WO2023035372A1 - 一种有限自我复制mRNA分子系统、制备方法及应用 - Google Patents

一种有限自我复制mRNA分子系统、制备方法及应用 Download PDF

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WO2023035372A1
WO2023035372A1 PCT/CN2021/126076 CN2021126076W WO2023035372A1 WO 2023035372 A1 WO2023035372 A1 WO 2023035372A1 CN 2021126076 W CN2021126076 W CN 2021126076W WO 2023035372 A1 WO2023035372 A1 WO 2023035372A1
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mrna
sequence
protein
replicase
seq
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PCT/CN2021/126076
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French (fr)
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王刚
于寅
黄健
易桦林
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臻赫医药(杭州)有限公司
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Definitions

  • the present application relates to the technical field of biomedicine, in particular to a limited self-replicating mRNA molecular system, preparation method and application.
  • Messenger RNA (mRNA) therapy is a novel therapeutic modality with potential for a wide range of clinical applications, including vaccines against infectious agents as well as treatments for cancer or genetic diseases, regenerative therapies and immunotherapy.
  • the advantages of messenger RNA therapy include that messenger RNA can synthesize proteins through the body's own cells, without the need for complex protein synthesis and purification processes or production lines; intracellular and membrane-bound proteins can be used as therapeutic targets ; It can be rapidly industrialized under cell-free GMP conditions, and the cycle from research and development to products is short.
  • messenger RNA therapy is limited by factors such as structural instability, innate immunogenicity, and low delivery efficiency in vivo.
  • the development direction of this technology is as follows: First, it must avoid rejection by the innate immune system. Messenger RNA is mistaken for non-self nucleic acid, resulting in rejection, which is especially important for repeated dosing of messenger RNA therapeutics, as immune memory may limit the effectiveness of drug products.
  • some studies believe that chemical modification of the nucleotide bases of messenger RNA can reduce innate immune rejection, thereby improving the translation efficiency of messenger RNA into protein, but how to carry out nucleoside modification, the ratio of modification and how to carry out nucleotide modification combination still not clear.
  • RNA is unstable, easily degraded, and its expression lasts for a short time. Some studies have shown that common messenger RNA can only be expressed in cells for 24 hours. Self-replicating messenger RNA, because it can replicate itself, can amplify the protein translation instructions of messenger RNA, and can enhance and prolong the expression of messenger RNA protein.
  • the self-replicating messenger RNA molecular system used in the prior art is derived from the genome framework of an alphavirus, wherein the partial framework encoding the viral RNA replicase is complete, and the structural protein framework encoding the virus is replaced by a sequence encoding the target protein.
  • the messenger RNA molecular system has the following defects: First, compared with non-self-replicating messenger RNA, the nucleotide sequence of self-replicating messenger RNA is much longer, and the burden on cells is heavy. It is technically difficult to synthesize messenger RNA by in vitro transcription, and the cost of industrial production is high; Secondly, the self-replicating messenger RNA molecular system is essentially an RNA pseudovirus capable of self-replication, and its viral properties are obvious, such as the number of replications cannot be predicted, and there is the possibility of infinite replication (pseudovirus reproduction in vivo), for example, vesicular stomatitis virus When antigens and rabies virus antigens are packaged into the above-mentioned self-replicating RNA, there is a possibility of amplifying its toxicity; third, the above-mentioned messenger RNA molecular system has high cytotoxicity, and because it cannot be modified by nucleosides, it will cause cell or body The
  • the purpose of this application is to provide a limited self-replication mRNA molecular system, preparation method and application to solve the technical problem that mRNA cannot achieve limited self-replication in the prior art.
  • the first aspect of the present application provides a limited self-replicating mRNA molecular system, comprising:
  • At least one second mRNA encoding a protein of interest at least one second mRNA encoding a protein of interest
  • the mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region.
  • the mutant replicase comprises a sequentially connected nsP1 region, nsP2 region, nsP3 region and nsP4 region, the amino acid sequence of the mutant replicase is shown in SEQ ID NO.1, and the mutant replicase
  • SEQ ID NO.1 amino acid sequence of the mutant replicase is shown in SEQ ID NO.1
  • the mutant replicase The mutation of serine S at position 796 shown in SEQ ID NO.1 to proline P and the mutation of arginine R at position 1187 shown in SEQ ID NO.1 to aspartic acid D are produced.
  • the first mRNA includes a mutant replicase coding sequence
  • the mutant replicase coding sequence includes an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.2;
  • Each of the second mRNAs includes a replicase 5' end-specific sequence, a target protein coding sequence and a replicase 3' end-specific sequence connected in sequence, and the replicase 5' end-specific sequence includes such as SEQ ID NO.
  • the RNA sequence corresponding to the nucleic acid sequence shown in 7 the replicase 3' end specific sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.8.
  • the first mRNA and the second mRNA further include: a 5' cap structure, a 5' UTR sequence, a 3' UTR sequence and a polyadenylation sequence;
  • the first mRNA includes the following elements in sequence according to the 5' ⁇ 3' direction: 5' cap structure, 5'UTR sequence, mutant replicase coding sequence, 3'UTR sequence and polyadenylation sequence;
  • Each second mRNA includes the following elements in sequence according to the 5' ⁇ 3' direction: 5' cap structure, 5'UTR sequence, replicase 5'-end specific sequence, target protein coding sequence, replicase 3'-end specificity sequence, 3'UTR sequence and polyadenylation sequence;
  • the 5'UTR sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.9
  • the 3'UTR sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.10
  • the 5' The cap structure is selected from at least one of 3'-O-Me-m7G, m 7 GpppG, m 2 7,3'-O GpppG, m 7 Gppp(5')N1 or m 7 Gppp(m 2'-O)N1 A sort of.
  • some or all of the uracils in the first mRNA or the second mRNA have been chemically modified to improve the stability of the first mRNA in vivo, and the chemical modification includes using N1- methylpseudouridine replaces at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the uracil in the first mRNA;
  • the first mRNA and the second mRNA are processed by RNase III, and the first mRNA and the second mRNA are purified by fast protein liquid chromatography.
  • the target protein comprises an antigenic polypeptide of SARS-CoV-2;
  • the target protein includes interleukin-2 and amino-free alpha-fetoprotein;
  • the target protein includes L1 protein of HPV6, L1 protein of HPV11, L1 protein of HPV16, L1 protein of HPV18 and E6 protein of HPV;
  • the target protein includes envelope glycoprotein E of HSV and envelope glycoprotein D of HSV;
  • the target protein comprises influenza virus HA antigen
  • the target protein comprises Gag antigen of HIV, EnV antigen of HIV and CD40L of HIV;
  • the target protein comprises the NL-S protein of African swine fever virus, the cd2v ep402r protein of African swine fever virus and the TK protein of African swine fever virus;
  • the target protein includes Taffazin protein
  • the target protein includes c-Myc protein, Klf4 protein, Sox2 protein, OCT4 protein and Lin28 protein;
  • the target protein includes Cas9 protein and DNAJC19 protein;
  • the target protein includes hydrolyzed GFP protein.
  • the second aspect of the present application provides a method for preparing a limited self-replicating mRNA molecular system, including:
  • the first mRNA encodes a mutant alphavirus replicase
  • the second mRNA encodes a target protein
  • the mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region.
  • the first mRNA and the second mRNA are purified by fast protein liquid chromatography.
  • said synthesizing the first mRNA includes:
  • mutant replicase DNA coding sequence includes the 5' untranslated region DNA sequence as shown in SEQ ID NO.9, the mutant replication as shown in SEQ ID NO.2 Enzyme coding sequence, 3' untranslated region DNA sequence as shown in SEQ ID NO.10;
  • the DNA synthesis template of the first mRNA is transcribed in vitro to synthesize the first mRNA.
  • said synthesizing the second mRNA includes:
  • the target protein DNA coding sequence of synthetic specificity modification comprises the 5' untranslated region DNA sequence as shown in SEQ ID NO.9, as shown in SEQ ID NO.7
  • the DNA synthesis template of the second mRNA is transcribed in vitro to synthesize the second mRNA.
  • the third aspect of the present application provides a biological material, the biological material is any one of A1) to A6):
  • A1 a nucleic acid molecule encoding the first mRNA
  • A2 a nucleic acid molecule encoding the second mRNA
  • A3 a recombinant vector containing the nucleic acid molecule of A1);
  • A4 a recombinant vector containing the nucleic acid molecule of A2)
  • a transgenic animal cell line containing the recombinant vector described in A4) A transgenic animal cell line containing the recombinant vector described in A4).
  • the fourth aspect of the present application provides a pharmaceutical composition, including at least one of the above-mentioned limited self-replicating mRNA molecular systems, and a delivery carrier.
  • the fifth aspect of the present application provides the use of the first mRNA encoding alphavirus mutant replicase in the preparation of an adjuvant for regulating the immune system, wherein the mutant replicase produces a mutation at position 259 of the nsP2 region and nsP2 Mutation at position 650 of the region.
  • the sixth aspect of the present application provides the use of the above-mentioned limited self-replicating mRNA molecular system or the above-mentioned biological material or the above-mentioned pharmaceutical composition in the preparation of cell reediting reagents, the use in the preparation of gene editing reagents, and the preparation of Barth syndrome
  • the limited self-replicating mRNA molecular system of the present application includes a first mRNA encoding alphavirus mutant replicase, and at least one second mRNA encoding a target protein, adjusted by specific mutations in the nsP2 subunit of mutant replicase, so that The limited self-replicating mRNA molecular system can realize limited self-replication and avoid cytotoxicity; by constructing different mRNAs with mutant replicase and different target proteins, the mutant replicase encoded by the first mRNA can simultaneously limitedly replicate multiple different The target protein can realize the continuous expression of multiple target proteins.
  • Fig. 1 is the cardiac ejection fraction effect diagram of the mouse Barth syndrome model treatment experiment of the present application
  • Fig. 2 is the staining diagram of the cardiac pathological evaluation of the mouse Barth syndrome model treatment experiment of the present application
  • Figure 3 is a diagram showing the functional half-life of the limited self-replicating mRNA molecular system of the present application and the results of cellular innate immune rejection;
  • Figure 4 is a graph showing the low cytotoxic effect of the limited self-replicating mRNA molecular system of the present application.
  • Figure 5 is a comparison chart of the application of the limited self-replicating mRNA molecular system of the present application to cell reprogramming;
  • Figure 6 is a staining result of the application of the limited self-replicating mRNA molecular system of the present application to cell reprogramming products;
  • Fig. 7 is a result diagram of the application of the limited self-replicating mRNA molecular system of the present application to gene editing;
  • Fig. 8 is a structural schematic diagram of the limited self-replicating mRNA molecular system of the present application.
  • Positive-strand RNA viral genomes are templates for translation and replication, resulting in multilevel interactions between host translation factors and RNA replication. All known positive-strand RNA viruses carry the gene for RNA-dependent RNA polymerase (RdRp) for genome replication. However, unlike other RNA viruses, positive-strand RNA viruses do not encapsidate the RNA polymerase. Thus, upon infection of a new cell, viral RNA replication does not begin until the genomic RNA is translated to produce RNA polymerase (and, for most positive-strand RNA viruses, replication factors). All characterized positive-strand RNA viruses assemble their RNA replication complexes to the inner cell membrane. Positive-strand RNA viruses produce negative-strand RNA, plus-strand RNA, double-stranded RNA (dsRNA), and subgenomic mRNA during replication, which themselves are potent inducers of the innate immune response pathway.
