WO2024160232A1 - 质粒包装系统及其应用 - Google Patents

质粒包装系统及其应用 Download PDF

Info

Publication number
WO2024160232A1
WO2024160232A1 PCT/CN2024/074973 CN2024074973W WO2024160232A1 WO 2024160232 A1 WO2024160232 A1 WO 2024160232A1 CN 2024074973 W CN2024074973 W CN 2024074973W WO 2024160232 A1 WO2024160232 A1 WO 2024160232A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmid
gene
nucleic acid
sequence
promoter
Prior art date
Application number
PCT/CN2024/074973
Other languages
English (en)
French (fr)
Inventor
肖溯
李斌
成传刚
李孟悦
Original Assignee
纽福斯(苏州)生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202310049101.7A external-priority patent/CN118460617A/zh
Application filed by 纽福斯(苏州)生物科技有限公司 filed Critical 纽福斯(苏州)生物科技有限公司
Publication of WO2024160232A1 publication Critical patent/WO2024160232A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
    • 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
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • sequence file submitted electronically with this specification are incorporated herein by reference in their entirety: Computer-readable format copy of the sequence file (file name: F24W1513PCT-new-sequence listing.xml, creation date: January 30, 2024, file size: 630kb).
  • the present invention relates to the field of biotechnology, in particular to a plasmid system and application thereof.
  • Recombinant adeno-associated virus can stably express foreign genes in vivo for a long time. It is a low immunogenic viral vector, so it has become the most promising delivery tool in the field of gene therapy and is widely used in basic and clinical research. There are hundreds of clinical trials currently underway, and eight AAV gene therapy drugs have been launched on the market, with a very broad commercial prospect.
  • the first AAV gene therapy drug Glybera was announced to be withdrawn from the market. The reason is that it has a high price but no market. The cost of a single treatment is as high as 1 million US dollars. The high production cost is the fundamental reason for its high price. Therefore, achieving large-scale production and obtaining high-titer and high-purity rAAV vectors has always been a major problem that biopharmaceutical companies need to solve.
  • rcAAV replication-competent adeno-associated virus
  • the AAV packaging system when constructing the AAV packaging system, separate the structural genes of AAV into transcription units of different plasmids, and minimize the similarity between Rep/Cap gene sequences and ITR sequences, reduce the risk of recombination to produce new infectious viruses or replicative viruses, fully consider the possibility of producing replicative viruses through homologous or non-homologous recombination during production and use, and the vector should avoid containing any known elements with human tumorigenicity risks.
  • rcAAV is one of the important sources of these two proteins, which will seriously affect the safety and quality of drugs. As an impurity in AAV vector preparations, rcAAV cannot be distinguished and removed during the downstream purification process. rcAAV residue detection is also a key inspection item required by regulations, so the rcAAV content must be reduced.
  • the main method for producing rAAV is the AAV Helper-Free System, which contains three plasmids: the first is a vector plasmid containing the gene-of-interest (GOI), which retains the terminal inverted repeat sequence (ITR) of AAV at both ends, which is a cis-acting element necessary for replication and packaging; the second is the RC plasmid (pRC) that determines the serotype, encoding the replication protein Rep and structural protein Cap of AAV itself; finally, a helper plasmid pHelper is required to provide the helper genes E2A, E4orf6 and VA RNA separated from adenovirus Ad (adenovirus).
  • a vector plasmid containing the gene-of-interest containing the gene-of-interest (GOI), which retains the terminal inverted repeat sequence (ITR) of AAV at both ends, which is a cis-acting element necessary for replication and packaging
  • the second is the RC plasmid (pRC
  • rAAV can be produced quickly and easily by transiently transfecting mammalian cells with three plasmids, but the cells used for production in this method are mostly adherent cells, which require fetal bovine serum culture, have strict requirements on growth conditions and downstream purification, and require the preparation of a large number of high-quality plasmids, so the production cost is very high and difficult to expand.
  • commercial suspension HEK293 cells are currently available, the advantage is that they are cultured in serum-free medium, which not only reduces the downstream purification steps, but also allows for scale-up.
  • the disadvantage is that they are affected by the kinetic factors of transfection complex formation, resulting in low transfection efficiency.
  • the yield of rAAV is not as high as that of adherent culture.
  • the Rep and Cap genes of the packaging RC plasmid used are on the same plasmid backbone and are arranged in basically the same sequence as the wtAAV gene, homologous or non-homologous recombination between the ITR and its surrounding sequences of the vector plasmid (pAAV-GOI) and the Rep-Cap sequence of the packaging plasmid (pRC) is very easy to occur during the production process to form rcAAV.
  • pAAV-GOI homologous or non-homologous recombination between the ITR and its surrounding sequences of the vector plasmid
  • pRC Rep-Cap sequence of the packaging plasmid
  • the first method to reduce rcAAV formation is to eliminate homologous sequences between the vector plasmid and the RC plasmid. Further, Rep and Cap can be placed under the control of heterologous promoters, replacing the natural promoters P5, P19 or P40 of AAV, or even transcribed in the opposite direction. There are literature reports that this method can reduce rcAAV to below the detection level. The disadvantage is that the rAAV yield may be greatly reduced.
  • Another method is to generate an oversized RC plasmid by inserting introns inside the Rep or Cap gene, so that the final sequence exceeds the packaging limit of AAV, but in this method, the introns are likely to be cut off during the packaging process, so the wtAAV gene will still be packaged in, and the residual rcAAV may not meet the regulatory requirements for clinical-grade AAV vector preparations.
  • a recombinant nucleic acid comprising a Rep gene and/or a Cap gene of an adeno-associated virus (AAV), and having one or more of the following characteristics:
  • the donor site of the original intron of the viral genome of the adeno-associated virus contains a mutation, and the donor site is equivalent to the nucleotide sites 1717-1722 of SEQ ID NO:7;
  • a non-parental serotype promoter is contained upstream of the Rep gene or downstream of the Cap gene, and is operably linked to the Rep gene and/or the Cap gene.
  • the recombinant nucleic acid comprises feature (1), and the insertion position of the P5 promoter located upstream of the Rep gene is immediately after the 120bp nucleotide site of the adeno-associated virus genome corresponding to SEQ ID NO:7, and is operably linked to the Rep gene. In some embodiments, the recombinant nucleic acid inserts a P5 promoter downstream of the Cap gene and is operably linked to the Cap gene.
  • the P5 promoter is spaced from the Rep gene or the Cap gene by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides, or there are no intervening nucleotides.
  • the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.
  • the recombinant nucleic acid comprises feature (2), and the correction of the TATA box mutation of the original P5 promoter comprises mutating T corresponding to nucleotide position 4469 of SEQ ID NO:7 to A, and/or mutating GGGGGGG corresponding to nucleotide positions 4477-4483 of SEQ ID NO:7 to TATTTAA.
  • the sequence of the original P5 promoter after correction is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:9.
  • the recombinant nucleic acid comprises feature (3), and the mutation of the donor site comprises mutating the CA corresponding to nucleotides 1721-1722 of SEQ ID NO:7 to GT, or mutating the donor site sequence corresponding to nucleotides 1717-1722 of SEQ ID NO:7 to GTACGT.
  • the mutated intron sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:11.
  • the recombinant nucleic acid comprises feature (4) and the liver-specific enhancer-promoter sequence is deleted.
  • the liver-specific enhancer-promoter sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 35.
  • the recombinant nucleic acid comprises feature (5), and the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.
  • the hGH intron is located downstream of the P5 promoter of feature (1) and upstream of the Rep gene of feature (1).
  • the hGH intron and the P5 promoter of feature (1) are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • the hGH intron and the start codon of the Rep gene are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides. In some embodiments, there are no intervening nucleotides between the hGH intron and the start codon of the Rep gene.
  • the recombinant nucleic acid comprises feature (6), and the sequence of the intron of the adeno-associated virus genome is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.
  • the intron of the adeno-associated virus genome is located downstream of the P5 promoter of feature (1) and upstream of the Rep gene of feature (1).
  • the intron of the adeno-associated virus genome and the P5 promoter of feature (1) are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • the intron of the adeno-associated virus genome and the start codon of the Rep gene are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • the recombinant nucleic acid comprises feature (7), and the sequence of the non-parental serotype promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:15.
  • the non-parental serotype promoter is located downstream of the Cap gene.
  • the non-parental serotype promoter is located upstream of the Rep gene, and the non-parental serotype promoter is located upstream of the hGH intron.
  • the non-parental serotype promoter is located at a position corresponding to nucleotides 4270-4271 of SEQ ID NO:7.
  • the recombinant nucleic acid does not contain a sequence corresponding to nucleotides 4368-4532 of SEQ ID NO:7, or does not contain a sequence corresponding to nucleotides 4373-4532 of SEQ ID NO:7.
  • the recombinant nucleic acid does not contain a sequence corresponding to nucleotides 121-130 of SEQ ID NO:7.
  • the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or AAV-DJ.
  • the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-DJ.
  • the serotype of the AAV is AAV2, AAV5, AAV8, or AAV9.
  • the serotype of the AAV is AAV2.
  • sequence of the Rep gene is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2.
  • sequence of the Cap gene is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3.
  • the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 48-57.
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 58-67.
  • the recombinant nucleic acid comprises the features:
  • the recombinant nucleic acid comprises the Rep gene and the Cap gene. In some embodiments, the recombinant nucleic acid comprises the Cap gene but does not comprise a nucleic acid sequence encoding the Rep gene. In some embodiments, the recombinant nucleic acid comprises the Rep gene but does not comprise a nucleic acid sequence encoding the Cap gene.
  • a plasmid comprising the recombinant nucleic acid described herein.
  • the plasmid is a packaging plasmid (RC plasmid) in a recombinant adeno-associated virus helper virus-free system, or a helper plasmid (pHelper plasmid), or a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • RC plasmid packaging plasmid
  • pHelper plasmid helper plasmid
  • the plasmid comprises the Cap gene, but does not comprise a nucleic acid sequence encoding a Rep gene, nor a nucleic acid sequence encoding an auxiliary gene for AAV production, nor an AAV inverted repeat sequence (ITR).
  • the plasmid is a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence, and comprises the Cap gene.
  • a plasmid in one aspect of the present invention, characterized in that it contains a Cap gene but does not contain a nucleic acid sequence encoding a Rep gene, a nucleic acid sequence encoding an auxiliary gene for AAV production, or an AAV inverted repeat sequence (ITR).
  • ITR AAV inverted repeat sequence
  • a plasmid in one aspect of the present invention, characterized in that the target gene plasmid contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • a helper plasmid comprises the recombinant nucleic acid comprising the Cap gene as described herein, and the helper plasmid comprises a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production.
  • AAV adeno-associated virus
  • a helper plasmid in one aspect of the present invention, contains a nucleic acid sequence encoding a Cap gene and a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production, wherein the Cap gene encodes a functional Cap protein.
  • AAV adeno-associated virus
  • the helper plasmid comprises a Rep gene.
  • the Rep gene expresses Rep78 and Rep52 proteins.
  • the Rep gene does not express Rep68 or Rep40 proteins.
  • the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • the auxiliary genes for AAV production include one or more genes selected from E2A, E4, and VA RNA. In some embodiments, the auxiliary genes for AAV production include two genes selected from E2A, E4, and VA RNA. In some embodiments, the auxiliary plasmid includes an E4 gene and a VA RNA gene. In some embodiments, the auxiliary genes include E2A, E4, and VA RNA.
  • the E2A, E4, and/or VA RNA is derived from adenovirus type 2 (Ad2).
  • the E2A gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 93.
  • the E4 gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 94.
  • the VA RNA gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 95.
