(摩尔比20:9) 脂质体进行包裹，然后，用含10% FBS的培养液进行稀释，每ml培养液含1 µg或5 µg DNA。用含DNA脂质体的培养液替换细胞培养液，继续置于培养箱培养。转染2天后用含不同浓度G418的培养液进行细胞换液，H1299（400 µg/ml）, H322（100 µg/ml），A549（100 µg/ml）。待细胞密度达到80%以上，用10%福尔马林固定，并用吉姆萨溶液进行染色，拍照，计数。细胞毒性由暴露于测试基因表达后剩余活细胞与eGFP阴性对照存活细胞的百分比确定（%细胞毒性 = 100 − [（实验组平均值/eGFP组平均值）×100]），其中实验组是受试5种基因之一，eGFP组是阴性对照。使用未配对的单尾学生 t-检验对观察到的差异进行统计评估，并在P≤0.05置信水平下建立显著性。
培养细胞中rRanp重组基因的集落形成抑制率如图12所示，实验表明，Bax和p53基因对这三种培养细胞株有不同细胞集落形成抑制率。Bax基因明显抑制A549细胞和H322细胞形成集落；p53基因抑制H1299细胞形成集落。rMQD-Ranp及rMSD-Ranp对这三种细胞形成集落均没有抑制作用，图12A-F。同时，BP-RNase重组基因也没有表现出明显的细胞集落生长抑制能力。
结论：基于对不同p53突变表型细胞株的集落生成抑制实验，rMQD-Ranp、rMSD-Ranp、及BP-RNase重组蛋白均没有显示出明显细胞毒性。
实施例8
为了评估rR3GE对mRNA-LNP保存稳定性的影响，并验证其是否延长mRNA保存时间，发明人对LNP制剂，在4℃和-20℃储存条件下，进行了示踪基因小鼠中表达强度的比较实验。试验使用经过oligo dT柱纯化的ѱFluc mRNA、和ѱFluc-rRanp mRNA。分别用LNP进行微流控芯片制备。制备好的mRNA-nLNP分别于4℃冰箱和-20℃冰箱中保存。保存后0W、1W、2W、4W、8W分别从4℃和-20℃各任选1支,加入400 uL 1XPBS稀释后（1:2稀释，mRNA浓度0.1667ug/uL）进行粒径测定和mRNA包封率测定。同时进行小鼠肌注给药示踪蛋白表达实验，在给药后0.25、1、2天后进行IVIS活体成像检测。
结果显示，4℃冰箱中保存4周后，ѱFluc-LNP的示踪蛋白表达总量出现明显降低，而ѱFluc-rRanp-LNP的基因表达水平没有显著改变，图13。在-20℃冰箱中保存两周后，ѱFluc-LNP和ѱFluc-rRanp-LNP的基因表达水平发生显著改变。rRanp能够促进mRNA-LNP稳定性。在-20℃冰箱储存4周后，ѱFluc-LNP递送的蛋白表达水平显著下降，而 ѱFluc-rRanp-LNP的蛋白表达水平下降不明显。
结论：rRanp基因片段显著延长了mRNA-rR3GE-LNP制剂在4℃或-20℃条件下的储存时长，延长储存时间至少一倍。
本发明为一段人工合成的重组基因片段，rR3GE，编码具有核酸酶Ⅲ活性的重组多肽，与药效mRNA共转染，通过抑制宿主细胞的先天免疫反应和mRNA降解，提高药效蛋白表达效率和表达时间。rR3GE可以整合于mRNA中，不改变mRNA的物理化学性质，因此适用于递送mRNA的所有递送方式和给药方式，而不改变mRNA的生物学功能及药效药理。
本发明已通过各个具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各个具体实施方式，普通技术人员在本发明的范围内可以作出各种改动或变形，并且在本说明书中各处提及的各技术特征可以相互组合，而仍不背离本发明的精神和范围。这样的改动和变形均在本发明的覆盖范围之内。

Claims (10)