  • dsRNA double-stranded RNA
  • the positive-strand RNA viral genome has the same polarity as the cellular mRNA, and the positive-strand RNA viral genomic RNA can be directly translated using the cellular translation system.
  • nonstructural proteins are synthesized as precursor polyproteins and cleaved into mature nonstructural proteins by viral proteases.
  • a complex including RNA polymerase (RdRp), additional nonstructural proteins, viral RNA, and host cytokines is assembled.
  • the assembled replication complex (RC) carries out the synthesis of viral RNA.
  • RNA-dependent RNA polymerase or "RdRp” is an enzyme, protein or peptide that has enzymatic activity that catalyzes the de novo synthesis of RNA from an RNA template.
  • Replicase is a complex of viral polyproteins or polyprotein processing products that has RdRp activity and catalyzes the replication of specific viral RNAs.
  • RdRp and replicase are usually encoded by viruses with RNA genomes. Thus, replicase not only provides the function of an RNA-dependent RNA polymerase, but further includes additional viral nonstructural polyprotein subunits that provide functions other than RdRp activity.
  • Recombinant vector refers to a DNA- or RNA-based vector or plasmid that carries genetic information in the form of nucleic acid sequences.
  • vector vector
  • expression vector expression vector
  • An embodiment of the present application provides a limited self-replicating mRNA molecular system, including a first mRNA and at least one second mRNA, wherein the first mRNA encodes an alphavirus mutant replicase, and each second mRNA encodes a target protein , to achieve limited replication of at least one protein of interest by a mutant replicase.
  • the mutant replicase produces a mutation at position 259 of the nsP2 region (mutation of serine S to proline P) and a mutation at position 650 of the nsP2 region (mutation of arginine R to aspartic acid D).
  • the mutant replicase includes the nsP1 region (537 amino acids), the nsP2 region (799 amino acids), the nsP3 region (482 amino acids) and the nsP4 region (1254 amino acids) connected in sequence, and the mutant replicase
  • the amino acid sequence of the enzyme is shown in SEQ ID NO.1, and the two mutation points of the mutant replicase are respectively produced at position 796 (serine S is mutated into proline P) shown in SEQ ID NO.1 and at The 1187 position shown in SEQ ID NO.1 (arginine R is mutated into aspartic acid D).
  • multiple second mRNAs may be included, and the multiple second mRNAs respectively encode the first target protein, the second target protein, . . . , the Nth target protein.
  • the first mRNA includes a mutant replicase coding sequence
  • the mutant replicase coding sequence includes an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.2.
  • the nucleic acid sequence shown in SEQ ID NO.2 is a DNA sequence with high GC content. Under the premise of not changing the corresponding amino acid sequence, the codon with high GC content is selected, which is 7-20 times higher than the wild-type replicase DNA sequence.
  • positions 1-1611 correspond to the high GC content DNA sequence of the nsP1 region
  • positions 1612-4008 correspond to the high GC content DNA sequence of the nsP2 region
  • positions 4009-5454 correspond to the DNA sequence of the nsP3 region
  • High GC content DNA sequence, 5455-9216 position corresponds to the high GC content DNA sequence of nsP4 region.
  • the nucleic acid sequence shown in SEQ ID NO.11 is the replicase DNA sequence of wild-type alphavirus, wherein, as shown in SEQ ID NO.11, positions 1-1611 correspond to the original DNA sequence of the nsP1 region, and positions 1612-4008 correspond to The original DNA sequence of the nsP2 region, 4009-5454 corresponds to the original DNA sequence of the nsP3 region, and 5455-9216 corresponds to the original DNA sequence of the nsP4 region.
  • each of the second mRNAs includes a specific sequence at the 5' end of replicase, a target protein coding sequence, and a specific sequence at the 3' end of replicase, which are sequentially connected, and the two ends of the target protein coding sequence are To improve the translation level of the target protein coding sequence, the specific sequences recognized by the replicase are connected to the ends, and achieve the same effect without retaining the entire alphavirus RNA framework system.
  • the replicase 5 The 'end-specific sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.7, and the 5'-end specific sequence of the replicase is derived from the first to the 221st position of the original DNA sequence corresponding to the nsP1 region of the replicase;
  • the 3' end-specific sequence of the replicase comprises the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.8, and the 3'-end specific sequence of the replicase is derived from the penultimate second of the original DNA sequence corresponding to the nsP4 region of the replicase to the 985th from the bottom.
  • the limited replication of the mRNA combination in this embodiment completely removes the virus attribute, and completely eliminates the possibility of in vivo reproduction of viruses currently using alphavirus vectors.
  • the first mRNA and the second mRNA also include: a 5'cap structure, a 5'UTR sequence, a 3'UTR sequence and a polyadenylation sequence; wherein, the first mRNA follows the sequence of 5' ⁇ The 3'direction includes the following elements in sequence: 5'cap structure, 5'UTR sequence, mutant replicase coding sequence, 3'UTR sequence and polyadenylation sequence.
  • each second mRNA includes the following elements in sequence according to the 5' ⁇ 3' direction: 5'cap structure, 5'UTR sequence, replicase 5' end-specific sequence, target protein coding sequence, replicase 3' terminal specific sequence, 3'UTR sequence and polyadenylation sequence.
  • the target protein coding sequence is preferably the RNA sequence corresponding to the open reading frame (ORF) in the target protein coding gene
  • the 5'UTR sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.9
  • the The 3'UTR sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.10
  • the 5' cap structure is selected from 3'-O-Me-m7G, m 7 GpppG, m 2 7,3'-O At least one of GpppG, m 7 Gppp(5')N1 or m 7 Gppp(m 2'-O)N1, preferably 3'-O-Me-m7G.
  • the polyadenylic acid sequence is a sequence comprising 60-200 adenine acid; preferably, the polyadenylic acid sequence is a sequence comprising 120 adenine acid.
  • part or all of the uracil in the first mRNA or the second mRNA is chemically modified to improve the stability of the first mRNA in vivo, and the chemical modification includes using N1- Methylpseudouridine replaces at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the uracil in said first mRNA.
  • N1-methylpseudouridine is used to replace 100% of uracil in the first mRNA or the second mRNA, so as to reduce innate immune rejection and improve the efficiency of mRNA translation into protein.
  • the first mRNA and the second mRNA obtained by in vitro transcription of the recombinant vector are first treated with RNase III, and then purified by fast protein liquid chromatography, which can further improve the efficiency of mRNA translation into protein.
  • the target protein can be any acceptable protein or polypeptide, for example:
  • the limited self-replicating mRNA molecular system includes a second mRNA encoding an antigenic polypeptide of SARS-CoV-2, which can be selected from the receptor binding domain RBD of SARS-CoV-2, the spike of SARS-CoV-2
  • the spike protein S1 subunit or the full-length sequence of the spike protein S of SARS-CoV-2; the above-mentioned spike protein is derived from the delta mutant strain of SARS-CoV-2 or the original strain of SARS-CoV-2.
  • the limited self-replicating mRNA molecule system is an mRNA vaccine.
  • the limited self-replicating mRNA molecular system includes two second mRNAs, one encoding interleukin-2 and the other encoding amino-free alpha-fetoprotein.
  • the limited self-replicating mRNA molecular system includes five second mRNAs, encoding the L1 protein of HPV6, the L1 protein of HPV11, the L1 protein of HPV16, the L1 protein of HPV18 and the E6 protein of HPV.
  • the limited self-replicating mRNA molecular system includes two second mRNAs, encoding HSV envelope glycoprotein E and HSV envelope glycoprotein D, respectively.
  • the limited self-replicating mRNA molecular system includes a second mRNA encoding the influenza virus HA antigen.
  • the limited self-replicating mRNA molecular system includes three second mRNAs, encoding HIV Gag antigen, HIV EnV antigen and HIV CD40L respectively.
  • the limited self-replicating mRNA molecular system includes three second mRNAs, which encode the NL-S protein of African swine fever virus, the cd2v ep402r protein of African swine fever virus and the TK protein of African swine fever virus, respectively.
  • the limited self-replicating mRNA molecular system includes a second mRNA encoding proteins including Taffazin.
  • the limited self-replicating mRNA molecular system includes five second mRNAs, which encode c-Myc protein, Klf4 protein, Sox2 protein, OCT4 protein and Lin28 protein, respectively.
  • the limited self-replicating mRNA molecular system includes two second mRNAs, which encode Cas9 protein and DNAJC19 protein, respectively.
  • the limited self-replicating mRNA molecular system includes a second mRNA encoding the hydrolyzed GFP protein.
  • the embodiment of the present application also provides a biological material, the biological material comprising: (i) a nucleic acid molecule encoding the first mRNA; and (ii) a nucleic acid molecule encoding the second mRNA.
  • the nucleic acid molecule encoding the first mRNA comprises the nucleic acid sequence shown in SEQ ID NO.2
  • the nucleic acid molecule encoding the second mRNA comprises the nucleic acid sequence shown in SEQ ID NO.7, the target protein DNA coding sequence connected in sequence And the nucleic acid sequence as shown in SEQ ID NO.8.
  • the nucleic acid molecule encoding the first mRNA comprises a nucleic acid sequence as shown in SEQ ID NO.9, a nucleic acid sequence as shown in SEQ ID NO.2, and a nucleic acid sequence as shown in SEQ ID NO.10 which are sequentially connected Nucleic acid sequences and polyA sequences are shown.
  • the nucleic acid molecule encoding the second mRNA includes a nucleic acid sequence as shown in SEQ ID NO.9, a nucleic acid sequence as shown in SEQ ID NO.7, a target protein DNA coding sequence, and a nucleic acid sequence as shown in SEQ ID NO.8, which are sequentially connected. Nucleic acid sequence, nucleic acid sequence and polyadenylic acid sequence as shown in SEQ ID NO.10.
  • the embodiment of the present application also provides a biological material, which includes: a first recombinant vector containing a nucleic acid molecule encoding a first mRNA; and a second recombinant vector containing a nucleic acid molecule encoding a second mRNA.
  • the embodiment of the present application also provides a biological material, which includes: a transgenic animal cell line containing the first recombinant vector; and a transgenic animal cell line containing the second recombinant vector.
  • Step 1 Using GeneArtTM Gibson HiFi reaction (Thermo Fisher, USA, A46624) synthesizes the mutant replicase DNA coding sequence (the nucleic acid molecule encoding the first mRNA does not contain polyadenylation sequence), and clones the mutant replicase DNA coding sequence in pcDNA3 after successful synthesis .3 Vector plasmid for industrial production.
  • Mutant replicase DNA coding sequence 5' untranslated region DNA sequence (SEQ ID NO.9), mutant replicase coding sequence (SEQ ID NO.2), 3' untranslated region DNA sequence (SEQ ID NO. 10), wherein, the mutant replicase coding sequence (SEQ ID NO.2) is divided into nsP1 region fragment (SEQ ID NO.3), nsP2 region fragment (SEQ ID NO.4), nsP3 region fragment (SEQ ID NO. 5), nsP4 region fragment (SEQ ID NO.6) four DNA fragments, all four DNA fragments are modified fragments with high GC content. The four DNA fragments were directly ordered from IDT Company in the United States in the form of gblock.