  • the E2A, E4, and/or VA RNA is derived from adenovirus type 2 (Ad2).
  • the plasmid is characterized in that it comprises at least one promoter selected from the group consisting of P5 promoter, P19 promoter, and P40 promoter, and the promoter is operably linked to the Cap gene. In some embodiments, it comprises P5 promoter, P19 promoter, and P40 promoter. In some embodiments, the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, sequences encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • the promoters and/or the promoter and the Cap gene are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides. In some embodiments, the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO: 1.
  • the sequence of the P19 promoter is T at a position relative to nucleotide 768 of SEQ ID NO: 1.
  • the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO: 1.
  • the sequence of the P40 promoter is GT at a position relative to nucleotides 1911-1912 of SEQ ID NO: 1.
  • the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO: 1.
  • the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO: 48-57.
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO: 58-67.
  • the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • the Cap gene and the polyA sequence are separated by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides. In some embodiments, there are no intervening nucleotides.
  • the polyA sequence has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with nucleotides 4411-4460 of SEQ ID NO:1.
  • the Cap gene encodes a functional Cap protein.
  • the plasmid is unable to express a functional Rep protein. In some embodiments, the plasmid does not contain a nucleic acid sequence encoding a Rep gene.
  • a helper plasmid in one aspect of the present invention, characterized in that it comprises a recombinant nucleic acid comprising the Rep gene as described herein, and the helper plasmid comprises a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production.
  • a helper plasmid is provided, characterized in that it comprises a nucleic acid sequence encoding the Rep gene and a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production,
  • the helper plasmid is characterized in that it contains a nucleic acid sequence encoding the Rep gene, and the plasmid (a) cannot express a functional Cap protein, and/or (b) does not contain a nucleic acid sequence encoding the Cap gene.
  • a plasmid in one aspect of the present invention, characterized in that it contains a nucleic acid sequence encoding the Rep gene, and the plasmid (a) cannot express a functional Cap protein, and/or (b) does not contain a nucleic acid sequence encoding the Cap gene.
  • a plasmid is provided, characterized in that it contains a nucleic acid sequence encoding the Rep gene, and the plasmid cannot express a functional Cap protein.
  • the plasmid does not contain a nucleic acid sequence encoding the Cap gene.
  • the plasmid cannot express a functional expression product of an auxiliary gene for AAV production.
  • the plasmid does not contain a nucleic acid sequence encoding an auxiliary gene for AAV production.
  • the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.
  • the sequence of the Rep gene comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • the Rep gene expresses Rep78 and Rep52 proteins. In some embodiments, the Rep gene does not express Rep68 or Rep40 proteins. In some embodiments, the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • the sequence of the Rep gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2196 of SEQ ID NO:1.
  • the Rep gene expresses Rep68 and Rep40 proteins. In some embodiments, the Rep gene does not express Rep78 or Rep52 proteins. In some embodiments, the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:103.
  • the recombinant nucleic acid or plasmid does not contain genes that express only Rep52 and Rep40 proteins.
  • the recombinant nucleic acid or plasmid comprising the Rep gene comprises a P5 promoter located upstream of the Rep gene and/or a P5 promoter located downstream of the Rep gene, and the P5 promoter is operably linked to the Rep gene.
  • the promoter and the Rep gene are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides.
  • sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO: 1, or to nucleotides 191-310 of SEQ ID NO: 1.
  • the Rep gene of the recombinant nucleic acid or plasmid comprising the Rep gene encodes a functional Rep protein.
  • the recombinant nucleic acid or plasmid comprising the Rep gene is characterized in that the nucleic acid sequence encoding the Rep gene encodes a polyA sequence, and the polyA sequence is located downstream of the Rep gene.
  • the Rep gene and the polyA sequence are separated by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides. In some embodiments, there are no intervening nucleotides.
  • the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • the nucleic acid sequence encoding the Rep gene in the plasmid comprises an hGH intron. In some embodiments, the intron is located before the start codon of the Rep gene.
  • the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.
  • the donor site of the original intron of the Rep gene comprises a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO: 1.
  • the mutation of the donor site comprises a mutation of CA equivalent to the nucleotides 1911-1912 of SEQ ID NO: 1 to GT, or a mutation of the donor site sequence equivalent to the nucleotides 1907-1912 of SEQ ID NO: 1 to GTACGT.
  • the plasmid further comprises a f1 origin site.
  • a three-plasmid system characterized in that it comprises:
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • it also comprises at least two AAV inverted repeat sequences (ITR), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence, wherein the plasmid described in (i) and/or (ii) comprises a recombinant nucleic acid encoding the Rep gene and/or the Cap gene as described herein.
  • the plasmid in (ii) comprises the Rep gene and the Cap gene. In some embodiments, the Rep gene and the Cap gene are on different plasmids. In some embodiments, the helper plasmid in (i) comprises the Rep gene, and the plasmid in (ii) comprises the Cap gene. In some embodiments, the plasmid in (ii) comprises the Rep gene, and the plasmid of the recombinant adeno-associated virus in (iii) comprises the Cap gene.
  • a three-plasmid system for adeno-associated virus (AAV) expression is provided, characterized in that one of the plasmids contains the Rep gene of AAV but does not contain the Cap gene of AAV, and another different plasmid contains the Cap gene of AAV but does not contain the Rep gene of AAV.
  • the three-plasmid system comprises an auxiliary plasmid (pHelper plasmid) containing the Rep gene, a second plasmid containing the Cap gene, and a third plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the third plasmid also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • the three-plasmid system comprises a helper plasmid (pHelper plasmid), a second plasmid comprising the Rep gene, and a third plasmid comprising the Cap gene and encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the third plasmid further comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • a three-plasmid system for adeno-associated virus (AAV) expression characterized in that it comprises:
  • helper plasmid described herein, comprising the Rep gene, and the helper plasmid (a) cannot express a functional Cap protein, and/or (b) does not comprise a nucleic acid sequence encoding a Cap gene;
  • a plasmid according to the present invention comprising the Cap gene, and the plasmid (a) cannot express a functional Rep protein, and/or (b) does not comprise a nucleic acid sequence encoding a Rep gene;
  • a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the plasmid further comprises at least two AAV inverted repeat sequences (ITRs), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • the plasmid system is used to produce a recombinant vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-DJ.
  • the AAV serotype is AAV2, AAV5, AAV8, or AAV9.
  • the AAV serotype is AAV2.
  • a three-plasmid system for adeno-associated virus (AAV) expression characterized in that it comprises:
  • helper plasmid (i) a helper plasmid (pHelper plasmid), wherein the helper plasmid comprises a recombinant nucleic acid encoding a Rep gene.
  • the helper plasmid (a) cannot express a functional Cap protein, and/or (b) does not comprise a nucleic acid sequence encoding a Cap gene;
  • plasmid (a) cannot express a functional Rep protein, and/or (b) does not comprise a nucleic acid sequence encoding a Rep gene;
  • a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the plasmid further comprises at least two AAV inverted repeat sequences (ITRs), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • a three-plasmid system for adeno-associated virus (AAV) expression characterized in that it comprises:
  • a plasmid according to the present invention comprising the Rep gene, and the plasmid (a) cannot express a functional Cap protein, and/or (b) does not contain a nucleic acid sequence encoding a Cap gene, and
  • a plasmid of a recombinant adeno-associated virus comprising the Cap gene and the heterologous nucleic acid sequence as described herein.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • it further comprises at least two AAV inverted repeat sequences (ITRs), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • a three-plasmid system for adeno-associated virus (AAV) expression characterized in that it comprises:
  • the plasmid (a) cannot express a functional Cap protein, and/or (b) does not comprise a nucleic acid sequence encoding a Cap gene;
  • the plasmid (a) cannot express a functional Rep protein, and/or (b) does not contain a nucleic acid sequence encoding a Rep gene.
  • the heterologous nucleic acid sequence encodes a heterologous gene. In some embodiments, it also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • a three-plasmid system for adeno-associated virus (AAV) expression characterized in that it comprises:
  • a three-plasmid system for adeno-associated virus (AAV) expression characterized in that it comprises:
  • helper plasmid (i) a helper plasmid (pHelper plasmid), wherein the helper plasmid comprises a recombinant nucleic acid encoding a Cap gene;
  • the (i) helper plasmid cannot express a functional Rep protein, and/or the (ii) plasmid encoding the Rep gene cannot express a functional Cap protein.
  • the (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence further comprises at least two AAV inverted repeats (ITRs), and the two AAV inverted repeats are located upstream and downstream of the heterologous nucleic acid sequence, respectively.
  • the (ii) plasmid encoding the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins
  • the (i) helper plasmid comprises a gene expressing Rep78 and/or Rep52 proteins.
  • the gene does not express Rep68 or Rep40.
  • the (ii) plasmid encoding the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins
  • the (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence comprises a gene expressing Rep78 and/or Rep52 proteins.
  • the gene does not express Rep68 or Rep40.
  • the (i) helper plasmid contains the E4 gene and the VA RNA gene; the (ii) plasmid encoding the Rep gene contains the E2A gene.
  • the plasmid system is used to produce a recombinant vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-DJ.
  • the AAV serotype is AAV2, AAV5, AAV8, or AAV9.
  • the serotype of AAV is AAV2.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • a two-plasmid system characterized in that it comprises (i) a first plasmid containing the Rep gene as described herein and (ii) a second plasmid containing the Cap gene as described herein.
  • a two-plasmid system characterized in that it comprises a first plasmid containing the Rep gene of AAV and a second plasmid containing the Cap gene of AAV, wherein the Rep gene and the Cap gene are located on different plasmids.
  • the Rep and the Cap genes are located on different plasmids.
  • the first plasmid containing the Rep gene is a helper plasmid (pHelper plasmid)
  • the second plasmid containing the Cap gene is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the second plasmid further comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • the second plasmid containing the Cap gene is a helper plasmid (pHelper plasmid), and the first plasmid containing the Rep gene is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the first plasmid further comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins. In some embodiments, the Rep gene expresses Rep78 and Rep52 proteins, but does not express Rep68 and Rep40 proteins. In some embodiments, the Rep gene expresses Rep68 and Rep40 proteins, but does not express Rep78 and Rep52 proteins. In some embodiments, the plasmid system does not contain genes that co-express Rep52 and Rep40 proteins.
  • the upstream of the Rep gene contains a hGH intron. In some embodiments, the upstream of the hGH intron contains a P5 promoter. In some embodiments, the downstream of the Rep gene contains a polyA sequence. In some embodiments, the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably connected to the Rep gene.
  • the upstream of the Cap gene contains three promoters, which are: P5 promoter, P19 promoter, and P40 promoter from 5' to 3', all of which are operably linked to the Cap gene, and the downstream of the Cap gene contains a polyA sequence.
  • the plasmid system comprises at least one polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO:16--28, 33-34, 40, 45-47, 68, 80-87, 96-98.
  • the plasmid system is used to produce a recombinant vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-DJ.
  • the AAV serotype is AAV2, AAV5, AAV8, or AAV9.
  • the AAV serotype is AAV2.
  • a method for modifying a plasmid system comprises one or more of the following modification methods:
  • the transformation method is characterized by:
  • the P5 promoter inserted upstream of the Rep gene as described in (1) has a sequence as shown in SEQ ID NO:4;
  • the mutated P5 promoter has the sequence shown in SEQ ID NO:8, and the P5 promoter inserted downstream of the Cap gene has the sequence of SEQ ID NO:4;
  • Mutating the donor site of the intron is to mutate GTACCA at the donor site of the intron to GTACGT, preferably, the intron before the mutation has a sequence as shown in SEQ ID NO: 10; and the intron after the mutation has a sequence as shown in SEQ ID NO: 11;
  • the hGH intron inserted in has the sequence shown in SEQ ID NO:13;
  • the modified wtAAV2 intron has a sequence as shown in SEQ ID NO:14; and/or
  • the non-parental serotype promoter is wtAAV7 P5 promoter, which has the sequence shown in SEQ ID NO:15.