  1. 增加共转染mRNA的蛋白表达水平和表达时长的重组基因，其特征在于：所述重组基因编码的多肽为具有核糖核酸酶Ⅲ活性、不受核糖核酸酶抑制剂抑制并且不降解单链RNA的多肽。
  2. 根据权利要求1所述的增加共转染mRNA的蛋白表达水平和表达时长的重组基因，其特征在于：所述多肽为重组豹蛙酶或缺失、取代、插入或增加一个或多个氨基酸的豹蛙酶重组衍生物；或其人类对应基因的重组衍生物。
  3. 根据权利要求2所述的增加共转染mRNA的蛋白表达水平和表达时长的重组基因，其特征在于：所述豹蛙酶重组衍生物为在豹蛙酶的N-端依次用氨基酸Met和Ser取代Gln，其具有如SEQ ID NO.1所示的氨基酸序列，所述其人类对应基因的重组衍生物具有如SEQ ID NO.2和SEQ ID NO.8所示的氨基酸序列。
  4. 根据权利要求1所述的增加共转染mRNA的蛋白表达水平和表达时长的重组基因，其特征在于：所述多肽为重组Amphinase或缺失、取代、插入或增加一个或多个氨基酸的Amphinase重组衍生物。
  5.  根据权利要求4所述的增加共转染mRNA的蛋白表达水平和表达时长的重组基因，其特征在于：所述Amphinase或其牛对应基因的重组衍生物为具有如SEQ ID NO.3-7所示的氨基酸序列。
  6.  利用权利要求1-5任一项所述的重组基因提高外源目标RNA在机体中表达水平的方法，其特征在于：所述重组基因和目标基因以串联方式构建于同一mRNA表达框架下，形成融合蛋白；或以2A肽、内部核糖体进入位点进行隔离，表达出独立的重组增强蛋白及目标蛋白。
  7. 根据权利要求6所述的方法，其特征在于：所述重组基因的核苷酸序列添加到任意在真核表达的RNA表达载体中，以独立转染或共转染的方式进行蛋白表达，提高共转染目的基因在机体和细胞中的表达水平和表达时长。
  8.  根据权利要求6所述的方法，其特征在于：所述重组基因的引入与核苷修饰相结合。
  9. 权利要求6或7或8的方法制得的mRNA制剂或RNA制剂，其特征在于：所述制剂为注射剂。
  10. 权利要求9所述的mRNA制剂在制备生物疫苗及基因药物中的应用。
PCT/CN2023/087369 2023-04-07 2023-04-10 增加共转染mRNA的蛋白表达水平和表达时长的重组基因及其应用 WO2024207543A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1212016A (zh) * 1996-02-21 1999-03-24 美国国有卫生与人类服务部 重组的核糖核酸酶蛋白质
CN1422610A (zh) * 2001-12-07 2003-06-11 上海福缘生化药学研发有限公司 人重组血管生成素的制备及皮肤增白产品
WO2011154611A2 (en) * 2010-06-11 2011-12-15 University Of Helsinki A method for enhanced protein synthesis
CN112481289A (zh) * 2020-12-04 2021-03-12 江苏普瑞康生物医药科技有限公司 一种转录环状rna的重组核酸分子及其在蛋白表达中的应用
CN113491643A (zh) * 2020-03-20 2021-10-12 湖州美易得生物科技有限公司 血管生成素的止痒用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1212016A (zh) * 1996-02-21 1999-03-24 美国国有卫生与人类服务部 重组的核糖核酸酶蛋白质
CN1422610A (zh) * 2001-12-07 2003-06-11 上海福缘生化药学研发有限公司 人重组血管生成素的制备及皮肤增白产品
WO2011154611A2 (en) * 2010-06-11 2011-12-15 University Of Helsinki A method for enhanced protein synthesis
CN113491643A (zh) * 2020-03-20 2021-10-12 湖州美易得生物科技有限公司 血管生成素的止痒用途
CN112481289A (zh) * 2020-12-04 2021-03-12 江苏普瑞康生物医药科技有限公司 一种转录环状rna的重组核酸分子及其在蛋白表达中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CAVAIUOLO MARINA, CHAGNEAU CARINE, LAALAMI SOUMAYA, PUTZER HARALD: "Impact of RNase E and RNase J on Global mRNA Metabolism in the Cyanobacterium Synechocystis PCC6803", FRONTIERS IN MICROBIOLOGY, FRONTIERS MEDIA, LAUSANNE, vol. 11, Lausanne , XP093221541, ISSN: 1664-302X, DOI: 10.3389/fmicb.2020.01055 *
CHEN, SHUMIN ET AL.: "Advances in the Research of Regnase-1 on Transcriptional Regulation and Immunity Homeostasis", INTERNATIONAL JOURNAL OF IMMUNOLOGY, vol. 42, no. 6, 30 November 2019 (2019-11-30), pages 632 - 639, XP009558129, ISSN: 1673-4394 *

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