  • Step 2 Adding the poly-(a) tail of the mRNA by PCR to obtain the DNA synthesis template of the first mRNA
  • the poly-(a) tail includes 120 adenine nucleotides.
  • the PCR product was recovered by cutting the gel (QIAquick PCR purification kit, Qiagen, cat.no.28106), and the final concentration of the tail template was adjusted to 100 ng/ ⁇ L, which was used as a DNA synthesis template for in vitro transcription and synthesis of the first mRNA.
  • Step 3 in vitro transcription and synthesis of the first mRNA
  • the expected total yield should be ⁇ 50ug (30-70ug range; 100 ⁇ L elution volume for a 40 ⁇ L IVT reaction is 300-700ng ⁇ L). Adjust the concentration to 100 ng/ ⁇ L by adding elution buffer or TE buffer (pH 7.0), or purify by FPLC.
  • Embodiment 2 the synthesis of the second mRNA
  • the synthesis steps of the second mRNA are similar to the first mRNA, including the following steps:
  • Step 1 Using GeneArtTM Gibson HiFi reaction (Thermo Fisher, USA, A46624) synthesizes specifically modified target protein DNA coding sequence (the nucleic acid molecule encoding the second mRNA does not contain polyadenylation sequence);
  • target protein DNA coding sequence 5' untranslated region DNA sequence (SEQ ID NO.9), replicase 5' specific DNA sequence (SEQ ID NO.7), target protein DNA coding sequence ( Please refer to Table 6), replicase 3' specific DNA sequence (SEQ ID NO.8), 3' untranslated region DNA sequence (SEQ ID NO.10).
  • Step 2 adding the poly-(a) tail of the mRNA to the specifically modified target protein DNA coding sequence by PCR to obtain the DNA synthesis template of the second mRNA;
  • Step 3 in vitro transcription and synthesis of the second mRNA.
  • the 26 kinds of second mRNAs shown in Table 6 were respectively synthesized according to the above method.
  • SEQ ID NO.14 to SEQ ID NO.39 and SEQ ID NO.47 have all undergone high GC modification on the basis of the corresponding original sequence without changing the original amino acid sequence.
  • This embodiment provides a pharmaceutical composition, a multiple molecular messenger RNA and a delivery carrier, wherein the multiple molecular messenger RNA includes the first mRNA prepared in Example 1 and the second mRNA-1 prepared in Example 2, and the delivery carrier is protamine protein.
  • the target protein is Taffazin protein.
  • mice Only males were used in this case, and doxycycline was placed in the drinking water of mice at a concentration of 2 mg/L, which also contained 10% sucrose.
  • Dilute 10 ⁇ L of protamine (Protamine Ipex5000 of MEDA Pharmaceutical Company) 5000IU/ml in 280 ⁇ L of water, press 280 ⁇ L+10 ⁇ L of protamine 5000, prepare 0.5 mg/ml of protamine solution, multiple molecular messenger RNA (multiple molecular molar ratio 1:1 solution) 0.5mg/ml, add an equivalent amount of protamine solution to the RNA solution, and quickly wash up and down at least 10 times, and place it at room temperature for 10 minutes to make 130nm protamine-RNA nanoparticles. And placed in the mouse subcutaneous pump (ALZET pump, https://www.alzet.com/guide-to-use/scid/) for continuous administration.
  • AZET pump https://www.alzet.com/guide-to-use/scid/
  • mice The Barth syndrome mice (TG) mice were divided into 6 groups: TG1, TG2, TG3, TG4, TG5, TG6;
  • Step 1 Induce TG1, TG2, TG3, TG4, TG5, and TG6 with doxycycline for 8 weeks, and detect cardiac ejection fraction FS%;
  • Step 2 Induce TG1, TG2, TG3, TG4, TG5, and TG6 with doxycycline for 10 weeks (continue to induce on the basis of step 1), and detect cardiac ejection fraction FS%;
  • Step 3 After TG1, TG2, TG3, and TG4 were treated with the pharmaceutical composition of Example 3 for 2 weeks, TG5, TG6 were not treated, and cardiac ejection fraction FS% was detected;
  • Step 4 After TG1, TG2, TG3, and TG4 were treated with the pharmaceutical composition of Example 3 for 3 weeks (continuing the treatment for 1 week on the basis of step 3), TG5, TG6 were not treated, and the cardiac ejection fraction FS% was detected ;
  • Step 5 After TG1, TG2, TG3, and TG4 were treated with the pharmaceutical composition of Example 3 for 6 weeks (continuing the treatment for 1 week on the basis of Step 4), the exercise capacity of the mice was detected.
  • mice 3.3 Evaluation of forced exercise ability of mice:
  • mice are performed on an enclosed motorized treadmill with adjustable speed and inclination, and equipped with an electric shock delivery grid with an electric shock intensity of 1 mA. Animals were acclimatized with an initial 30 min rest on the treadmill, and the test was started with a 10% incline and a speed of 5 m/min. Gradually increase by 5 m/min every 5 minutes to a final speed of 25 m/min.
  • Group A Wild-type mice were induced with doxycycline for 8 weeks;
  • Group B Barth syndrome mice (TG) were induced with doxycycline for 8 weeks, and treated with a common pharmaceutical composition (common messenger RNA system + delivery system) for 6 weeks, wherein the common messenger RNA system encodes Taffazin protein; among them, common messenger RNA
  • TG Barth syndrome mice
  • a common pharmaceutical composition common messenger RNA system + delivery system
  • the common messenger RNA system encodes Taffazin protein; among them, common messenger RNA
  • the system is prepared according to the method described in the prior art CN201910014953.6.
  • Group C Barth syndrome mice (TG) were induced with doxycycline for 8 weeks, and treated with the pharmaceutical composition of Example 3 for 6 weeks;
  • Groups A, B and C were evaluated pathologically with Sirius red staining for cardiac fibrosis.
  • the limited replication multiple molecular messenger RNA system encoding Taffazin protein treatment improves the cardiac function of mice with congenital cardiomyopathy Barth syndrome, specifically, Barth syndrome mice (TG) have lost the function of Taffazin protein induced by doxycycline , the symptoms of Barth syndrome appeared cardiomyopathy disease phenotype, cardiac function index - ejection fraction decreased, no treatment TG5, TG6 cardiac function decreased, compared with multiple molecular messenger RNA treatment for 2 weeks (TG1, 2, 3, 4), after 2 to 3 weeks of treatment, the heart function improved.
  • TG Barth syndrome mice
  • This example provides a limited self-replicating mRNA molecular system, including the first mRNA prepared in Example 1 and the second mRNA-2 prepared in Example 2, and the target protein is hydrolyzed GFP protein.
  • Example 4 transfection with common mRNA encoding hydrolyzed GFP protein (first group), bimolecular mRNA (second group) of Example 4 and full-length self-replicating mRNA encoding hydrolyzed GFP (third group)
  • first group common mRNA encoding hydrolyzed GFP protein
  • second group bimolecular mRNA
  • third group full-length self-replicating mRNA encoding hydrolyzed GFP
  • the following expression reporter gene hydrolyzes GFP (expressed GFP will be rapidly degraded by its own hydrolase, which can instantly reflect the duration and expression level of multiple messenger RNA molecules).
  • Example 4 The limited self-replicating mRNA molecular system transfection steps of Example 4 are as follows:
  • the half-life of mRNA in the first group of cells By detecting the fluorescence intensity of GFP protein, the half-life of mRNA in the first group of cells, the half-life of mRNA in the second group of cells and the half-life of mRNA in the third group of cells were respectively detected, and the cell congenital immune response.
  • the limited self-replicating mRNA molecular system of Example 4 encodes reporter gene hydrolyzed GFP has no difference in half-life compared with full-chain self-replicating messenger RNA, but has weak cytotoxicity, less immunogenicity, and longer half-life than ordinary messenger RNA .
  • the limited self-replicating mRNA molecular system of Example 4 has a longer functional half-life and low cellular innate immune rejection, and the half-life of the limited self-replicating mRNA molecular system of Example 4 is significantly higher than that of ordinary messengers RNA, similar to full-length self-replicating messenger RNA, but the cellular innate immune response (INFA, interferon A) was significantly lower than that of full-length self-replicating messenger RNA.
  • IFA cellular innate immune response
  • the limited self-replicating mRNA molecular system of embodiment 4 (limited replication multiple messenger RNA molecular system) has low cytotoxic effect, and the cytotoxicity of the messenger RNA produced by the limited self-replicating mRNA molecular system of embodiment 4 is the same as Common messenger RNA is similar, but significantly lower than that of full-length self-replicating messenger RNA.
  • This example provides a limited self-replicating mRNA molecular system, including the first mRNA prepared in Example 1 and the second mRNA-22, the second mRNA-23, the second mRNA-24, the second mRNA- 25.
  • the second mRNA-26, the target proteins are c-Myc protein, Klf4 protein, Sox2 protein, OCT4 protein and Lin28 protein respectively.
  • NuFF feeder cells Newborn human foreskin fibroblasts (GlobalStem, cat. no. GSC-3001G), thaw a bottle of mitotically inactivated NuFFs and seed the cells on gelatin cell plates.
  • the limited replication multiple messenger RNA molecular system simultaneously amplifies 5 encoding cell reprogramming factors Otc4, Sox2, Klf4, c-Myc, Lin28 (OSKML) to complete cell reprogramming efficiently; compared with ordinary messenger
  • the RNA system has longer protein expression and higher cell reprogramming (the number of iPS clones is an indicator); the cell reprogramming product produced by the limited self-replicating mRNA molecular system (limited replication multiple messenger RNA molecular system) of Example 5- iPS cells show typical pluripotency; the limited self-replicating mRNA molecular system (limited replication multiple messenger RNA molecular system) of Example 5 encodes 5 reprogramming factors OSKML to complete the product after cell reprogramming-iPS cells show the appearance of classic pluripotent stem cell clones, multiple Positive staining of potential marker Oct4 can form teratomas in vivo.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • This embodiment provides a limited self-replicating mRNA molecular system, including the first mRNA prepared in Example 1 and the second mRNA-3 prepared in Example 2, and the target protein is Cas9 protein.
  • the limited self-replicating mRNA molecular system (multiple molecular messenger RNA) of Example 6 is used for DNAJC19 gene editing or Taffazin gene editing in human induced stem cells (Induced Pluripotent Stem Cells).
  • Electrotransfection of human induced stem cells Assemble the gene editing reaction system as shown in Table 7, Taffazin gene gRNA sequence (SEQ ID NO.40, directly ordered from IDT Company), DNAJC19 gRNA sequence (SEQ ID NO.17, directly ordered from IDT Corporation).
  • DNAJC19 gene fragment was amplified by PCR.
  • the limited self-replicating mRNA molecular system (limited replication multiple messenger RNA molecular system) of Example 6 encodes the CRISPR protein Cas9, which efficiently edits DNAJC19 and human Taffazin genes.
  • the DNAJC19 gene was successfully gene-edited to generate a gene mutation, and was identified and cut by Surveyor. Three typical bands appeared, indicating that the efficient gene editing was completed.