  • a method for producing a recombinant adeno-associated virus characterized in that the plasmid described herein or all plasmids in the plasmid system are transfected into a host cell, cultured, and isolated to obtain the recombinant adeno-associated virus.
  • the production method is characterized in that the ratio of the plasmid containing the Cap gene to the plasmid containing the Rep gene is at least 1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, or at least 4:1. In some embodiments, at least 3:1.
  • the production method is characterized in that the ratio of the plasmid containing the Cap gene to the plasmid containing the Rep gene is about 1: 1, about 1.5: 1, about 2: 1, about 2.5: 1, about 3: 1, about 3.5: 1, or about 4: 1. In some embodiments, about 3: 1.
  • the production method is characterized in that the ratio of the plasmid containing the Rep gene to the plasmid containing the recombinant adeno-associated virus encoding the heterologous nucleic acid sequence is at least 1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, or at least 4:1. In some embodiments, at least 2.5:1.
  • the production method is characterized in that the ratio of the plasmid containing the Rep gene to the plasmid containing the recombinant adeno-associated virus encoding the heterologous nucleic acid sequence is about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, or about 4:1. In some embodiments, about 2.5:1.
  • the production method is characterized in that, compared with the production method based on the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid, the recombinant adeno-associated virus produced by the method is:
  • the solidity is higher; optionally, the solidity value is at least 0.05, at least 0.10, at least 0.15, at least 0.20, at least 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, or at least 0.80 higher;
  • the genomic titer is higher; optionally, the genomic titer is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher;
  • the residual impurities associated with the Cap encoding sequence are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventional methods; and/or
  • the residual rcAAV is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventional methods.
  • a recombinant adeno-associated virus is provided, characterized in that it is obtained by the production method described herein.
  • the host cell culture method includes adherent culture or suspension culture.
  • the recombinant adeno-associated virus is compared with the recombinant adeno-associated virus produced by the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid:
  • the solidity is higher; optionally, the solidity value is at least 0.05, at least 0.10, at least 0.15, at least 0.20, at least 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, or at least 0.80 higher;
  • the genomic titer is higher; optionally, the genomic titer is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher;
  • the residual impurities associated with the Cap coding sequence are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventionally produced recombinant adeno-associated viruses; and/or
  • the residual rcAAV is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventionally produced recombinant adeno-associated viruses.
  • a host cell in one aspect of the present invention, characterized in that the host cell contains the plasmid described herein or all the plasmids in the plasmid system.
  • the host cell is selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.
  • the present invention provides a use of the recombinant nucleic acid described herein in the preparation of a drug. In one aspect, the present invention provides a use of the plasmid described herein in the preparation of a drug. In one aspect, the present invention provides a use of the plasmid system described herein in the preparation of a drug. In one aspect, the present invention provides a use of the recombinant adeno-associated virus described herein in the preparation of a drug. In one aspect, the present invention provides a use of the host cell described herein in the preparation of a drug.
  • a pharmaceutical preparation characterized in that the drug comprises the recombinant adeno-associated virus described herein, and a pharmaceutically acceptable carrier and excipient.
  • the present invention provides a use of the pharmaceutical preparation described herein in treating a disease.
  • a method for treating a disease comprises administering the recombinant adeno-associated virus or the pharmaceutical preparation described herein to a patient in need thereof.
  • the disease is caused by a gene mutation and/or gene deletion in a target gene in the human body.
  • a method which comprises the recombinant nucleic acid, plasmid, plasmid system, recombinant adeno-associated virus, and/or host cell described herein.
  • the present invention provides a series of technical solutions for the existing defects of AAV expression system, in order to achieve the purpose of increasing the yield, saving the high manufacturing cost, reducing the contamination impurities in the production process (such as replication-competent AAV (rcAAV), etc.), including but not limited to:
  • a variety of novel three-plasmid systems which place the Rep gene or the Cap gene on a helper plasmid or a target gene plasmid, and another plasmid expresses Rep or Cap alone.
  • no plasmid in the system contains both the Rep gene and the Cap gene.
  • a variety of novel two-plasmid systems are provided, which separate the Rep gene and the Cap gene, with one placed on a helper plasmid and the other placed on a target gene plasmid.
  • the AAV plasmid such as the traditional packaging plasmid, or the plasmid in the novel three-plasmid system or the novel two-plasmid system
  • modifying adding, replacing the P5 promoter, deleting non-essential sequences, inserting introns or changing the intron donor structure, and regulating the expression level and ratio of the Rep and Cap gene products
  • the specific modification method is as follows:
  • the P5 promoter upstream of the Rep gene; preferably, the P5 promoter has a sequence as shown in SEQ ID NO:4.
  • the mutated P5 promoter located downstream of the Cap gene or inserting the P5 promoter downstream of the Cap gene; preferably, the mutated P5 promoter has a sequence as shown in SEQ ID NO:8; more preferably, the corrected P5 promoter has a sequence as shown in SEQ ID NO:9; inserting a sequence having SEQ ID NO:4 downstream of the Cap gene.
  • GTACCA at the donor site of the intron is mutated to GTACGT; preferably, the intron before mutation has a sequence as shown in SEQ ID NO:10; more preferably, the intron after mutation of donor has a sequence as shown in SEQ ID NO:11.
  • LSP liver specific enhancer promoter
  • the hGH intron has a sequence as shown in SEQ ID NO:13.
  • the plasmid transformation includes the following modifications (the transformation method number is consistent with the aforementioned specific transformation method number):
  • the inserted AAV promoter (e.g., P5 promoter) is derived from the same parental AAV serotype as the source of the corresponding Rep gene and/or Cap gene to which it is operably linked (e.g., a P5 promoter derived from AAV2 is operably linked to the corresponding AAV2 Rep gene and/or AAV2 Cap gene).
  • the inserted AAV promoter (e.g., P5 promoter) is derived from a non-parental AAV serotype that is different from the source of the corresponding Rep gene and/or Cap gene to which it is operably linked (e.g., a P5 promoter derived from AAV7 is operably linked to the AAV2 Rep gene and/or AAV2 Cap gene).
  • the plasmid comprises a recombinant nucleic acid as described herein.
  • the plasmid is a packaging plasmid (RC plasmid) in a recombinant adeno-associated virus without a helper virus system.
  • the plasmid is a plasmid formed after the Rep or Cap gene of the packaging plasmid is split.
  • the plasmid is a helper plasmid (pHelper plasmid).
  • the plasmid is a plasmid (target gene plasmid) encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • a helper plasmid capable of expressing Cap protein is provided, characterized in that it contains a nucleic acid sequence encoding the Cap gene and a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production.
  • AAV adeno-associated virus
  • the auxiliary plasmid comprises at least one promoter selected from the group consisting of a P5 promoter, a P19 promoter, and a P40 promoter, and the promoter is operably linked to the Cap gene.
  • the auxiliary plasmid comprises a P5 promoter, a P19 promoter, and a P40 promoter.
  • the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, a sequence encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • the promoters are spaced apart by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides. In some embodiments, there are no spaced nucleotides between the promoters. In some embodiments, the promoter and the Cap gene are spaced apart by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides. In some embodiments, there are no spaced nucleotides between the promoters.
  • sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO: 1. In some embodiments, the sequence of the P19 promoter is T at a position relative to nucleotide 768 of SEQ ID NO: 1. In some embodiments, the sequence of the P19 promoter is 100% identical to nucleotides 741-890 of SEQ ID NO: 1 (except nucleotide 768).
  • the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO: 1. In some embodiments, the sequence of the P40 promoter is GT at a position relative to nucleotides 1911-1912 of SEQ ID NO: 1. In some embodiments, the sequence of the P40 promoter is 100% identical to nucleotides 1701-2202 of SEQ ID NO: 1 (except nucleotides 1911-1912).
  • the Cap gene encodes a functional Cap protein.
  • the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO: 1.
  • the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO: 48-57.
  • the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • the Cap gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the Cap gene and the polyA sequence. In some embodiments, the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • the helper plasmid is optimized for expression of the Cap gene in the manner described herein, including but not limited to one or more of the following optimization methods:
  • a non-parental serotype promoter is contained downstream of the Cap gene and is operably linked to the Cap gene.
  • the helper plasmid cannot express a functional Rep protein. In some embodiments, the helper plasmid does not contain a nucleic acid sequence encoding a Rep gene.
  • the helper plasmid can only express specific Rep proteins, but cannot express other Rep proteins.
  • the helper plasmid can only express Rep78 and Rep52 proteins, but cannot express Rep68 and Rep40 proteins.
  • the helper plasmid contains a helper gene for AAV production comprising one or more genes selected from E2A, E4, and VA RNA. In some embodiments, the helper plasmid contains a helper gene for AAV production comprising a gene selected from E2A, E4, and VA RNA. In some embodiments, the helper plasmid contains an E2A gene. In some embodiments, the helper plasmid contains an E4 gene. In some embodiments, the helper plasmid contains a VA RNA gene.
  • the auxiliary genes for AAV production contained in the auxiliary plasmid include at least two genes selected from E2A, E4, and VA RNA. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E2A, E4 genes, but do not include VA RNA genes. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E2A, VA RNA genes, but do not include E4 genes. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E4, VA RNA genes, but do not include E2A genes.
  • the auxiliary gene not contained in the auxiliary plasmid is present in another plasmid in the same AAV plasmid system. In some embodiments, the auxiliary gene not contained in the auxiliary plasmid is present in another plasmid expressing Rep protein in the same AAV plasmid system.
  • the helper plasmid contains helper genes for AAV production including E2A, E4, and VA RNA genes.
  • a helper plasmid capable of expressing Rep protein is provided, characterized in that it contains a nucleic acid sequence encoding the Rep gene and a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production.
  • AAV adeno-associated virus
  • the Rep gene encodes a functional Rep protein.
  • the sequence of the Rep gene comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO: 1.
  • the sequence of the Rep gene comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.
  • the nucleic acid sequence encoding the Rep gene encodes a polyA sequence, and the polyA sequence is located downstream of the Rep gene.
  • the Rep gene and the polyA sequence are separated by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides.
  • the polyA sequence has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with nucleotides 4411-4460 of SEQ ID NO:1.
  • the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.
  • the Rep gene expresses Rep78 and Rep52 proteins, but does not express Rep68 and Rep40 proteins. In some embodiments, the Rep gene expresses Rep68 and Rep40 proteins, but does not express Rep78 and Rep52 proteins.
  • the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably linked to the Rep gene.
  • the helper plasmid is optimized for expression of the Rep gene in the manner described herein, including but not limited to one or more of the following optimization methods:
  • the donor site of the original intron of the Rep gene contains a mutation, and the donor site corresponds to the nucleotide sites 1717-1722 of SEQ ID NO:7;
  • a non-parental serotype promoter is contained upstream of the Rep gene and is operably linked to the Rep gene.
  • the helper plasmid cannot express a functional Cap protein. In some embodiments, the helper plasmid does not contain a nucleic acid sequence encoding a Cap gene.
  • the helper plasmid contains a helper gene for AAV production comprising one or more genes selected from E2A, E4, and VA RNA. In some embodiments, the helper plasmid contains a helper gene for AAV production comprising a gene selected from E2A, E4, and VA RNA. In some embodiments, the helper plasmid contains an E2A gene. In some embodiments, the helper plasmid contains an E4 gene. In some embodiments, the helper plasmid contains a VA RNA gene.