  • the Taffazin gene was successfully gene-edited The gene mutation generated by editing was recognized and cut by Surveyor, and three typical bands appeared, indicating that the efficient gene editing was completed.
  • Embodiment 7 is a diagrammatic representation of Embodiment 7:
  • This embodiment provides an mRNA vaccine, comprising the first mRNA of embodiment 1, the second mRNA-5 of embodiment 2, and protamine, which are delivered in the form of 130nm protamine RNA particles.
  • the target protein is the antigenic polypeptide of SARS-CoV-2 (wild-type spike protein S).
  • This embodiment also provides an mRNA vaccine, comprising the first mRNA of embodiment 1, the second mRNA-28 of embodiment 2, and protamine, formed into 130nm protamine RNA particles for delivery.
  • the target protein is the antigenic polypeptide of SARS-CoV-2 (the spike protein S of the Delta strain).
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • This embodiment provides an mRNA vaccine, comprising the first mRNA of Example 1, the second mRNA-8, the second mRNA-9, the second mRNA-10, the second mRNA-11, the second mRNA- 12 and protamine, forming 130 nm protamine RNA particles for delivery.
  • the target protein is L1 protein of HPV6, L1 protein of HPV11, L1 protein of HPV16, L1 protein of HPV18 and E6 protein of HPV.
  • Embodiment 9 is a diagrammatic representation of Embodiment 9:
  • This embodiment provides an mRNA vaccine, comprising the first mRNA of Example 1, the second mRNA-13, the second mRNA-14 of Example 2, and protamine, which are delivered in the form of 130nm protamine RNA particles.
  • the target proteins are envelope glycoprotein E of HSV and envelope glycoprotein D of HSV.
  • This embodiment provides an mRNA vaccine, comprising the first mRNA of embodiment 1, the second mRNA-15 of embodiment 2, and protamine, which are delivered in the form of 130nm protamine RNA particles.
  • the target protein is influenza virus HA antigen.
  • the present embodiment provides a kind of mRNA vaccine, comprises the first mRNA of embodiment 1, the second mRNA-16 of embodiment 2, the second mRNA-17, the second mRNA-18 and protamine, forms 130 nanometer protamine RNA particles for delivery.
  • the target proteins are Gag antigen of HIV, EnV antigen of HIV and CD40L of HIV.
  • the present embodiment provides a kind of mRNA vaccine, comprises the first mRNA of embodiment 1, the second mRNA-19 of embodiment 2, the second mRNA-20, the second mRNA-21 and protamine, form 130 nanometer protamine RNA particles for delivery.
  • the target proteins are NL-S protein of African swine fever virus, cd2v ep402r protein of African swine fever virus and TK protein of African swine fever virus.
  • This embodiment provides a pharmaceutical composition for the treatment of colon cancer, comprising the first mRNA of Example 1, the second mRNA-6, the second mRNA-7 of Example 2, and protamine to form 130 nanometer protamine protein RNA particles for delivery.
  • the target proteins are interleukin-2 and alpha-fetoprotein without amino groups.
  • This embodiment provides an mRNA vaccine, comprising the first mRNA of embodiment 1, the second mRNA-27 of embodiment 2 and protamine, which are delivered by forming 130nm protamine RNA particles.
  • the target protein is a rabies antigen (rabies glycoprotein).

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Abstract

一种有限自我复制mRNA分子系统、制备方法及其应用，所述有限自我复制mRNA分子系统包括编码甲病毒属突变型复制酶的第一mRNA，以及至少一个编码目标蛋白的第二mRNA，通过在突变型复制酶的nsP2亚单元产生特异突变调整，使该有限自我复制mRNA分子系统能够实现有限自我复制，避免产生细胞毒性；通过将突变型复制酶及不同的目标蛋白构建不同的mRNA，第一mRNA编码的突变型复制酶能够同时有限复制多个不同的目标蛋白，实现多重目标蛋白的持续表达。

Description

一种有限自我复制mRNA分子系统、制备方法及应用 【技术领域】
本申请涉及生物医药技术领域，尤其涉及一种有限自我复制mRNA分子系统、制备方法及应用。
【背景技术】
信使RNA(mRNA)疗法是一种全新的治疗方式，具有广泛的临床应用潜力，包括针对传染源的疫苗以及针对癌症或遗传疾病的治疗、再生疗法和免疫疗法。与基于蛋白质的生物制剂相比，信使RNA疗法的优势包括信使RNA可以通过机体自身的细胞合成蛋白，不需要复杂的蛋白质合成及纯化工艺或生产线；可以将细胞内和膜结合蛋白作为治疗靶点；可以快速无细胞GMP条件下工业化生产，研发到产品的周期短等。
但是信使RNA疗法的应用受到结构不稳定、先天免疫原性、体内递送低效等因素的限制，该技术发展的方向在于：第一，它必须避免先天免疫系统排斥，先天免疫系统会将治疗性信使RNA误认为非自身核酸，从而产生排斥，这对于信使RNA治疗药物的重复给药尤其重要，因为免疫记忆可能会限制药物产品的有效性。目前有研究认为通过化学修饰信使RNA的核苷酸碱基可以降低先天免疫排斥，从而提高信使RNA的翻译为蛋白的效率，但是如何进行核苷修饰，修饰的比例和如何进行核苷酸修饰组合尚不清楚。第二，普通信使RNA不稳定，容易降解且表达持续的时间不长，有研究表明普通信使RNA在细胞中仅能表达24小时。自我复制的信使RNA，因其可以自我复制，可以放大信使RNA的蛋白翻译指令，可以增强和延长信使RNA蛋白的表达。现有技术中采用的自我复制信使RNA分子系统源自甲病毒的基因组骨架，其中编码病毒RNA复制酶部分骨架是完整的，编码病毒的结构蛋白骨架被取代为编码目标蛋白序列。该信使RNA分子系统有如下缺陷：首先，相对于非自我复制的信使RNA，自我复制信 使RNA核苷酸序列要长很多，细胞负担重，体外转录合成信使RNA有技术难度，工业化生产成本高；其次，自我复制信使RNA分子系统实质为能够自我复制的RNA假病毒，其病毒属性明显，例如无法预测其复制的次数，存在无限复制的可能(体内假病毒繁殖)，例如，水泡性口炎病毒抗原和狂犬病病毒抗原被包装为上述的自我复制RNA时存在从而放大其毒性的可能；第三，上述的信使RNA分子系统的细胞毒性大，而且由于无法对其进行核苷修饰而导致细胞或者机体的免疫反应大大超过非自我复制信使RNA。
因此，有必要提供一种有限自我复制的信使RNA分子系统。
【发明内容】
本申请的目的在于提供一种有限自我复制mRNA分子系统、制备方法及应用，以解决现有技术中mRNA无法实现有限自我复制的技术问题。
本申请第一方面提供了一种有限自我复制mRNA分子系统，包括：
编码甲病毒属突变型复制酶的第一mRNA；以及
至少一个编码目标蛋白的第二mRNA；
其中，所述突变型复制酶产生nsP2区域的第259位的突变以及nsP2区域的第650位的突变。
可选地，所述突变型复制酶包括依次连接的nsP1区域、nsP2区域、nsP3区域以及nsP4区域，所述突变型复制酶的氨基酸序列如SEQ ID NO.1所示，所述突变型复制酶产生在SEQ ID NO.1所示的796位点的丝氨酸S突变为脯氨酸P以及在SEQ ID NO.1所示的1187位点的精氨酸R突变为天冬氨酸D。
可选地，所述第一mRNA包括突变型复制酶编码序列，所述突变型复制酶编码序列包括如SEQ ID NO.2所示的核酸序列对应的RNA序列；
每个所述第二mRNA包括依次连接的复制酶5’端特异性序列、目标蛋白编码序列以及复制酶3’端特异性序列，所述复制酶5’端特异性序列包括如SEQ ID NO.7所示的核酸序列对应的RNA序列，所述复制酶3’端特异性序列包括如SEQ ID NO.8所示的核酸序列对应的RNA序列。