  • the auxiliary genes for AAV production contained in the auxiliary plasmid include at least two genes selected from E2A, E4, and VA RNA. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E2A, E4 genes, but do not include VA RNA genes. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E2A, VA RNA genes, but do not include E4 genes. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E4, VA RNA genes, but do not include E2A genes.
  • the auxiliary gene not contained in the auxiliary plasmid is present in another plasmid in the same AAV plasmid system. In some embodiments, the auxiliary gene not contained in the auxiliary plasmid is present in another plasmid expressing Rep protein in the same AAV plasmid system.
  • the helper plasmid contains helper genes for AAV production including E2A, E4, and VA RNA genes.
  • the helper plasmid described herein contains helper genes for AAV production.
  • the helper genes include one or more genes selected from E2A, E4, and VA RNA.
  • the helper plasmid contains a gene selected from E2A, E4, and VA RNA for the production of AAV.
  • the helper plasmid contains an E2A gene.
  • the helper plasmid contains an E4 gene.
  • the helper plasmid contains a VA RNA gene.
  • the auxiliary genes for AAV production contained in the auxiliary plasmid include at least two genes selected from E2A, E4, and VA RNA. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E2A, E4 genes, but do not include VA RNA genes. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E2A, VA RNA genes, but do not include E4 genes. In some embodiments, the auxiliary genes for AAV production contained in the auxiliary plasmid include E4, VA RNA genes, but do not include E2A genes.
  • the auxiliary gene not contained in the auxiliary plasmid is present in another plasmid in the same AAV plasmid system. In some embodiments, the auxiliary gene not contained in the auxiliary plasmid is present in another plasmid expressing Rep protein in the same AAV plasmid system.
  • the helper plasmid contains helper genes for AAV production including E2A, E4, and VA RNA genes.
  • the helper plasmid cannot express a functional Cap protein. In some embodiments, the helper plasmid does not contain a nucleic acid sequence encoding a Cap gene.
  • the helper plasmid cannot express a functional Rep protein. In some embodiments, the helper plasmid does not contain a nucleic acid sequence encoding a Rep gene.
  • a plasmid (pRep) for expressing Rep protein is provided, characterized in that it comprises a nucleic acid sequence encoding the Rep gene.
  • the plasmid cannot express a functional Cap protein.
  • the plasmid does not comprise a nucleic acid sequence encoding the Cap gene.
  • the plasmid for expressing the Rep protein cannot express a functional expression product of an auxiliary gene for AAV production. In some embodiments, the plasmid does not contain a nucleic acid sequence encoding an auxiliary gene for AAV production.
  • the plasmid for expressing the Rep protein comprises no more than one auxiliary gene for AAV production (e.g., when the auxiliary plasmid in the corresponding plasmid system lacks an essential auxiliary gene).
  • the plasmid for expressing the Rep protein comprises the auxiliary gene E2A.
  • the plasmid for expressing the Rep protein comprises the auxiliary gene E4.
  • the plasmid for expressing the Rep protein comprises the auxiliary gene VA RNA.
  • the plasmid used to express the Rep protein does not contain an AAV inverted repeat sequence (ITR).
  • ITR AAV inverted repeat sequence
  • the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.
  • the sequence of the Rep gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • the Rep gene expresses Rep78 and Rep52 proteins. In some embodiments, the Rep gene does not express Rep68 or Rep40 proteins. In some embodiments, the sequence of the Rep gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2196 of SEQ ID NO:1.
  • the Rep gene expresses Rep68 and Rep40 proteins. In some embodiments, the Rep gene does not express Rep78 or Rep52 proteins. In some embodiments, the gene expressing Rep68 and Rep40 proteins (Rep68/40) comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69.
  • the gene expressing Rep68 and Rep40 proteins comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:103.
  • the plasmid comprises a P5 promoter located upstream of the Rep gene, and the P5 promoter is operably linked to the Rep gene. In some embodiments, the plasmid comprises a P5 promoter located downstream of the Rep gene, and the P5 promoter is operably linked to the Rep gene. In some embodiments, the plasmid comprises a P5 promoter located upstream of the Rep gene and a P5 promoter located downstream of the Rep gene, and the two P5 promoters are operably linked to the Rep gene.
  • the promoter and the Rep gene are separated by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides. In some embodiments, there are no intervening nucleotides between the promoter and the Rep gene.
  • the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1. In some embodiments, the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-310 of SEQ ID NO:1.
  • the nucleic acid sequence encoding the Rep gene comprises an hGH intron.
  • the intron is located before the start codon of the Rep gene.
  • the P5 promoter is spaced from the hGH intron by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the P5 promoter and the hGH intron.
  • the hGH intron is spaced from the Rep gene by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the hGH intron and the Rep gene. In some embodiments, the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.
  • the donor site of the original intron of the Rep gene comprises a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO: 1.
  • the mutation of the donor site comprises mutating the CA equivalent to the nucleotides 1911-1912 of SEQ ID NO: 1 to GT.
  • the mutation of the donor site comprises mutating the donor site sequence equivalent to the nucleotides 1907-1912 of SEQ ID NO: 1 to GTACGT.
  • the nucleic acid sequence encoding the Rep gene encodes a polyA sequence, and the polyA sequence is located downstream of the Rep gene.
  • the Rep gene and the polyA sequence are separated by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides.
  • the polyA sequence has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with nucleotides 4411-4460 of SEQ ID NO:1.
  • the pRep plasmid comprises, from 5' to 3' direction, a P5 promoter, an hGH intron sequence, a Rep gene, and a polyA sequence.
  • the Rep gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • the Rep gene expresses four proteins, Rep78/68/52/40.
  • the GTACCA at the intron donor site in the Rep gene is mutated to GTACGT, specifically, the CA relative to nucleotides 1911-1912 of SEQ ID NO:1 is mutated to GT.
  • the hGH intron sequence comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13.
  • the polyA sequence comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO: 1.
  • the pRep plasmid comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 46 (pRep6).
  • the pRep plasmid is unable to express a functional Cap protein, or does not comprise a nucleic acid sequence encoding a Cap gene.
  • a non-limiting example of such a pRep plasmid is a pRep6 plasmid.
  • a plasmid (pCap) for expressing Cap protein characterized in that it comprises a nucleic acid sequence encoding a Cap gene.
  • the plasmid cannot express a functional Rep protein.
  • the plasmid does not comprise a nucleic acid sequence encoding a Rep gene.
  • the plasmid for expressing the Cap protein cannot express a functional expression product of an auxiliary gene for AAV production. In some embodiments, the plasmid does not contain a nucleic acid sequence encoding an auxiliary gene for AAV production.
  • the plasmid for expressing the Cap protein contains no more than one auxiliary gene for AAV production (e.g., when the auxiliary plasmid in the corresponding plasmid system lacks an essential auxiliary gene).
  • the plasmid for expressing the Rep protein contains the auxiliary gene E2A.
  • the plasmid for expressing the Cap protein contains the auxiliary gene E4.
  • the plasmid for expressing the Cap protein contains the auxiliary gene VA RNA.
  • the plasmid used to express the Cap protein does not contain an AAV inverted repeat sequence (ITR).
  • ITR AAV inverted repeat sequence
  • a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence
  • the heterologous nucleic acid sequence encodes a heterologous gene of interest (GOI).
  • the plasmid further comprises at least two AAV inverted repeat sequences (ITRs), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • the plasmid is responsible for producing a polynucleotide encoding a gene of interest packaged in a recombinant adeno-associated virus.
  • the heterologous target gene encodes an ND4 protein.
  • the ND4 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:112.
  • the heterologous target gene comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:104.
  • the heterologous target gene comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:105.
  • the heterologous target gene encodes an ND6 protein.
  • the ND6 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:113.
  • the heterologous target gene comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:106.
  • the heterologous target gene comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:107.
  • the heterologous gene of interest encodes an ND1 protein.
  • the ND1 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:114.
  • the heterologous gene of interest comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:108.
  • the heterologous gene of interest comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:109.
  • the heterologous target gene encodes an Opa1 protein.
  • the Opa1 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:115.
  • the heterologous target gene comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:110.
  • the heterologous target gene comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:111.
  • the heterologous gene of interest encodes an NRF2 protein.
  • the NRF2 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:116.
  • a plasmid comprising a Cap gene and encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence
  • the heterologous nucleic acid sequence encodes a heterologous gene of interest (GOI).
  • the plasmid further comprises at least two AAV inverted repeat sequences (ITRs), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • the plasmid is responsible for producing a polynucleotide encoding a gene of interest packaged in a recombinant adeno-associated virus.
  • the pGOI-Cap plasmid comprises at least one promoter selected from the group consisting of a P5 promoter, a P19 promoter, and a P40 promoter, and the promoter is operably linked to the Cap gene.
  • the pGOI-Cap plasmid comprises a P5 promoter, a P19 promoter, and a P40 promoter.
  • the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, sequences encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • the promoters are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the promoters. In some embodiments, the promoter and the Cap gene are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the promoters.
  • sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO: 1. In some embodiments, the sequence of the P19 promoter is T at a position relative to nucleotide 768 of SEQ ID NO: 1. In some embodiments, the sequence of the P19 promoter is 100% identical to nucleotides 741-890 of SEQ ID NO: 1 (except nucleotide 768).
  • the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO: 1. In some embodiments, the sequence of the P40 promoter is GT at a position relative to nucleotides 1911-1912 of SEQ ID NO: 1. In some embodiments, the sequence of the P40 promoter is 100% identical to nucleotides 1701-2202 of SEQ ID NO: 1 (except nucleotides 1911-1912).
  • the Cap gene encodes a functional Cap protein.
  • the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO: 1.
  • the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO: 48-57.
  • the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • the Cap gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the Cap gene and the polyA sequence. In some embodiments, the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • the pGOI-Cap plasmid is optimized for expression of the Cap gene in the manner described herein, including but not limited to one or more of the following optimization methods:
  • a non-parental serotype promoter is contained downstream of the Cap gene and is operably linked to the Cap gene.
  • the pGOI-Cap plasmid cannot express a functional Rep protein. In some embodiments, the pGOI-Cap plasmid does not contain a nucleic acid sequence encoding a Rep gene.
  • the pGOI-Cap plasmid can only express specific Rep proteins, but cannot express other Rep proteins.
  • the pGOI-Cap plasmid can only express Rep78 and Rep52 proteins, but cannot express Rep68 and Rep40 proteins.
  • the three-plasmid system described herein comprises (i) a helper plasmid, (ii) a packaging plasmid comprising a Rep gene and a Cap gene; and (iii) a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence.
  • a helper plasmid for example, a traditional three-plasmid system as shown in FIG. 1 .
  • the Rep gene and/or the Cap gene of this plasmid system are optimized and designed and modified in the manner described herein to obtain technical effects such as increased yield, cost savings, and reduced contamination impurities.
  • Rep and Cap become a separate plasmid, and together with the auxiliary plasmid and the vector plasmid, they form a four-plasmid system.
  • adding plasmids will increase the investment cost. Studies have found that only 5% of the initial plasmids are transfected into cells during the transfection process, and the plasmids that eventually enter the cell nucleus account for about 0.6% of the total input plasmids. Therefore, the probability of four plasmids entering the same cell nucleus at the same time is even lower, which is very unfavorable for the production of rAAV.