可选地，所述第一mRNA和所述第二mRNA还包括：5’帽结构、5’UTR序列、3’UTR序列以及多聚腺苷酸序列；
其中，所述第一mRNA按照5’→3’方向依次包括如下元件：5’帽结构、5’UTR序列、突变型复制酶编码序列、3’UTR序列和多聚腺苷酸序列；
每个所述第二mRNA按照5’→3’方向依次包括如下元件：5’帽结构、5’UTR序列、复制酶5’端特异性序列、目标蛋白编码序列、复制酶3’端特异性序列、3’UTR序列和多聚腺苷酸序列；
所述5’UTR序列包括如SEQ ID NO.9所示的核酸序列对应的RNA序列，所述3’UTR序列包括如SEQ ID NO.10所示的核酸序列对应的RNA序列，所述5’帽结构选自3′-O-Me-m7G、m 7 GpppG、m 2 7,3′-O GpppG、m 7 Gppp(5')N1或m 7 Gppp(m 2′-O)N1中的至少一种。
可选地，所述第一mRNA或所述第二mRNA中部分或全部的尿嘧啶进行了能够提高所述第一mRNA在生物体内稳定性的化学改性，所述化学改性包括利用N1-甲基假尿苷置换所述第一mRNA中的至少50％、至少60％、至少70％、至少80％、至少90％或100％的尿嘧啶；
或，所述第一mRNA和所述第二mRNA被RNase III处理，所述第一mRNA和所述第二mRNA经快速蛋白质液相色谱提纯。
可选地，所述目标蛋白包括SARS-CoV-2的抗原性多肽；
或，所述目标蛋白包括白细胞介素-2和不含氨基的甲胎蛋白；
或，所述目标蛋白包括HPV6的L1蛋白、HPV11的L1蛋白、HPV16的L1蛋白、HPV18的L1蛋白和HPV的E6蛋白；
或，所述目标蛋白包括HSV的包膜糖蛋白E和HSV的包膜糖蛋白D；
或，所述目标蛋白包括流感病毒HA抗原；
或，所述目标蛋白包括HIV的Gag抗原、HIV的EnV抗原和HIV的CD40L；
或，所述目标蛋白包括非洲猪瘟病毒的NL-S蛋白、非洲猪瘟病毒的cd2v ep402r蛋白和非洲猪瘟病毒的TK蛋白；
或，所述目标蛋白包括Taffazin蛋白；
或，所述目标蛋白包括c-Myc蛋白、Klf4蛋白、Sox2蛋白、OCT4蛋白和Lin28蛋白；
或，所述目标蛋白包括Cas9蛋白和DNAJC19蛋白；
或，所述目标蛋白包括水解GFP蛋白。
本申请第二方面提供了一种有限自我复制mRNA分子系统的制备方法，包括：
合成第一mRNA；
合成至少一个第二mRNA；
其中，第一mRNA编码甲病毒属突变型复制酶，第二mRNA编码目标蛋白，所述突变型复制酶产生nsP2区域的第259位的突变以及nsP2区域的第650位的突变。
可选地，还包括：
利用RNase III对所述第一mRNA和所述第二mRNA进行处理；
利用快速蛋白质液相色谱对所述第一mRNA和所述第二mRNA进行提纯。
可选地，所述合成第一mRNA，包括：
合成突变型复制酶DNA编码序列，其中，所述突变型复制酶DNA编码序列包括如SEQ ID NO.9所示的5’非翻译区DNA序列、如SEQ ID NO.2所示的突变型复制酶编码序列、如SEQ ID NO.10所示的3’非翻译区DNA序列；
通过PCR在所述突变型复制酶DNA编码序列上添加mRNA的poly-(a)尾巴得到第一mRNA的DNA合成模版；
将所述第一mRNA的DNA合成模版进行体外转录合成第一mRNA。
可选地，所述合成第二mRNA，包括：
合成特异性修饰的目标蛋白DNA编码序列，其中，所述特异性修饰的目标蛋白DNA编码序列包括如SEQ ID NO.9所示的5’非翻译区DNA序列、如SEQ ID NO.7所示的复制酶5’端特异性DNA序列、目标蛋白DNA编码序列、如SEQ ID NO.8所示的复制酶3’端特异性DNA序列、如SEQ  ID NO.10所示的3’非翻译区DNA序列；
通过PCR在所述特异性修饰的目标蛋白DNA编码序列上添加mRNA的poly-(a)尾巴得到第二mRNA的DNA合成模版；
将所述第二mRNA的DNA合成模版进行体外转录合成第二mRNA。
本申请第三方面提供了一种生物材料，所述生物材料为A1)至A6)中的任一种：
A1)编码所述第一mRNA的核酸分子；
A2)编码所述第二mRNA的核酸分子；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述核酸分子的重组载体；
A5)含有A3)所述重组载体以及的转基因动物细胞系；
A6)含有A4)所述重组载体的转基因动物细胞系。
本申请第四方面提供了一种药物组合物，包括上述的有限自我复制mRNA分子系统中的至少一种，以及递送载体。
本申请第五方面提供了上述编码甲病毒属突变型复制酶的第一mRNA在制备调节免疫系统的佐剂的用途，其中，所述突变型复制酶产生nsP2区域的第259位的突变以及nsP2区域的第650位的突变。
本申请第六方面提供了上述的有限自我复制mRNA分子系统或上述的生物材料或上述的药物组合物在制备细胞重编辑试剂中的用途、在制备基因编辑试剂中的用途、在制备Barth综合征治疗药物中的用途、在制备感染性疾病疫苗中的用途或在制备肿瘤疫苗中的用途。
本申请的有限自我复制mRNA分子系统包括编码甲病毒属突变型复制酶的第一mRNA，以及至少一个编码目标蛋白的第二mRNA，通过在突变型复制酶的nsP2亚单元产生特异突变调整，使该有限自我复制mRNA分子系统能够实现有限自我复制，避免产生细胞毒性；通过将突变型复制酶及不同的目标蛋白构建不同的mRNA，第一mRNA编码的突变型复制酶能够同时有限复制多个不同的目标蛋白，实现多重目标蛋白的持续表达。
【附图说明】
图1为本申请小鼠Barth综合症模型治疗实验的心脏射血份数效果图；
图2为本申请小鼠Barth综合症模型治疗实验的心脏病理评价染色图；
图3为本申请有限自我复制mRNA分子系统功能半衰期及细胞先天免疫排斥结果图；
图4为本申请有限自我复制mRNA分子系统低细胞毒性效应结果图；
图5为本申请有限自我复制mRNA分子系统应用于细胞重编程结果对比图；
图6为本申请有限自我复制mRNA分子系统应用于细胞重编程产物染色结果图；
图7为本申请有限自我复制mRNA分子系统应用于基因编辑的结果图；
图8为本申请有限自我复制mRNA分子系统的结构原理图。
【具体实施方式】
下面将结合本申请实施例中的附图，对发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本申请的一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
在本文中提及“实施例”意味着，结合实施例描述的特定特征、结构或特性可以包含在本申请的至少一个实施例中。在说明书中的各个位置出现该短语并不一定均是指相同的实施例，也不是与其它实施例互斥的独立的或备选的实施例。本领域技术人员显式地和隐式地理解的是，本文所描述的实施例可以与其它实施例相结合。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
正链RNA病毒基因组是翻译和复制的模板，其导致宿主翻译因子(host translation factor)与RNA复制之间在多水平相互作用。所有已知的正链 RNA病毒均携带用于基因组复制的RNA依赖性RNA聚合酶(RdRp)的基因。然而，不同于其它RNA病毒，正链RNA病毒不将该RNA聚合酶壳体化。因此，在新细胞感染时，直至基因组RNA进行翻译以产生RNA聚合酶(对于大部分正链RNA病毒而言还有复制因子)时，才开始病毒RNA复制。所有特征化的正链RNA病毒均将其RNA复制复合体装配到细胞内膜上。正链RNA病毒在复制过程中产生负链RNA、正链RNA、双链RNA(dsRNA)和亚基因组mRNA，这些物质自身即是先天性免疫应答途径的强诱导剂。
正链RNA病毒基因组具有与细胞mRNA相同的极性，并且正链RNA病毒基因组RNA能够利用细胞翻译体系进行直接翻译。首先，合成非结构蛋白作为前体多蛋白，并通过病毒蛋白酶将其剪切成成熟的非结构蛋白。然后，在翻译和多蛋白处理后，组装包括RNA聚合酶(RdRp)、附加的非结构蛋白、病毒RNA和宿主细胞因子的复合体。组装形成的复制复合体(RC)进行病毒RNA的合成。
“RNA依赖型RNA聚合酶”或“RdRp”为一种具有催化由RNA模板从头合成RNA的酶活性的酶、蛋白质或肽。复制酶是一种具有RdRp活性，并催化特定病毒RNA复制的病毒多蛋白或多蛋白加工产物的复合体。RdRp和复制酶通常由具有RNA基因组的病毒编码。因此，复制酶不但提供RNA依赖型RNA聚合酶的功能，而且还进一步包括提供除RdRp活性以外的其它功能的额外的病毒非结构性多蛋白亚单元。
“重组载体”是指以核酸序列形式携带基因信息的基于DNA或RNA的载体或质粒。术语“质粒”、“载体”、“重组载体”和/或“表达载体”在本文中可互用。
本申请实施例提供了一种有限自我复制mRNA分子系统，包括一个第一mRNA以及至少一个第二mRNA，其中，第一mRNA编码甲病毒属突变型复制酶，每个第二mRNA编码一个目标蛋白，通过突变型复制酶实现至少一个目标蛋白的有限复制。
其中，所述突变型复制酶产生nsP2区域的第259位的突变(丝氨酸S 突变为脯氨酸P)以及nsP2区域的第650位的突变(精氨酸R突变为天冬氨酸D)。具体地，所述突变型复制酶包括依次连接的nsP1区域(537个氨基酸)、nsP2区域(799个氨基酸)、nsP3区域(482个氨基酸)以及nsP4区域(1254个氨基酸)，所述突变型复制酶的氨基酸序列如SEQ ID NO.1所示，所述突变型复制酶的两个突变点分别产生在SEQ ID NO.1所示的796位点(丝氨酸S突变为脯氨酸P)以及在SEQ ID NO.1所示的1187位点(精氨酸R突变为天冬氨酸D)。
在本实施例中，请参阅图8所示，可以包括多个第二mRNA，多个第二mRNA分别编码第一个目标蛋白、第二个目标蛋白，…，第N个目标蛋白。
在一个可选的实施方式中，所述第一mRNA包括突变型复制酶编码序列，所述突变型复制酶编码序列包括如SEQ ID NO.2所示的核酸序列对应的RNA序列。如SEQ ID NO.2所示的核酸序列为高GC含量的DNA序列，在不改变对应氨基酸序列的前提下，选用GC含量高的密码子，比野生型复制酶DNA序列GC含量高7～20％，具体地，如SEQ ID NO.2所示的1-1611位对应nsP1区域的高GC含量DNA序列、1612-4008位对应nsP2区域的高GC含量DNA序列、4009-5454位对应nsP3区域的高GC含量DNA序列、5455-9216位对应nsP4区域的高GC含量DNA序列。如SEQ ID NO.11所示的核酸序列为野生型甲病毒的复制酶DNA序列，其中，如SEQ ID NO.11所示的1-1611位对应nsP1区域的原始DNA序列、1612-4008位对应nsP2区域的原始DNA序列、4009-5454位对应nsP3区域的原始DNA序列、5455-9216位对应nsP4区域的原始DNA序列。
在一个可选的实施方式中，每个所述第二mRNA包括依次连接的复制酶5’端特异性序列、目标蛋白编码序列以及复制酶3’端特异性序列，在目标蛋白编码序列的两端分别连接了复制酶识别的特异性序列，以提高目标蛋白编码序列的翻译水平，在不保留整个甲病毒RNA框架体系的前提下，实现与之同样的效果，具体地，所述复制酶5’端特异性序列包括如SEQ ID NO.7所示的核酸序列对应的RNA序列，该复制酶5’端特异性序列来源于 复制酶nsP1区域对应原始DNA序列的第1位至第221位；所述复制酶3’端特异性序列包括如SEQ ID NO.8所示的核酸序列对应的RNA序列，该复制酶3’端特异性序列来源于复制酶nsP4区域对应原始DNA序列的倒数第2位至倒数第985位。本实施例的mRNA组合有限复制，彻底去除病毒属性，彻底杜绝目前使用甲病毒载体的病毒体内繁殖可能。
进一步地，所述第一mRNA和所述第二mRNA还包括：5’帽结构、5’UTR序列、3’UTR序列以及多聚腺苷酸序列；其中，所述第一mRNA按照5’→3’方向依次包括如下元件：5’帽结构、5’UTR序列、突变型复制酶编码序列、3’UTR序列和多聚腺苷酸序列。同样地，每个所述第二mRNA按照5’→3’方向依次包括如下元件：5’帽结构、5’UTR序列、复制酶5’端特异性序列、目标蛋白编码序列、复制酶3’端特异性序列、3’UTR序列和多聚腺苷酸序列。具体地，目标蛋白编码序列优选为目标蛋白编码基因中开放阅读框(ORF)对应的RNA序列，所述5’UTR序列包括如SEQ ID NO.9所示的核酸序列对应的RNA序列，所述3’UTR序列包括如SEQ ID NO.10所示的核酸序列对应的RNA序列，所述5’帽结构选自3′-O-Me-m7G、m 7 GpppG、m 2 7,3′-O GpppG、m 7 Gppp(5')N1或m 7 Gppp(m 2′-O)N1中的至少一种，优选为3′-O-Me-m7G。多聚腺苷酸序列为包含60～200个腺苷酸的序列；优选地，多聚腺苷酸序列为包含120个腺苷酸的序列。
在本实施例中，所述第一mRNA或第二mRNA中部分或全部的尿嘧啶进行了能够提高所述第一mRNA在生物体内稳定性的化学改性，所述化学改性包括利用N1-甲基假尿苷置换所述第一mRNA中的至少50％、至少60％、至少70％、至少80％、至少90％或100％的尿嘧啶。进一步地，在本实施例中，利用N1-甲基假尿苷置换所述第一mRNA或第二mRNA中100％的尿嘧啶，降低先天免疫排斥，提高mRNA翻译为蛋白的效率。
在本实施例中，经重组载体体外转录所得第一mRNA和第二mRNA首先经过RNase III处理，随后经快速蛋白质液相色谱提纯，能够进一步提高mRNA翻译为蛋白的效率。
在本实施例中，理论上目标蛋白可以为任意可接受的蛋白或多肽，例 如：
有限自我复制mRNA分子系统包括一个第二mRNA，编码SARS-CoV-2的抗原性多肽，该抗原性多肽可以选自SARS-CoV-2的受体结合结构域RBD，SARS-CoV-2的刺突蛋白S1亚基或SARS-CoV-2的刺突蛋白S全长序列；上述刺突蛋白来源于SARS-CoV-2德尔塔突变株或SARS-CoV-2原始株。此时，该有限自我复制mRNA分子系统为mRNA疫苗。
有限自我复制mRNA分子系统包括两个第二mRNA，其中一个编码白细胞介素-2，另一个编码不含氨基的甲胎蛋白。
有限自我复制mRNA分子系统包括五个第二mRNA，分别编码HPV6的L1蛋白、HPV11的L1蛋白、HPV16的L1蛋白、HPV18的L1蛋白和HPV的E6蛋白。
有限自我复制mRNA分子系统包括两个第二mRNA，分别编码HSV的包膜糖蛋白E和HSV的包膜糖蛋白D。
有限自我复制mRNA分子系统包括一个第二mRNA，编码流感病毒HA抗原。
有限自我复制mRNA分子系统包括三个第二mRNA，分别编码HIV的Gag抗原、HIV的EnV抗原和HIV的CD40L。
有限自我复制mRNA分子系统包括三个第二mRNA，分别编码非洲猪瘟病毒的NL-S蛋白、非洲猪瘟病毒的cd2v ep402r蛋白和非洲猪瘟病毒的TK蛋白。
有限自我复制mRNA分子系统包括一个第二mRNA，编码包括Taffazin蛋白。
有限自我复制mRNA分子系统包括五个第二mRNA，分别编码c-Myc蛋白、Klf4蛋白、Sox2蛋白、OCT4蛋白和Lin28蛋白。
有限自我复制mRNA分子系统包括两个第二mRNA，分别编码Cas9蛋白和DNAJC19蛋白。
有限自我复制mRNA分子系统包括一个第二mRNA，编码水解GFP蛋白。
本申请实施例还提供了一种生物材料，所述生物材料包括：(i)编码所述第一mRNA的核酸分子；以及(ii)编码所述第二mRNA的核酸分子。
其中，编码第一mRNA的核酸分子包括如SEQ ID NO.2所示的核酸序列，编码第二mRNA的核酸分子包括依次连接的如SEQ ID NO.7所示的核酸序列、目标蛋白DNA编码序列以及如SEQ ID NO.8所示的核酸序列。
在一个可选的实施方式中，编码第一mRNA的核酸分子包括依次连接的如SEQ ID NO.9所示的核酸序列、如SEQ ID NO.2所示的核酸序列、如SEQ ID NO.10所示的核酸序列以及多聚腺苷酸序列。编码第二mRNA的核酸分子包括依次连接的如SEQ ID NO.9所示的核酸序列、如SEQ ID NO.7所示的核酸序列、目标蛋白DNA编码序列、如SEQ ID NO.8所示的核酸序列、如SEQ ID NO.10所示的核酸序列以及多聚腺苷酸序列。
本申请实施例还提供了一种生物材料，所述生物材料包括：含有编码第一mRNA的核酸分子的第一重组载体；以及含有编码第二mRNA的核酸分子的第二重组载体。
本申请实施例还提供了一种生物材料，所述生物材料包括：含有所述第一重组载体的转基因动物细胞系；以及含有所述第二重组载体的转基因动物细胞系。
实施例1：第一mRNA的合成
步骤一、利用GeneArtTM Gibson
Figure PCTCN2021126076-appb-000001
HiFi reaction(美国Thermo Fisher，A46624)合成突变型复制酶DNA编码序列(编码第一mRNA的核酸分子不含多聚腺苷酸序列)，合成成功后将该突变型复制酶DNA编码序列克隆于pcDNA3.3载体质粒中以备工业化生产。
1.1突变型复制酶DNA编码序列：5’非翻译区DNA序列(SEQ ID NO.9)、突变型复制酶编码序列(SEQ ID NO.2)、3’非翻译区DNA序列(SEQ ID NO.10)，其中，突变型复制酶编码序列(SEQ ID NO.2)分为nsP1区域片段(SEQ ID NO.3)、nsP2区域片段(SEQ ID NO.4)、nsP3区域片段(SEQ ID NO.5)、nsP4区域片段(SEQ ID NO.6)四个DNA片段，四个DNA片段均为经过修饰后的高GC含量片段。四个DNA片段以gblock形 式直接订购于美国IDT公司。
具体包括如下步骤：按照表1组装Gibson反应，在PCR仪器中50℃反应60分钟，获得PCR产物。
表1 Gibson反应体系
Figure PCTCN2021126076-appb-000002
1.2将PCR产物转化One ShotTM TOP10化学感受态大肠杆菌细胞，具体包括如下步骤：
用无核酸酶水按照1:5稀释上述Gibson反应体系(PCR产物)，12μL无核酸酶水和3μLGibson反应体系，混匀，冰上反应；
将1μL稀释液加入One ShotTM TOP10化学感受态大肠杆菌细胞中并混合，转化混合物在冰上孵育20～30分钟；
将细胞在42℃孵育30秒，不要摇晃；
立即将反应管转移到冰上并在冰上孵育2分钟；
加入450μL室温S.O.C.培养液(美国Life Technology)；
37℃下以300rpm摇动1小时；
取100μL，涂细菌培养板(100μg/mL ampicillin or 50μg/mL kanamycin.)；
37℃度过夜，挑选细菌克隆，37℃摇菌，一代测序挑选含正确序列的双突变复制酶序列质粒。
步骤二、通过PCR添加mRNA的poly-(a)尾巴得到第一mRNA的DNA合成模版
其中，poly-(a)尾巴包括120个腺苷酸。
按照表2制备PCR预混液(总体积为200μL，八个反应各25μL)；
表2 PCR预混液的组成
组分 用量 终浓度
Kapa PRC mix(2X) 100μL 1X
加尾引物-F1 10um(SEQ ID NO.12) 6μL 0.3uM
加尾引物-T120 10um(SEQ ID NO.13) 6μL 0.3uM
水 80μL  
双突变复制酶线性化质粒10ug/μL 8μL 40-400pg/μL
按照表3所示反应条件进行PCR反应；
表3 PCR反应条件
循环次数 变性 退火 扩展
1 95℃,2–3min    
2-31 98℃,20s 60℃,15s 72℃,60s
32 72℃,3min    
通过凝胶电泳检查PCR产物的质量；
切胶回收PCR产物(QIAquick PCR purification kit,Qiagen,cat.no.28106)，将尾模板的最终浓度调整为100ng/μL，作为体外转录合成第一mRNA的DNA合成模版。
步骤三、体外转录合成第一mRNA
1、按照表4组装mRNA帽结构和核苷酸混合物：
帽子结构3′-O-Me-m7G(5′)ppp(5′)G RNA cap analog(New England Biolabs,cat.no.S1411S),-Methylcytidine-5′-triphosphate(Me-CTP；Trilink,cat.no.N1014)，N1-methyl-pseudo-UTP(Trilink,cat.no.N1019)，其他组分均来自MEGAscript T7试剂盒(Ambion,cat.no.AM1334)。
表4 mRNA帽结构和核苷酸混合物
Figure PCTCN2021126076-appb-000003
2、按照表5组装第一mRNA体外转录体系：
表5 第一mRNA体外转录体系
组分 用量(ml) 终浓度
DNase/RNase-free water 1.2  
Custom NTP(from last step) 14.8  
Tailed PCR product,100ug/μL 16 40ng/μL
T7 buffer,10X((from MEGAscript T7kit) 4.0 1X
T7 enzyme mix,10×(from MEGAscript T7kit) 4.0 1X
3、将反应置于PCR仪器在37℃孵育3～6h。
4、向每个样品中添加2μL Turbo DNase(来自MEGAscript T7试剂盒,Ambion,cat.no.AM1334)。
5、轻轻混合并在37℃下孵育15min。
6、使用MEGAclear试剂盒(Ambion,cat.no.AM1908)，纯化经过DNase和RNAa seIII处理的反应；用总共100μL的洗脱缓冲液洗脱修饰的mRNA(50μL的洗脱缓冲液洗脱两次)。
7、使用磷酸酶(Antarctic phosphatase(New England Biolabs,cat.no.M0289S)处理纯化的修饰mRNA。
8、向每个样品(～100μL)中，添加11μL 10×磷酸酶缓冲液，然后添加2μL磷酸酶；轻轻混合样品并在37℃下孵育0.5～1h。
9、洗脱后，在NanoDrop分光光度计中测量修饰的第一mRNA的浓度。预期的总产量应为～50ug(30～70ug范围；一次40μL IVT反应的100μL洗脱体积为300～700ngμL)。通过添加洗脱缓冲液或TE缓冲液(pH7.0)，将浓度调节至100ng/μL，或者FPLC提纯。
实施例2：第二mRNA的合成
第二mRNA的合成步骤与第一mRNA类似，包括如下步骤：
步骤一、利用GeneArtTM Gibson
Figure PCTCN2021126076-appb-000004
HiFi reaction(美国Thermo Fisher，A46624)合成特异性修饰的目标蛋白DNA编码序列(编码第二mRNA的核酸分子不含多聚腺苷酸序列)；
其中，特异性修饰的目标蛋白DNA编码序列：5’非翻译区DNA序列(SEQ ID NO.9)、复制酶5’端特异性DNA序列(SEQ ID NO.7)、目标蛋白DNA编码序列(请参见表6)、复制酶3’端特异性DNA序列(SEQ ID NO.8)、3’非翻译区DNA序列(SEQ ID NO.10)。
步骤二、通过PCR在特异性修饰的目标蛋白DNA编码序列上添加mRNA的poly-(a)尾巴得到第二mRNA的DNA合成模版；
步骤三、体外转录合成第二mRNA。
按照上述方法分别合成表6所示的26种第二mRNA。
表6 不同第二mRNA的DNA合成模版
Figure PCTCN2021126076-appb-000005
Figure PCTCN2021126076-appb-000006