  • Novel three-plasmid system In one aspect of the present invention, a variety of novel three-plasmid systems are provided, which place the Rep gene or the Cap gene on a helper plasmid or a carrier plasmid, and another plasmid expresses Rep or Cap alone. In some embodiments, no plasmid in the system contains both the Rep gene and the Cap gene.
  • a three-plasmid system characterized in that it comprises: (i) an auxiliary plasmid (pHelper plasmid), (ii) a plasmid comprising a Rep gene or a Cap gene, and (iii) a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence, wherein one plasmid comprises a recombinant nucleic acid encoding a Rep gene as described herein, and the other plasmid comprises a recombinant nucleic acid encoding a Cap gene as described herein.
  • pHelper plasmid a auxiliary plasmid
  • a plasmid comprising a Rep gene or a Cap gene
  • a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence
  • one plasmid comprises a recombinant nucleic acid
  • the auxiliary plasmid described in (i) comprises the Rep gene, and the plasmid described in (ii) comprises the Cap gene.
  • the plasmid described in (ii) comprises the Rep gene, and the plasmid of the recombinant adeno-associated virus described in (iii) comprises the Cap gene.
  • the auxiliary plasmid described in (i) comprises the Cap gene, and the plasmid described in (ii) comprises the Rep gene.
  • the auxiliary plasmid contains Rep, so that the new auxiliary plasmid can become a public plasmid.
  • Rep When other projects are carried out, it will not increase production costs, nor will it need to change the vector plasmid where the target gene is located, which is safer.
  • the Rep or Cap gene is placed on an auxiliary plasmid or a carrier plasmid, and another plasmid expresses Rep or Cap alone.
  • the novel plasmid system described herein splits Cap onto an auxiliary plasmid, and Rep is a separate plasmid.
  • no single plasmid in the system contains both a Rep gene and a Cap gene.
  • the novel three-plasmid system described herein is composed of pHelper-Rep plasmid, pCap plasmid, and pGOI plasmid. In some embodiments, the novel three-plasmid system has a structure as shown in FIG. 2A .
  • the novel three-plasmid system described herein is composed of pHelper plasmid, pRep plasmid, and pGOI-Cap plasmid. In some embodiments, the novel three-plasmid system has a structure as shown in FIG. 2B .
  • the novel three-plasmid system described herein is composed of pHelper-Cap plasmid, pRep plasmid, and pGOI plasmid. In some embodiments, the novel three-plasmid system has a structure as shown in FIG. 24 .
  • one or more of all the essential accessory genes required for expression of AAV are contained on the plasmid expressing Rep, while the remaining essential accessory genes are contained on the plasmid expressing Cap.
  • the three-plasmid system described herein has one or more of the following modifications:
  • the P5 promoter upstream of the Rep gene; preferably, the P5 promoter has a sequence as shown in SEQ ID NO:4.
  • the mutated P5 promoter located downstream of the Cap gene or inserting the P5 promoter downstream of the Cap gene; preferably, the mutated P5 promoter has a sequence as shown in SEQ ID NO:8; more preferably, the corrected P5 promoter has a sequence as shown in SEQ ID NO:9; more preferably, a sequence having SEQ ID NO:4 is inserted downstream of the Cap gene.
  • GTACCA at the donor site of the intron is mutated to GTACGT; preferably, the intron before mutation has a sequence as shown in SEQ ID NO:10; more preferably, the intron after mutation of donor has a sequence as shown in SEQ ID NO:11.
  • LSP liver specific enhancer promoter
  • the hGH intron has a sequence as shown in SEQ ID NO:13.
  • the novel three-plasmid system comprises the following modifications (the modification method sequence number is consistent with the aforementioned specific modification method sequence number):
  • a three-plasmid system for adeno-associated virus (AAV) expression comprises: (i) a helper plasmid containing the Rep gene (pHelper-Rep helper plasmid); (ii) a plasmid containing the Cap gene (pCap plasmid), and (iii) a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence (pGOI plasmid).
  • the helper plasmid of the three-plasmid system cannot express a functional Cap protein, and/or the plasmid does not contain a Cap gene.
  • the plasmid containing the Cap gene in the three-plasmid system cannot express a functional Rep protein, and/or the plasmid does not contain the Rep gene.
  • the auxiliary plasmid containing the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins.
  • the pCap plasmid (ii) of the three-plasmid system can express Rep78 and/or Rep52 proteins but does not express Rep68 and Rep40 proteins.
  • the plasmid (iii) of the three-plasmid system containing a heterologous nucleic acid sequence can express Rep78 and/or Rep52 proteins but does not express Rep68 and Rep40 proteins.
  • the auxiliary plasmid containing the Rep gene expresses Rep78 and Rep52 proteins but does not express Rep68 or Rep40 proteins.
  • the pCap plasmid (ii) of the three-plasmid system can express Rep68 and/or Rep40 proteins, but does not express Rep78 and Rep52 proteins.
  • the plasmid (iii) of the three-plasmid system containing a heterologous nucleic acid sequence can express Rep68 and/or Rep40 proteins, but does not express Rep78 and Rep52 proteins.
  • the helper gene for AAV production contained in (i) the helper plasmid of the three-plasmid system comprises one or more genes selected from E2A, E4, and VA RNA.
  • the helper gene for AAV production contained in the helper plasmid comprises a gene selected from E2A, E4, and VA RNA.
  • the helper plasmid contains the E2A gene.
  • the helper plasmid contains the E4 gene.
  • the helper plasmid contains the VA RNA gene.
  • the auxiliary genes for AAV production contained in the (i) helper plasmid of the three-plasmid system include at least two genes selected from E2A, E4, and VA RNA.
  • the auxiliary genes for AAV production contained in the helper plasmid include E2A, E4 genes, but do not include VA RNA genes.
  • the auxiliary genes for AAV production contained in the helper plasmid include E2A, VA RNA genes, but do not include E4 genes.
  • the auxiliary genes for AAV production contained in the helper plasmid include E4, VA RNA genes, but do not include E2A genes.
  • the auxiliary gene not contained in the helper plasmid is present in another plasmid in the plasmid system. In some embodiments, the auxiliary gene not contained in the helper plasmid is present in a plasmid containing a Cap gene. In some embodiments, the auxiliary gene not contained in the helper plasmid is present in a pGOI plasmid containing a heterologous nucleic acid sequence.
  • the plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence in the three-plasmid system further comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • the three-plasmid system is characterized in that the serotype of AAV is AAV2.
  • Embodiment A1 A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that it comprises:
  • helper plasmid which contains a recombinant nucleic acid encoding a Rep gene
  • plasmid comprising a Cap gene; optionally, the plasmid (a) is unable to express a functional Rep protein, and/or (b) does not comprise a nucleic acid sequence encoding a Rep gene; and
  • Embodiment A2 The plasmid system according to Embodiment A1 is characterized in that the auxiliary plasmid cannot express a functional Cap protein and/or does not contain a nucleic acid sequence encoding a Cap gene.
  • Embodiment A3 A plasmid system according to any one of Embodiments A1-A2, characterized in that the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.
  • Embodiment A4 According to the plasmid system described in Embodiment A3, it is characterized in that the sequence of the Rep gene contains a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • the plasmid system is characterized in that the Rep gene expresses Rep78 and Rep52 proteins; optionally, the Rep gene does not express Rep68 or Rep40 proteins; optionally, the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • Embodiment A6 A plasmid system according to any one of Embodiments A1-A2, characterized in that the Rep gene expresses Rep68 and Rep40 proteins; optionally, the Rep gene does not express Rep78 or Rep52 proteins.
  • Embodiment A7 A plasmid system according to Embodiment A6, characterized in that the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:103.
  • Embodiment A8 The plasmid system according to any one of Embodiments A1 to A7, characterized in that the plasmid system does not contain genes for co-expressing Rep52 protein and Rep40 protein.
  • Embodiment A9 A plasmid system according to any one of Embodiments A1 to A8, characterized in that the upstream of the Rep gene contains an hGH intron.
  • Embodiment A10 The plasmid system according to Embodiment A9, characterized in that the upstream of the hGH intron contains a P5 promoter.
  • Embodiment A11 A plasmid system according to any one of embodiments A9-A10, characterized in that the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13.
  • Embodiment A12 A plasmid system according to any one of Embodiments A10-A11, characterized in that there is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides between the P5 promoter and the hGH intron, and/or between the hGH intron and the Rep gene; preferably, there are no intervening nucleotides.
  • Embodiment A13 A plasmid system according to any one of Embodiments A1-A12, characterized in that the downstream of the Rep gene contains a polyA sequence; optionally, the Rep gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment A14 A plasmid system according to any one of embodiments A12-A13, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment A15 The plasmid system according to any one of Embodiments A1 to A14, characterized in that the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably linked to the Rep gene.
  • Embodiment A16 A plasmid system according to embodiment A15, characterized in that the spacing between the promoter and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment A17 The plasmid system according to any one of embodiments A1-A16 is characterized in that the donor site of the original intron of the Rep gene contains a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO:1.
  • Embodiment A18 According to the plasmid system described in Embodiment A17, it is characterized in that the mutation of the donor site comprises mutating CA equivalent to nucleotides at positions 1911-1912 of SEQ ID NO:1 to GT, or mutating the donor site sequence equivalent to nucleotides at positions 1907-1912 of SEQ ID NO:1 to GTACGT.
  • Embodiment A19 The plasmid system according to any one of Embodiments A1 to A18, characterized in that it comprises at least one promoter selected from the group consisting of a P5 promoter, a P19 promoter, and a P40 promoter, and the promoter is operably linked to the Cap gene.
  • Embodiment A20 The plasmid system according to Embodiment A19 is characterized in that it comprises a P5 promoter, a P19 promoter, and a P40 promoter; preferably, the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, a sequence encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • Embodiment A21 A plasmid system according to any one of Embodiments A19-A20, characterized in that the spacing between the promoters and/or between the promoter and the Cap gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment A22 A plasmid system according to any one of embodiments A19-A21, characterized in that the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • Embodiment A23 A plasmid system according to any one of embodiments A19-A22, characterized in that the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO:1; preferably, the sequence of the P19 promoter is T at the position relative to nucleotide 768 of SEQ ID NO:1.
  • Embodiment A24 A plasmid system according to any one of embodiments A19-A23, characterized in that the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO:1; preferably, the sequence of the P40 promoter is GT relative to nucleotides 1911-1912 of SEQ ID NO:1.
  • Embodiment A25 A plasmid system according to any one of embodiments A1-A24, characterized in that the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO:1.
  • Embodiment A26 A plasmid system according to any one of embodiments A1-A24, characterized in that the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:48-57.
  • Embodiment A27 A plasmid system according to embodiment A26, characterized in that the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:58-67.
  • Embodiment A28 A plasmid system according to any one of Embodiments A1 to A27, characterized in that the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • Embodiment A29 A plasmid system according to embodiment A28, characterized in that the Cap gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment A30 A plasmid system according to any one of embodiments A28-A29, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment A31 The plasmid system according to any one of Embodiments A1-A30 is characterized in that it further comprises a P5 promoter downstream of the Cap gene, which is operably connected to the Cap gene; optionally, the P5 promoter is located downstream of the polyA sequence downstream of the Cap gene; optionally, the P5 promoter and the Cap gene, or the P5 promoter and the polyA sequence, are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • Embodiment A32 A plasmid system according to any one of embodiments A1-A31, characterized in that the (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment A33 A plasmid system according to any one of Embodiments A1 to A32, characterized in that the heterologous nucleic acid sequence encodes a heterologous gene.
  • Embodiment A34 A plasmid system according to any one of Embodiments A1 to A33, characterized in that the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or AAV-DJ; preferably, the serotype of the AAV is AAV2, AAV5, AAV8, or AAV9; more preferably, the serotype of the AAV is AAV2.
  • Three-plasmid system including pHelper plasmid, pRep plasmid, and pGOI-Cap plasmid
  • a three-plasmid system for adeno-associated virus (AAV) expression comprises: (i) a helper plasmid; (ii) a plasmid containing the Rep gene (pRep plasmid), and (iii) a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence and containing the Cap gene (pGOI-Cap plasmid) as described herein.
  • the pGOI-Cap plasmid of the three-plasmid system cannot express a functional Rep protein, and/or the plasmid does not contain Rep.
  • the plasmid comprising the Rep gene in the three-plasmid system cannot express a functional Cap protein, and/or the plasmid does not comprise the Cap gene.
  • the plasmid comprising the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins.
  • the auxiliary plasmid (i) of the three-plasmid system can express Rep78 and/or Rep52 proteins but does not express Rep68 and Rep40 proteins.
  • the plasmid (iii) of the three-plasmid system comprising a heterologous nucleic acid sequence and a Cap gene can express Rep78 and/or Rep52 proteins but does not express Rep68 and Rep40 proteins.
  • the plasmid comprising the Rep gene expresses Rep78 and Rep52 proteins but does not express Rep68 or Rep40 proteins.
  • the (i) auxiliary plasmid of the three-plasmid system can express Rep68 and/or Rep40 proteins, but does not express Rep78 and Rep52 proteins.
  • the (iii) plasmid comprising a heterologous nucleic acid sequence and a Cap gene of the three-plasmid system can express Rep68 and/or Rep40 proteins, but does not express Rep78 and Rep52 proteins.
  • the helper gene for AAV production contained in (i) the helper plasmid of the three-plasmid system comprises one or more genes selected from E2A, E4, and VA RNA.
  • the helper gene for AAV production contained in the helper plasmid comprises a gene selected from E2A, E4, and VA RNA.
  • the helper plasmid contains the E2A gene.
  • the helper plasmid contains the E4 gene.
  • the helper plasmid contains the VA RNA gene.
  • the auxiliary genes for AAV production contained in the (i) helper plasmid of the three-plasmid system include at least two genes selected from E2A, E4, and VA RNA.
  • the auxiliary genes for AAV production contained in the helper plasmid include E2A, E4 genes, but do not include VA RNA genes.
  • the auxiliary genes for AAV production contained in the helper plasmid include E2A, VA RNA genes, but do not include E4 genes.
  • the auxiliary genes for AAV production contained in the helper plasmid include E4, VA RNA genes, but do not include E2A genes.
  • the auxiliary gene not contained in the helper plasmid is present in another plasmid in the plasmid system. In some embodiments, the auxiliary gene not contained in the helper plasmid is present in a plasmid containing the Rep gene. In some embodiments, the auxiliary gene not contained in the helper plasmid is present in a pGOI plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence and containing a Cap gene.
  • the plasmid (iii) of the three-plasmid system encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence and containing a Cap gene further contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment B A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that it comprises:
  • Embodiment B2 The plasmid system according to Embodiment B1, characterized in that the plasmid (ii) cannot express a functional Cap protein and/or does not contain a nucleic acid sequence encoding a Cap gene.
  • Embodiment B3 A plasmid system according to any one of Embodiments B1-B2, characterized in that the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.
  • Embodiment B4 A plasmid system according to embodiment B3, characterized in that the sequence of the Rep gene comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • Embodiment B5. A plasmid system according to any one of Embodiments B1-B2, characterized in that the Rep gene expresses Rep78 and Rep52 proteins; optionally, the Rep gene does not express Rep68 or Rep40 proteins; optionally, the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • Embodiment B6 A plasmid system according to any one of Embodiments B1-B2, characterized in that the Rep gene expresses Rep68 and Rep40 proteins; optionally, the Rep gene does not express Rep78 or Rep52 proteins.
  • Embodiment B7 A plasmid system according to Embodiment B6, characterized in that the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:103.
  • Embodiment B8 A plasmid system according to any one of Embodiments B1 to B7, characterized in that the plasmid system does not contain genes for co-expressing Rep52 protein and Rep40 protein.
  • Embodiment B9 A plasmid system according to any one of Embodiments B1-B8, characterized in that the upstream of the Rep gene contains an hGH intron.
  • Embodiment B10 A plasmid system according to Embodiment B9, characterized in that the upstream of the hGH intron contains a P5 promoter.
  • Embodiment B11 A plasmid system according to any one of embodiments B9-B10, characterized in that the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13.
  • Embodiment B12 A plasmid system according to any one of Embodiments B10-B11, characterized in that the spacing between the P5 promoter and the hGH intron, and/or between the hGH intron and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment B13 A plasmid system according to any one of Embodiments B1-B12, characterized in that the downstream of the Rep gene contains a polyA sequence; optionally, the Rep gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment B14 A plasmid system according to any one of embodiments B12-B13, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment B15 The plasmid system according to any one of Embodiments B1 to B14, characterized in that the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably connected to the Rep gene.
  • Embodiment B16 A plasmid system according to Embodiment B15, characterized in that the spacing between the promoter and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment B17 The plasmid system according to any one of Embodiments B1-B16, characterized in that the donor site of the original intron of the Rep gene contains a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO:1.
  • Embodiment B18 According to the plasmid system described in Embodiment B17, it is characterized in that the mutation of the donor site comprises mutating CA equivalent to nucleotides at positions 1911-1912 of SEQ ID NO:1 to GT, or mutating the donor site sequence equivalent to nucleotides at positions 1907-1912 of SEQ ID NO:1 to GTACGT.
  • Embodiment B19 The plasmid system according to any one of Embodiments B1 to B18, characterized in that it comprises at least one promoter selected from the group consisting of a P5 promoter, a P19 promoter, and a P40 promoter, and the promoter is operably linked to the Cap gene.
  • Embodiment B20 The plasmid system according to Embodiment B19 is characterized in that it comprises a P5 promoter, a P19 promoter, and a P40 promoter; preferably, the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, a sequence encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • Embodiment B21 A plasmid system according to any one of Embodiments B19-B20, characterized in that the spacing between the promoters and/or between the promoter and the Cap gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment B22 A plasmid system according to any one of embodiments B19-B21, characterized in that the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • Embodiment B23 A plasmid system according to any one of embodiments B19-B22, characterized in that the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO:1; preferably, the sequence of the P19 promoter is T at the position relative to nucleotide 768 of SEQ ID NO:1.
  • Embodiment B24 A plasmid system according to any one of embodiments B19-B23, characterized in that the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO:1; preferably, the sequence of the P40 promoter is GT relative to nucleotides 1911-1912 of SEQ ID NO:1.
  • Embodiment B25 A plasmid system according to any one of embodiments B1-B24, characterized in that the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO:1.
  • Embodiment B26 A plasmid system according to any one of Embodiments B1-B24, characterized in that the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:48-57.
  • Embodiment B27 A plasmid system according to Embodiment B26, characterized in that the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:58-67.
  • Embodiment B28 A plasmid system according to any one of Embodiments B1 to B27, characterized in that the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • Embodiment B29 A plasmid system according to embodiment B28, characterized in that the Cap gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment B30 A plasmid system according to any one of embodiments B28-B29, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment B31 The plasmid system according to any one of Embodiments B1-B30, characterized in that it further comprises a P5 promoter downstream of the Cap gene, which is operably connected to the Cap gene; optionally, the P5 promoter is located downstream of the polyA sequence downstream of the Cap gene; optionally, the P5 promoter and the Cap gene, or the P5 promoter and the polyA sequence, are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • Embodiment B32 A plasmid system according to any one of embodiments B1-B31, characterized in that the (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment B33 A plasmid system according to any one of Embodiments B1 to B32, characterized in that the heterologous nucleic acid sequence encodes a heterologous gene.
  • Embodiment B34 A plasmid system according to any one of Embodiments B1 to B33, characterized in that the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or AAV-DJ; preferably, the serotype of the AAV is AAV2, AAV5, AAV8, or AAV9; more preferably, the serotype of the AAV is AAV2.
  • Three-plasmid system including pHelper-Cap plasmid, pRep plasmid, and pGOI plasmid
  • a three-plasmid system for adeno-associated virus (AAV) expression comprises: (i) a helper plasmid containing a Cap gene (pHelper-Cap helper plasmid); (ii) a plasmid containing a Rep gene (pRep plasmid), and (iii) a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence (pGOI plasmid).
  • the Cap gene is placed on the auxiliary plasmid pHelper, and another plasmid expresses the Rep plasmid alone.
  • these two plasmids can become common plasmids. When carrying out other projects, the cost of plasmid production will not be increased, and there is no need to add Rep or Cap sequences to the vector plasmid where the target gene is located, thereby reducing the risk of packaging these two elements.
  • the two plasmids can also be modified separately to regulate the expression of the Rep and Cap genes respectively, so that the yield is doubled.
  • the large-scale expression of the three auxiliary genes of the Cap gene and the pHelper plasmid is very beneficial to the increase in yield, and it is easy to control the feeding on one plasmid.
  • the three-plasmid system is conducive to the packaging of recombinant adeno-associated virus (rAAV).
  • the helper plasmid of the three-plasmid system cannot express a functional Rep protein, and/or the helper plasmid does not contain a Rep gene.
  • the plasmid comprising the Rep gene in the three-plasmid system cannot express a functional Cap protein, and/or the plasmid does not comprise the Cap gene.
  • the plasmid comprising the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins.
  • the auxiliary plasmid (i) of the three-plasmid system can express Rep78 and/or Rep52 proteins but does not express Rep68 and Rep40 proteins.
  • the plasmid (iii) of the three-plasmid system comprising a heterologous nucleic acid sequence can express Rep78 and/or Rep52 proteins but does not express Rep68 and Rep40 proteins.
  • the plasmid comprising the Rep gene expresses Rep78 and Rep52 proteins but does not express Rep68 or Rep40 proteins.
  • the auxiliary plasmid (i) of the three-plasmid system can express Rep68 and/or Rep40 proteins but does not express Rep78 and Rep52 proteins.
  • the plasmid (iii) comprising a heterologous nucleic acid sequence of the three-plasmid system can express Rep68 and/or Rep40 proteins but does not express Rep78 and Rep52 proteins.
  • the helper gene for AAV production contained in (i) the helper plasmid of the three-plasmid system comprises one or more genes selected from E2A, E4, and VA RNA.
  • the helper gene for AAV production contained in the helper plasmid comprises a gene selected from E2A, E4, and VA RNA.
  • the helper plasmid contains the E2A gene.
  • the helper plasmid contains the E4 gene.
  • the helper plasmid contains the VA RNA gene.
  • the auxiliary genes for AAV production contained in the (i) helper plasmid of the three-plasmid system include at least two genes selected from E2A, E4, and VA RNA.
  • the auxiliary genes for AAV production contained in the helper plasmid include E2A, E4 genes, but do not include VA RNA genes.
  • the auxiliary genes for AAV production contained in the helper plasmid include E2A, VA RNA genes, but do not include E4 genes.
  • the auxiliary genes for AAV production contained in the helper plasmid include E4, VA RNA genes, but do not include E2A genes.
  • the auxiliary gene not contained in the helper plasmid is present in another plasmid in the plasmid system. In some embodiments, the auxiliary gene not contained in the helper plasmid is present in a plasmid containing the Rep gene. In some embodiments, the auxiliary gene not contained in the helper plasmid is present in the pGOI plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the plasmid expressing Rep may also contain one or more auxiliary genes.