上述的SEQ ID NO.14至SEQ ID NO.39以及SEQ ID NO.47均在不改变原始氨基酸序列的前提下，在对应的原始序列的基础上进行了高GC修饰。
实施例3：
本实施例提供一种药物组合物，多重分子信使RNA和递送载体，其中，多重分子信使RNA包括实施例1制备的第一mRNA以及实施例2制备的第二mRNA-1，递送载体为鱼精蛋白。本实施例中目标蛋白为Taffazin蛋白。
实施例3应用-小鼠Barth综合症模型治疗实验
3.1小鼠Barth综合症模型及诱导:
小鼠基因组引入强力霉素诱导Taffazin蛋白Knock down，建立小鼠Barth综合症模型，通过PCR分析确定进行基因分型DNA，引物：
5’CCATGGAATTCGAACGCTGACGTC 3’(SEQ ID NO.45)；
3’TATGGGCTATGAACTAATGACCC 5’(SEQ ID NO.46)；
本案例只使用雄性，强力霉素以2mg/L浓度置于小鼠饮用水中，同时含有10％蔗糖。
3.2多重分子信使RNA治疗:
在280μL水中稀释10μL的鱼精蛋白(MEDA制药公司的Protamine Ipex5000)5000IU/ml，按280μL+10μL鱼精蛋白5000，制备0.5mg/ml的鱼精蛋白溶液，多重分子信使RNA(多重分子摩尔比为1:1溶液)0.5mg/ml，在RNA溶液中加入等量的鱼精蛋白溶液，并快速上下吹洗至少10次，室温放置10分钟，制成130纳米鱼精蛋白-RNA纳米颗粒，并置于小鼠皮下泵(ALZET pump,https://www.alzet.com/guide-to-use/scid/)持续给药。
将Barth综合症小鼠(TG)小鼠分为6组：TG1、TG2、TG3、TG4、TG5、TG6；
步骤1：将TG1、TG2、TG3、TG4、TG5、TG6采用强力霉素诱导8周，检测心脏射血份数FS％；
步骤2：将TG1、TG2、TG3、TG4、TG5、TG6采用强力霉素诱导10周(在步骤1的基础上继续诱导)，检测心脏射血份数FS％；
步骤3：将TG1、TG2、TG3、TG4采用实施例3的药物组合物治疗2周后，TG5、TG6无治疗，检测心脏射血份数FS％；
步骤4：将TG1、TG2、TG3、TG4采用实施例3的药物组合物治疗3周后(在步骤3的基础上继续治疗1周)，TG5、TG6无治疗，检测心脏射血份数FS％；
步骤5：将TG1、TG2、TG3、TG4采用实施例3的药物组合物治疗6周后(在步骤4的基础上继续治疗1周)，检测小鼠运动能力。
3.3小鼠强制运动能力评价：
老鼠是在封闭的电动跑步机上进行的可调节速度和倾角,并配备了电击传送网，电冲击强度1毫安。最初在跑步机上休息30分钟让动物适应环境，测试以10％的坡度和5米/分钟的速度开始。每5分钟逐步增加 5米/分钟到最终速度25m/min。
3.4 Barth模型鼠心脏功能用超声评价和心脏纤维化用天狼星红染色病 理评价
试验分组
A组：野生型小鼠强力霉素诱导8周；
B组：Barth综合症小鼠(TG)强力霉素诱导8周，普通药物组合物(普通信使RNA系统+递送系统)治疗6周，其中，普通信使RNA系统编码Taffazin蛋白；其中，普通信使RNA系统按照现有技术CN201910014953.6中记载的方法制备。
C组：Barth综合症小鼠(TG)强力霉素诱导8周，实施例3的药物组合物治疗6周；
分别对A组、B组和C组进行心脏纤维化用天狼星红染色病理评价。
3.5实验结果及分析：
3.5.1心脏功能指标检测结果
多重分子信使RNA治疗Barth综合症心脏射血份数超声波结果提示多重分子信使RNA治疗Barth综合症可以提高其心脏功能。
图1所示，有限复制多重分子信使RNA系统编码Taffazin蛋白治疗提高先天性心肌病Barth综合症小鼠心脏功能，具体地，Barth综合症小鼠(TG)在强力霉素诱导下Taffazin蛋白功能缺失，出现Barth综合症的症状出现心肌病疾病表型，心脏功能指标-射血份数下降，无治疗的TG5，TG6心脏功能下降，相比之下多重分子信使RNA治疗2周(TG1、2、3、4)，治疗2～3周，心功能好转。
3.5.2小鼠运动能力试验结果
多重分子信使RNA治疗Barth综合症强制运动结果提示多重分子信使RNA治疗Barth综合症可以提高其运动能力。动物最初在跑步机上休息30分钟让动物适应环境，测试以10％的坡度和5米/分钟的速度开始。每5分钟逐步增加5米/分钟到最终速度，25m/min。因此，运动的持续时间为36.8分钟并且行进的距离是507.4m，结果提示模型鼠未能在皮带 上保持15m/min和10％的倾斜度，并且没有一个模型鼠能够维持运行，跑步机速度超过20米/分钟，而多重分子信使RNA治疗治疗6周后模型鼠均能够维持运，说明多重分子信使RNA治疗可以明显提高模型鼠运动能力。
3.5.3病理分析结果
图2所示，有限复制多重分子信使RNA系统编码Taffazin蛋白治疗先天性心肌病Barth综合症病理分析。
Barth综合症小鼠(TG)在强力霉素诱导下Taffazin蛋白功能缺失，出现Barth综合症的症状出现心肌病疾病表型，心脏病理提示心脏纤维化，多重分子信使RNA治疗8周明显改善心脏纤维化的程度，且优于普通信使RNA治疗效果。
实施例4：
本实施例提供一种有限自我复制mRNA分子系统，包括实施例1制备的第一mRNA以及实施例2制备的第二mRNA-2，目标蛋白为水解GFP蛋白。
实施例4的应用：分别采用编码水解GFP蛋白的普通mRNA(第一组)、实施例4的双分子mRNA(第二组)以及编码水解GFP的全长自我复制mRNA(第三组)转染细胞，步骤见i-vii，下述表达报告基因水解GFP(表达的GFP会被自带的水解酶迅速降解，可以即时反应多重信使RNA分子的持续时间和表达水平)。
实施例4的有限自我复制mRNA分子系统转染细胞步骤如下：
(i)解冻10μl实施例4的有限自我复制mRNA分子系统(第一mRNA和第二mRNA-2的摩尔比例为6:4)，加入40μl OPTI-MEM，轻轻混合。
(ii)在另外一个试管中，加入45μlOPTI-MEM，加5μl Lipofectamine RNAiMax，轻轻混合。
重复移液。
(iii)将稀释后的Lipofectamine RNAiMax加入稀释后的实施例4的有 限自我复制mRNA分子系统中，反复轻轻混匀。
(iv)将混合物在室温下孵育15分钟。
(v)用100μl实施例4的有限自我复制mRNA分子系统/转染试剂复合物均匀加入六孔板的一个孔。
(vi)轻轻地左右摇动板子，以确保转染复合物的均匀扩散。
(vii)放回37℃、5％CO 2、5％O 2细胞培养箱。
通过检测GFP蛋白荧光强度，分别检测第一组细胞中mRNA半衰期、第二组细胞中mRNA半衰期以及第三组细胞中mRNA半衰期，并对第一组、第二组和第三组进行细胞先天性免疫反应。
检测第一组、第二组和第三组转染后的细胞数。
实验结果及分析：
请参阅图3所示，实施例4的有限自我复制mRNA分子系统编码报告基因水解GFP相比全链自我复制信使RNA半衰期无差异，但是细胞毒性弱，免疫原性少，比普通信使RNA半衰期长。
请继续参阅图3所示，实施例4的有限自我复制mRNA分子系统有较长的功能半衰期和低的细胞先天性免疫排斥，实施例4的有限自我复制mRNA分子系统的半衰期明显高于普通信使RNA，同全长自我复制信使RNA相似，但细胞先天性免疫反应(INFA，干扰素A)显著低于同全长自我复制信使RNA。
请参阅图4所示，实施例4的有限自我复制mRNA分子系统(有限复制多重信使RNA分子系统)具备低细胞毒性效应，实施例4的有限自我复制mRNA分子系统产生的信使RNA的细胞毒性同普通信使RNA相似，但明显低于同全长自我复制信使RNA。
实施例5：
本实施例提供一种有限自我复制mRNA分子系统，包括实施例1制备的第一mRNA以及实施例2制备的第二mRNA-22、第二mRNA-23、第二mRNA-24、第二mRNA-25、第二mRNA-26，目标蛋白分别为c-Myc蛋白、Klf4蛋白、Sox2蛋白、OCT4蛋白和Lin28蛋白。
实施例5的应用-细胞重编程试验
细胞重编程步骤：
1,用0.1％(wt/vol)明胶，于六孔板每孔加入1ml；
在室温下至少放置1小时。或者，4摄氏度下过夜，接种人类NuFF饲养细胞前1天，通过吸去除明胶并让板在室温下干燥。
2，NuFF饲养细胞(Newborn human foreskin fibroblasts(GlobalStem,cat.no.GSC-3001G)，解冻一瓶有丝分裂灭活的NuFFs并将细胞接种在明胶细胞板上。
3，接种重编程目标成纤维细胞后6-12小时，用Pluriton(stemgent)完全重编程培养基替换成纤维细胞培养基(Pluriton含B18R(eBioscience,cat.no.34-8185-85，200ng/ml))，每孔使用2ml然后，在37℃，5％CO2、5％O2培养基中孵育细胞过夜。
4，使用Lipofectamine RNAiMax(Invitrogen,cat.no.56532))进行转染；
(i)解冻10μl修饰的mRNA混合物(双突变复制酶，OCT4,KLF4,c-MYC,SOX2,LIN28A摩尔比例6:1:1:1:1:1,respectively.100ng/μl)，加入40μl OPTI-MEM，轻轻混合。
(ii)在另外一个试管中，加入45μl OPTI-MEM，加5μl Lipofectamine RNAiMax，轻轻混合。
重复移液。
(iii)将稀释后的Lipofectamine RNAiMax加入稀释后的修饰mRNA中，反复轻轻混匀。
(iv)将混合物在室温下孵育15分钟。
(v)用100μl修饰的mRNA/转染试剂复合物均匀加入六孔板的一个孔
(vi)轻轻地左右摇动板子，以确保转染复合物的均匀扩散。
(vii)放回37℃、5％CO 2、5％O 2细胞培养箱。
5，每72小时重复上述i-vii步骤，直到重编程细胞克隆出现。
请参阅图5和图6所示，有限复制多重信使RNA分子系统同时放大5个编码细胞重编程因子Otc4,Sox2,Klf4,c-Myc,Lin28(OSKML)重高效完成细胞重编程；对比普通信使RNA该系统有更长蛋白表达，和更高的细胞重编程(iPS克隆数为指标)；实施例5的有限自我复制mRNA分子系统(有限复制多重信使RNA分子系统)产生的细胞重编程产物-iPS细胞显示典型多潜能；实施例5的有限自我复制mRNA分子系统(有限复制多重信使RNA分子系统)编码5重编程因子OSKML完成细胞重编程后产物-iPS细胞显示经典多潜能干细胞克隆外形，多潜能标志物Oct4染色阳性，可以在体内形成畸胎瘤。
实施例6：
本实施例提供一种有限自我复制mRNA分子系统，包括实施例1制备的第一mRNA以及实施例2制备的第二mRNA-3，目标蛋白为Cas9蛋白。
实施例6的应用：基因编辑试验
实施步骤：
实施例6的有限自我复制mRNA分子系统(多重分子信使RNA)在人诱导性干细胞(Induced Pluripotent Stem Cells)中进行DNAJC19基因编辑或Taffazin基因编辑。
1、人诱导性干细胞电转染：按下表7组装基因编辑反应体系，Taffazin基因gRNA序列(SEQ ID NO.40，直接订购于IDT公司)，DNAJC19gRNA序列(SEQ ID NO.17，直接订购于IDT公司)。
表7 基因编辑反应体系
Figure PCTCN2021126076-appb-000007
2、按引物
F:TAAGCTAACCTGTCACCCCA(SEQ ID NO.41)；
R:AGAGCACAGAGGCGAGGCTT(SEQ ID NO.42)；
PCR扩增Taffazin基因片段；
或者，按引物
F：CTCAAAAGACTTCTGTTCTTGAGC(SEQ ID NO.43)；
R：CACTGAACACTGTGATAATCTGCT(SEQ ID NO.44)；
PCR扩增DNAJC19基因片段。
3、Surveyor酶评价人诱导干细胞Taffazin基因编辑或DNAJC19基因编辑((IDT,cat.no.706025)，按下表8组装反应体系，
表8 反应体系
组分 体积(μL)
0.15M MgCl2 4
Surveyor enhancer S 1
Surveyor nuclease S 2
4、混合均匀并在42℃孵育60分钟。
5、加入Surveyor Mutation Detection Kit的1/10体积终止溶液以终止反应和1/6体积DNA。
6、通过4–20％TBE凝胶在200V下电泳约60分钟，分析的Surveyor核酸酶消化产物。
7、在1×TBE中用0.5g/ml溴化乙锭染色凝胶10分钟。在水中清洗凝胶10分钟。
8、使用紫外线透射仪对凝胶进行成像。
实验结果及分析：
请参阅图7所示，实施例6的有限自我复制mRNA分子系统(有限复制多重信使RNA分子系统)编码CRISPR蛋白Cas9，高效编辑DNAJC19和人Taffazin基因。具体地，请参阅图7左，DNAJC19基因成功被基因编辑产生基因突变，并被Surveyor识别剪切，出现3条典型条带，说明高效基因编辑完成，请参阅图7右，Taffazin基因成功被基因编辑产生基因突变，并被Surveyor识别剪切，出现3条典型条带，说明高效基因编辑完成。
实施例7：
本实施例提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例2的第二mRNA-5以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为SARS-CoV-2的抗原性多肽(野生型的刺突蛋白S)。
本实施例还提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例2的第二mRNA-28以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为SARS-CoV-2的抗原性多肽(德尔塔株的刺突蛋白S)。
实施例8：
本实施例提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例2的第二mRNA-8、第二mRNA-9、第二mRNA-10、第二mRNA-11、第二mRNA-12以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为HPV6的L1蛋白、HPV11的L1蛋白、HPV16的L1蛋白、HPV18的L1蛋白和HPV的E6蛋白。
实施例9：
本实施例提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例2的第二mRNA-13、第二mRNA-14以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为HSV的包膜糖蛋白E和HSV的包膜糖蛋白D。
实施例10：
本实施例提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例2的第二mRNA-15以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为流感病毒HA抗原。
实施例11：
本实施例提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例2的第二mRNA-16、第二mRNA-17、第二mRNA-18以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为HIV的Gag抗原、HIV的EnV抗原和HIV的CD40L。
实施例12：
本实施例提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例 2的第二mRNA-19、第二mRNA-20、第二mRNA-21以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为非洲猪瘟病毒的NL-S蛋白、非洲猪瘟病毒的cd2v ep402r蛋白和非洲猪瘟病毒的TK蛋白。
实施例13：
本实施例提供一种药物组合物，用于治疗结肠癌，包括实施例1的第一mRNA、实施例2的第二mRNA-6、第二mRNA-7以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为白细胞介素-2和不含氨基的甲胎蛋白。
实施例14：
本实施例提供一种mRNA疫苗，包括实施例1的第一mRNA、实施例2的第二mRNA-27以及鱼精蛋白，形成130纳米鱼精蛋白RNA粒子进行递送。目标蛋白为狂犬病抗原(狂犬病糖蛋白)。
以上所述的仅是本申请的实施方式，在此应当指出，对于本领域的普通技术人员来说，在不脱离本申请创造构思的前提下，还可以做出改进，但这些均属于本申请的保护范围。

Claims (14)

  1. 一种有限自我复制mRNA分子系统，其特征在于，包括：
    编码甲病毒属突变型复制酶的第一mRNA；以及
    至少一个编码目标蛋白的第二mRNA；
    其中，所述突变型复制酶产生nsP2区域的第259位的突变以及nsP2区域的第650位的突变。
  2. 根据权利要求1所述的有限自我复制mRNA分子系统，其特征在于，所述突变型复制酶包括依次连接的nsP1区域、nsP2区域、nsP3区域以及nsP4区域，所述突变型复制酶的氨基酸序列如SEQ ID NO.1所示，所述突变型复制酶产生在SEQ ID NO.1所示的796位点的丝氨酸S突变为脯氨酸P以及在SEQ ID NO.1所示的1187位点的精氨酸R突变为天冬氨酸D。
  3. 根据权利要求1所述的有限自我复制mRNA分子系统，其特征在于，所述第一mRNA包括突变型复制酶编码序列，所述突变型复制酶编码序列包括如SEQ ID NO.2所示的核酸序列对应的RNA序列；
    每个所述第二mRNA包括依次连接的复制酶5’端特异性序列、目标蛋白编码序列以及复制酶3’端特异性序列，所述复制酶5’端特异性序列包括如SEQ ID NO.7所示的核酸序列对应的RNA序列，所述复制酶3’端特异性序列包括如SEQ ID NO.8所示的核酸序列对应的RNA序列。
  4. 根据权利要求3所述的有限自我复制mRNA分子系统，其特征在于，所述第一mRNA和所述第二mRNA还包括：5’帽结构、5’UTR序列、3’UTR序列以及多聚腺苷酸序列；
    其中，所述第一mRNA按照5’→3’方向依次包括如下元件：5’帽结构、5’UTR序列、突变型复制酶编码序列、3’UTR序列和多聚腺苷酸序列；
    每个所述第二mRNA按照5’→3’方向依次包括如下元件：5’帽结构、5’UTR序列、复制酶5’端特异性序列、目标蛋白编码序列、复制酶3’端特异性序列、3’UTR序列和多聚腺苷酸序列；
    所述5’UTR序列包括如SEQ ID NO.9所示的核酸序列对应的RNA序列，所述3’UTR序列包括如SEQ ID NO.10所示的核酸序列对应的RNA序列，所述5’帽结构选自3′-O-Me-m7G、m 7 GpppG、m 2 7,3′-O GpppG、m 7 Gppp(5')N1或m 7 Gppp(m 2′-O)N1中的至少一种。
  5. 根据权利要求1所述的有限自我复制mRNA分子系统，其特征在于，所述第一mRNA或所述第二mRNA中部分或全部的尿嘧啶进行了能够提高所述第一mRNA在生物体内稳定性的化学改性，所述化学改性包括利用N1-甲基假尿苷置换所述第一mRNA中的至少50％、至少60％、至少70％、至少80％、至少90％或100％的尿嘧啶；
    或，所述第一mRNA和所述第二mRNA被RNase III处理，所述第一mRNA和所述第二mRNA经快速蛋白质液相色谱提纯。
  6. 根据权利要求1所述的有限自我复制mRNA分子系统，其特征在于，所述目标蛋白包括SARS-CoV-2的抗原性多肽；
    或，所述目标蛋白包括白细胞介素-2和不含氨基的甲胎蛋白；
    或，所述目标蛋白包括HPV6的L1蛋白、HPV11的L1蛋白、HPV16的L1蛋白、HPV18的L1蛋白和HPV的E6蛋白；
    或，所述目标蛋白包括HSV的包膜糖蛋白E和HSV的包膜糖蛋白D；
    或，所述目标蛋白包括流感病毒HA抗原；
    或，所述目标蛋白包括HIV的Gag抗原、HIV的EnV抗原和HIV的CD40L；
    或，所述目标蛋白包括非洲猪瘟病毒的NL-S蛋白、非洲猪瘟病毒的cd2v ep402r蛋白和非洲猪瘟病毒的TK蛋白；
    或，所述目标蛋白包括Taffazin蛋白；
    或，所述目标蛋白包括c-Myc蛋白、Klf4蛋白、Sox2蛋白、OCT4蛋白和Lin28蛋白；
    或，所述目标蛋白包括Cas9蛋白和DNAJC19蛋白；
    或，所述目标蛋白包括水解GFP蛋白。
  7. 一种有限自我复制mRNA分子系统的制备方法，其特征在于，包 括：
    合成第一mRNA；
    合成至少一个第二mRNA；
    其中，第一mRNA编码甲病毒属突变型复制酶，第二mRNA编码目标蛋白，所述突变型复制酶产生nsP2区域的第259位的突变以及nsP2区域的第650位的突变。
  8. 根据权利要求7所述的制备方法，其特征在于，还包括：
    利用RNase III对所述第一mRNA和所述第二mRNA进行处理；
    利用快速蛋白质液相色谱对所述第一mRNA和所述第二mRNA进行提纯。
  9. 根据权利要求7所述的制备方法，其特征在于，所述合成第一mRNA，包括：
    合成突变型复制酶DNA编码序列，其中，所述突变型复制酶DNA编码序列包括如SEQ ID NO.9所示的5’非翻译区DNA序列、如SEQ ID NO.2所示的突变型复制酶编码序列、如SEQ ID NO.10所示的3’非翻译区DNA序列；
    通过PCR在所述突变型复制酶DNA编码序列上添加mRNA的poly-
    (a)尾巴得到第一mRNA的DNA合成模版；
    将所述第一mRNA的DNA合成模版进行体外转录合成第一mRNA。
  10. 根据权利要求7所述的制备方法，其特征在于，所述合成第二mRNA，包括：
    合成特异性修饰的目标蛋白DNA编码序列，其中，所述特异性修饰的目标蛋白DNA编码序列包括如SEQ ID NO.9所示的5’非翻译区DNA序列、如SEQ ID NO.7所示的复制酶5’端特异性DNA序列、目标蛋白DNA编码序列、如SEQ ID NO.8所示的复制酶3’端特异性DNA序列、如SEQ ID NO.10所示的3’非翻译区DNA序列；
    通过PCR在所述特异性修饰的目标蛋白DNA编码序列上添加mRNA的poly-(a)尾巴得到第二mRNA的DNA合成模版；
    将所述第二mRNA的DNA合成模版进行体外转录合成第二mRNA。
  11. 一种生物材料，其特征在于，所述生物材料为A1)至A6)中的任一种：
    A1)编码所述第一mRNA的核酸分子；
    A2)编码所述第二mRNA的核酸分子；
    A3)含有A1)所述核酸分子的重组载体；
    A4)含有A2)所述核酸分子的重组载体；
    A5)含有A3)所述重组载体以及的转基因动物细胞系；
    A6)含有A4)所述重组载体的转基因动物细胞系。
  12. 一种药物组合物，其特征在于，包括权利要求1～6中任一项所述的有限自我复制mRNA分子系统中的至少一种，以及递送载体。
  13. 编码甲病毒属突变型复制酶的第一mRNA在制备调节免疫系统的佐剂的用途，其中，所述突变型复制酶产生nsP2区域的第259位的突变以及nsP2区域的第650位的突变。
  14. 权利要求1～6中任一项所述的有限自我复制mRNA分子系统或权利要求7所述的生物材料或权利要求8所述的药物组合物在制备细胞重编辑试剂中的用途、在制备基因编辑试剂中的用途、在制备Barth综合征治疗药物中的用途、在制备感染性疾病疫苗中的用途或在制备肿瘤疫苗中的用途。
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