  • one or more of all the necessary auxiliary genes required for expressing AAV are contained on the plasmid expressing Rep, and the remaining necessary auxiliary genes are contained on the plasmid expressing Cap.
  • the plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence in the three-plasmid system further comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • the three-plasmid system comprises (i) a helper plasmid containing a Cap gene (pHelper-Cap helper plasmid); (ii) a plasmid containing a Rep gene (pRep plasmid), and (iii) a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence (pGOI plasmid).
  • the auxiliary plasmid containing the Cap gene comprises, from 5' to 3' direction, P5 promoter (e.g. 191-320 nt of wtAAV2), P19 promoter (e.g. 741-890 nt of wtAAV2, wherein the 768G is mutated to T), P40 promoter (e.g. 1701-2202 nt of wtAAV2, wherein the GTACCA at the intron donor site is mutated to GTACGT, specifically the 1911-1912 nt CA is mutated to GT), Cap gene, polyA sequence (e.g. 4411-4460 nt of wtAAV2);
  • P5 promoter e.g. 191-320 nt of wtAAV2
  • P19 promoter e.g. 741-890 nt of wtAAV2, wherein the 768G is mutated to T
  • P40 promoter e.g. 1701-2202 nt of wtAAV2, where
  • the plasmid (ii) containing the Rep gene contains the P5 promoter and the Rep gene (relative to the sequence of 191-2300 nt of wtAAV2, expressing four proteins Rep78/68/52/40, wherein the GTACCA at the intron donor site is mutated to GTACGT, specifically, the 1911-1912 nt CA is mutated to GT), and then the hGH intron sequence (for example, SEQ ID NO: 13) is inserted before the start codon AUG of the Rep gene (relative to the 321 nt of wtAAV2), and a polyA sequence is added after the 2300 nt of wtAAV2 (4411-4460 nt of wtAAV2).
  • Embodiment C A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that it comprises:
  • helper plasmid (i) a helper plasmid (pHelper plasmid), wherein the helper plasmid comprises a recombinant nucleic acid encoding a Cap gene;
  • Embodiment C2 A plasmid system according to Embodiment C1, characterized in that the (i) auxiliary plasmid cannot express a functional Rep protein, and/or the (ii) plasmid encoding the Rep gene cannot express a functional Cap protein.
  • Embodiment C3 A plasmid system according to any one of Embodiments C1-C2, characterized in that the upstream of the Cap gene contains three promoters, which from 5' to 3' direction are: P5 promoter, P19 promoter, and P40 promoter, all of which are operably linked to the Cap gene, and the downstream of the Cap gene contains a polyA sequence.
  • Embodiment C4 A plasmid system according to any one of embodiments C1-C3, characterized in that the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or AAV-DJ; preferably, the serotype of the AAV is AAV2, AAV5, AAV8, or AAV9; more preferably, the serotype of the AAV is AAV2.
  • Implementation C5. The plasmid system according to any one of Implementation Options C1-C4, characterized in that the donor site of the original intron of the Rep gene contains a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO:1.
  • Embodiment C6 According to the plasmid system described in Embodiment C5, it is characterized in that the mutation of the donor site comprises mutating CA equivalent to nucleotides at positions 1911-1912 of SEQ ID NO:1 to GT, or mutating the donor site sequence equivalent to nucleotides at positions 1907-1912 of SEQ ID NO:1 to GTACGT.
  • Embodiment C7 A plasmid system according to any one of Embodiments C1-C6, characterized in that the Rep gene expresses Rep78, Rep68, Rep52 and Rep40 proteins.
  • Embodiment C8 According to the plasmid system described in Embodiment C7, it is characterized in that the sequence of the Rep gene contains a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • Embodiment C9 A plasmid system according to any one of embodiments C1-C8, characterized in that the Rep gene expresses Rep78 and Rep52 proteins; optionally, the Rep gene does not express Rep68 or Rep40 proteins; optionally, the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • Embodiment C10 According to the plasmid system described in embodiments C1-C9, it is characterized in that the sequence of the Rep gene contains a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2196 of SEQ ID NO:1.
  • Embodiment C11 A plasmid system according to any one of Embodiments C1-C10, characterized in that the Rep gene expresses Rep68 and Rep40 proteins; optionally, the Rep gene does not express Rep78 or Rep52 proteins.
  • Embodiment C12 A plasmid system according to embodiment C11, characterized in that the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:103.
  • Embodiment C13 A plasmid system according to any one of Embodiments C1 to C12, characterized in that the plasmid system does not contain genes for co-expressing Rep52 protein and Rep40 protein.
  • Embodiment C14 A plasmid system according to any one of Embodiments C1-C13, characterized in that the (ii) plasmid encoding the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins, and the (i) auxiliary plasmid contains a gene expressing Rep78 and/or Rep52 proteins; optionally, the gene does not express Rep68 or Rep40.
  • Embodiment C15 A plasmid system according to any one of embodiments C1 to C14, characterized in that the (ii) plasmid encoding the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins, and the (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence contains a gene expressing Rep78 and/or Rep52 proteins; optionally, the gene does not express Rep68 or Rep40.
  • Embodiment C16 A plasmid system according to any one of Embodiments C1-C15, characterized in that the (i) auxiliary plasmid contains the E4 gene and the VA RNA gene; and the (ii) plasmid encoding the Rep gene contains the E2A gene.
  • Embodiment C17 A plasmid system according to any one of Embodiments C1 to C16, characterized in that the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably linked to the Rep gene.
  • Embodiment C18 A plasmid system according to embodiment C17, characterized in that the spacing between the promoter and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment C19 According to any one of embodiments C17-C18, the plasmid system is characterized in that the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1, or to nucleotides 191-310 of SEQ ID NO:1.
  • Embodiment C20 A plasmid system according to any one of Embodiments C1-C19, characterized in that the upstream of the Rep gene contains an hGH intron.
  • Embodiment C21 A plasmid system according to embodiment C20, characterized in that the upstream of the hGH intron contains a P5 promoter.
  • Embodiment C22 A plasmid system according to any one of embodiments C20-C21, characterized in that the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13.
  • Embodiment C23 A plasmid system according to any one of Embodiments C21-C22, characterized in that there is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides between the P5 promoter and the hGH intron, and/or between the hGH intron and the Rep gene; preferably, there are no intervening nucleotides.
  • Embodiment C24 A plasmid system according to any one of Embodiments C1 to C23, characterized in that the downstream of the Rep gene contains a polyA sequence.
  • Embodiment C25 A plasmid system according to embodiment C24, characterized in that the interval between the Rep gene and the polyA sequence is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment C26 A plasmid system according to any one of embodiments C24-C25, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment C27 A plasmid system according to any one of Embodiments C1 to C26, characterized in that the heterologous nucleic acid sequence encodes a heterologous gene.
  • Embodiment C28 A plasmid system according to any one of embodiments C1-C27, characterized in that the (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment C29 A plasmid system according to any one of Embodiments C1-C28, characterized in that it comprises a P5 promoter, a P19 promoter, and a P40 promoter; preferably, the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, a sequence encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • Embodiment C30 A plasmid system according to Embodiment C29, characterized in that there are no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides between the promoters and/or between the promoter and the Cap gene; preferably, there are no intervening nucleotides.
  • Embodiment C31 A plasmid system according to any one of embodiments C29-C30, characterized in that the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • Embodiment C32 A plasmid system according to any one of embodiments C29-C31, characterized in that the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO:1; preferably, the sequence of the P19 promoter is T at the position relative to nucleotide 768 of SEQ ID NO:1.
  • Embodiment C33 A plasmid system according to any one of embodiments C29-C32, characterized in that the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO:1; preferably, the sequence of the P40 promoter is GT relative to nucleotides 1911-1912 of SEQ ID NO:1.
  • Embodiment C34 A plasmid system according to any one of embodiments C1-C33, characterized in that the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO:1.
  • Embodiment C35 A plasmid system according to any one of embodiments C1-C34, characterized in that the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:48-57.
  • Embodiment C36 A plasmid system according to embodiment C35, characterized in that the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:58-67.
  • Embodiment C37 A plasmid system according to any one of Embodiments C1 to C36, characterized in that the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • Embodiment C38 A plasmid system according to embodiment C37, characterized in that the Cap gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment C39 A plasmid system according to any one of embodiments C37-C38, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment C40 A plasmid system according to any one of Embodiments C1-C39, characterized in that it further comprises a P5 promoter downstream of the Cap gene, which is operably connected to the Cap gene; optionally, the P5 promoter is located downstream of the polyA sequence downstream of the Cap gene; optionally, the P5 promoter and the Cap gene, or the P5 promoter and the polyA sequence, are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • a two-plasmid system characterized in that it comprises a first plasmid containing the Rep gene and a second plasmid containing the Cap gene, and the Rep and Cap genes are located on different plasmids, respectively.
  • the two-plasmid system described herein splits the Rep and Cap genes into auxiliary plasmids and vector plasmids, respectively.
  • the system places Rep on an auxiliary plasmid (i.e., forming a pHelper-Rep plasmid) and places Cap on a vector plasmid.
  • this plasmid system is simpler and more flexible in form, and pHelper-Rep can be used as a common plasmid, which not only improves the economy of production and manufacturing, but also does not need to change the vector plasmid where the target gene is located, thereby enhancing the safety and quality attributes of the vector preparation.
  • a two-plasmid system characterized in that it comprises a first plasmid containing the Rep gene of AAV and a second plasmid containing the Cap gene of AAV, and the Rep gene and the Cap gene are located on different plasmids.
  • the two-plasmid system is characterized in that the first plasmid containing the Rep gene is a helper plasmid (pHelper plasmid), and the second plasmid containing the Cap gene is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the second plasmid containing the Cap gene is a helper plasmid (pHelper plasmid)
  • the first plasmid containing the Rep gene is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the Rep and Cap genes are located on two different plasmids, respectively.
  • any plasmid contains only one of the Rep gene and the Cap gene, but does not contain both the Rep gene and the Cap gene.
  • the two-plasmid system consists of a pHelper-Rep plasmid or a pGOI-Cap plasmid.
  • the pHelper-Rep plasmid is a helper plasmid containing a Rep gene
  • the pGOI-Cap plasmid is a plasmid containing a Cap gene and encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the two-plasmid system consists of pGOI-Rep and pHelper-Cap.
  • the pHelper-Cap plasmid is a helper plasmid containing the Cap gene
  • the pGOI-Rep plasmid is a plasmid containing the Rep gene and encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a heterologous gene.
  • the two-plasmid system has one or more of the following modifications:
  • the P5 promoter upstream of the Rep gene; preferably, the P5 promoter has a sequence as shown in SEQ ID NO:4.
  • the mutated P5 promoter located downstream of the Cap gene or inserting the P5 promoter downstream of the Cap gene; preferably, the mutated P5 promoter has a sequence as shown in SEQ ID NO:8; more preferably, the corrected P5 promoter has a sequence as shown in SEQ ID NO:9; inserting a sequence having SEQ ID NO:4 downstream of the Cap gene.
  • GTACCA at the donor site of the intron is mutated to GTACGT; preferably, the intron before mutation has a sequence as shown in SEQ ID NO:10; more preferably, the intron after mutation of donor has a sequence as shown in SEQ ID NO:11.
  • LSP liver specific enhancer promoter
  • the hGH intron has a sequence as shown in SEQ ID NO:13.
  • the two-plasmid system includes the following modifications (the modification method number is consistent with the aforementioned specific modification method number):
  • Embodiment D1 A two-plasmid system, characterized in that it comprises a first plasmid containing the Rep gene of AAV and a second plasmid containing the Cap gene of AAV, wherein the Rep gene and the Cap gene are located on different plasmids, respectively, the first plasmid containing the Rep gene is a helper plasmid (pHelper plasmid), and the second plasmid containing the Cap gene is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • pHelper plasmid helper plasmid
  • the second plasmid containing the Cap gene is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • Embodiment D2 according to the plasmid system described in Embodiment D1, is characterized in that the auxiliary plasmid contains one or more auxiliary genes selected from E2A, E4, and VA RNA.
  • Embodiment D3 A plasmid system according to any one of Embodiments D1-D2, characterized in that the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.
  • Embodiment D4 According to the plasmid system described in Embodiment D3, it is characterized in that the sequence of the Rep gene contains a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • Embodiment D5 A plasmid system according to any one of Embodiments D1-D2, characterized in that the Rep gene expresses Rep78 and Rep52 proteins; optionally, the Rep gene does not express Rep68 or Rep40 proteins; optionally, the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • Embodiment D6 A plasmid system according to any one of Embodiments D1-D2, characterized in that the Rep gene expresses Rep68 and Rep40 proteins; optionally, the Rep gene does not express Rep78 or Rep52 proteins.
  • Embodiment D7 A plasmid system according to Embodiment D6, characterized in that the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:103.
  • Embodiment D8 A plasmid system according to any one of Embodiments D1-D7, characterized in that the plasmid system does not contain genes for co-expressing Rep52 protein and Rep40 protein.
  • Embodiment D9 A plasmid system according to any one of embodiments D1-D8, characterized in that the upstream of the Rep gene contains an hGH intron.
  • Embodiment D10 A plasmid system according to embodiment D9, characterized in that the upstream of the hGH intron contains a P5 promoter.
  • Embodiment D11 A plasmid system according to any one of embodiments D9-D10, characterized in that the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13.
  • Embodiment D12 A plasmid system according to any one of embodiments D10-D11, characterized in that the spacing between the P5 promoter and the hGH intron, and/or between the hGH intron and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment D13 A plasmid system according to any one of embodiments D1-D12, characterized in that the downstream of the Rep gene contains a polyA sequence; optionally, the Rep gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment D14 A plasmid system according to any one of embodiments D12-D13, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment D15 The plasmid system according to any one of Embodiments D1 to D14, characterized in that the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably connected to the Rep gene.
  • Embodiment D16 A plasmid system according to embodiment D15, characterized in that the spacing between the promoter and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment D17 The plasmid system according to any one of Embodiments D1-D16 is characterized in that the donor site of the original intron of the Rep gene contains a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO:1.
  • Embodiment D18 According to the plasmid system described in Embodiment D17, it is characterized in that the mutation of the donor site comprises mutating CA equivalent to nucleotides at positions 1911-1912 of SEQ ID NO:1 to GT, or mutating the donor site sequence equivalent to nucleotides at positions 1907-1912 of SEQ ID NO:1 to GTACGT.
  • Embodiment D19 The plasmid system according to any one of Embodiments D1-D18, characterized in that it comprises at least one promoter selected from the group consisting of P5 promoter, P19 promoter, and P40 promoter, and the promoter is operably linked to the Cap gene.
  • Embodiment D20 According to the plasmid system described in Embodiment D19, it is characterized in that it comprises a P5 promoter, a P19 promoter, and a P40 promoter; preferably, the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, a sequence encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • Embodiment D21 A plasmid system according to any one of Embodiments D19-D20, characterized in that the spacing between the promoters and/or between the promoter and the Cap gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment D22 A plasmid system according to any one of embodiments D19-D21, characterized in that the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • Embodiment D23 A plasmid system according to any one of embodiments D19-D22, characterized in that the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO:1; preferably, the sequence of the P19 promoter is T at the position relative to nucleotide 768 of SEQ ID NO:1.
  • Embodiment D24 A plasmid system according to any one of embodiments D19-D23, characterized in that the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO:1; preferably, the sequence of the P40 promoter is GT relative to nucleotides 1911-1912 of SEQ ID NO:1.
  • Embodiment D25 A plasmid system according to any one of embodiments D1-D24, characterized in that the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO:1.
  • Embodiment D26 A plasmid system according to any one of embodiments D1-D24, characterized in that the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:48-57.
  • Embodiment D27 A plasmid system according to embodiment D26, characterized in that the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:58-67.
  • Embodiment D28 A plasmid system according to any one of Embodiments D1-D27, characterized in that the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • Embodiment D29 A plasmid system according to embodiment D28, characterized in that the Cap gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment D30 A plasmid system according to any one of embodiments D28-D29, characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment D31 The plasmid system according to any one of Embodiments D1-D30 is characterized in that it further comprises a P5 promoter downstream of the Cap gene, which is operably connected to the Cap gene; optionally, the P5 promoter is located downstream of the polyA sequence downstream of the Cap gene; optionally, the P5 promoter and the Cap gene, or the P5 promoter and the polyA sequence, are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • Embodiment D32 A plasmid system according to any one of embodiments D1-D31, characterized in that the (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment D33 A plasmid system according to any one of Embodiments D1-D32, characterized in that the heterologous nucleic acid sequence encodes a heterologous gene.
  • Embodiment D34 A plasmid system according to any one of embodiments D1-D33, characterized in that the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or AAV-DJ; preferably, the serotype of the AAV is AAV2, AAV5, AAV8, or AAV9; more preferably, the serotype of the AAV is AAV2.
  • a recombinant nucleic acid or a plasmid comprising the recombinant nucleic acid which comprises the Rep gene and/or the Cap gene of an adeno-associated virus (AAV) and has one or more of the following characteristics:
  • the donor site of the original intron of the viral genome of the adeno-associated virus contains a mutation, and the donor site is equivalent to the nucleotide sites 1717-1722 of SEQ ID NO:7;
  • a non-parental serotype promoter is contained upstream of the Rep gene or downstream of the Cap gene, and is operably linked to the Rep gene and/or the Cap gene.
  • the recombinant nucleic acid or plasmid comprises the features:
  • the recombinant nucleic acid or plasmid comprises both a Rep gene and a Cap gene.
  • the recombinant nucleic acid or plasmid comprises only one of the Rep gene and the Cap gene (eg, the pHelper-Cap helper plasmid and the pRep plasmid in some embodiments).
  • the recombinant nucleic acid or plasmid comprises feature (1), and the insertion position of the P5 promoter located upstream of the Rep gene is immediately after the 120 bp nucleotide site of the adeno-associated virus genome corresponding to SEQ ID NO: 7, and is operably linked to the Rep gene.
  • the P5 promoter is spaced from the Rep gene by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there is no spacer of nucleotides.
  • sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.
  • the recombinant nucleic acid or plasmid comprises feature (2), and the correction of the TATA box mutation of the original P5 promoter comprises mutating T corresponding to nucleotide position 4469 of SEQ ID NO:7 to A, and/or mutating GGGGGGG corresponding to nucleotide positions 4477-4483 of SEQ ID NO:7 to TATTTAA.
  • the sequence of the original P5 promoter after correction is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:9.
  • the recombinant nucleic acid or plasmid has a P5 promoter inserted downstream of the Cap gene, operably linked to the Cap gene, and the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.
  • the recombinant nucleic acid or plasmid comprises feature (3), and the mutation of the donor site comprises mutating the CA corresponding to nucleotides 1721-1722 of SEQ ID NO:7 to GT, or mutating the donor site sequence corresponding to nucleotides 1717-1722 of SEQ ID NO:7 to GTACGT.
  • the mutated intron sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:11.
  • the recombinant nucleic acid or plasmid comprises feature (4), and the liver-specific enhancer-promoter sequence is deleted.
  • the liver-specific enhancer-promoter sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:12 or SEQ ID NO:35.
  • the recombinant nucleic acid or plasmid comprises feature (5), and the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.
  • the hGH intron is located downstream of the P5 promoter of feature (1) and upstream of the Rep gene of feature (1).
  • the hGH intron and the P5 promoter of feature (1) are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • the hGH intron of the recombinant nucleic acid or plasmid is spaced no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there is no intervening nucleotide. In some embodiments, there is no intervening nucleotide between the hGH intron and the Rep gene start codon.
  • the recombinant nucleic acid or plasmid comprises feature (6), and the sequence of the intron of the adeno-associated virus genome is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:14.
  • the intron of the adeno-associated virus genome is located downstream of the P5 promoter of feature (1) and upstream of the Rep gene of feature (1). In some embodiments, the intron of the adeno-associated virus genome and the P5 promoter of feature (1) are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • the spacing between the intron of the adeno-associated virus genome and the start codon of the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • the recombinant nucleic acid or plasmid comprises feature (7), and the sequence of the non-parental serotype promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:15.
  • the non-parental serotype promoter is located downstream of the Cap gene. In some embodiments, the non-parental serotype promoter is located at a position corresponding to nucleotides 4270-4271 of SEQ ID NO:7.
  • the recombinant nucleic acid or plasmid does not contain a sequence corresponding to nucleotides 4368-4532 of SEQ ID NO:7, or does not contain a sequence corresponding to nucleotides 4373-4532 of SEQ ID NO:7.
  • the non-parental serotype promoter is located upstream of the Rep gene, and the non-parental serotype promoter is located upstream of the hGH intron.
  • the recombinant nucleic acid or plasmid does not contain a sequence corresponding to nucleotides 121-130 of SEQ ID NO:7.
  • a method for modifying a plasmid system comprises one or more of the following modification methods:
  • the method for modifying the plasmid system is characterized by:
  • the P5 promoter inserted upstream of the Rep gene as described in (1) has a sequence as shown in SEQ ID NO:4;
  • the mutated P5 promoter has the sequence shown in SEQ ID NO:8, and the P5 promoter inserted downstream of the Cap gene has the sequence of SEQ ID NO:4;
  • Mutating the donor site of the intron is to mutate GTACCA at the donor site of the intron to GTACGT, preferably, the intron before the mutation has a sequence as shown in SEQ ID NO: 10; and the intron after the mutation has a sequence as shown in SEQ ID NO: 11;
  • the hGH intron inserted in has the sequence shown in SEQ ID NO:13;
  • the modified wtAAV2 intron has a sequence as shown in SEQ ID NO: 14;
  • the non-parental serotype promoter is wtAAV7 P5 promoter, which has the sequence shown in SEQ ID NO:15.
  • a recombinant adeno-associated virus is provided, characterized in that it is obtained using the production method of the recombinant adeno-associated virus described herein.
  • a method for producing a recombinant adeno-associated virus characterized in that the plasmid described herein or all plasmids in the plasmid system described herein are transfected into a host cell, cultured, and isolated to obtain the recombinant adeno-associated virus.
  • the ratio of the (i) auxiliary plasmid to the (ii) plasmid encoding the Rep gene is at least 1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, or at least 4:1; preferably, at least 3:1.
  • the ratio of the (i) auxiliary plasmid to the (ii) plasmid encoding the Rep gene is about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, or about 4:1; preferably, about 3:1.
  • the ratio of the plasmid encoding the Rep gene (ii) to the plasmid encoding the recombinant adeno-associated virus (iii) comprising a heterologous nucleic acid sequence is at least 1: 1, at least 1.5: 1, at least 2: 1, at least 2.5: 1, at least 3: 1, at least 3.5: 1, or at least 4: 1; preferably, at least 2.5: 1.
  • the ratio of the plasmid encoding the Rep gene (ii) to the plasmid encoding the recombinant adeno-associated virus (iii) comprising a heterologous nucleic acid sequence is about 1: 1, about 1.5: 1, about 2: 1, about 2.5: 1, about 3: 1, about 3.5: 1, or about 4: 1; preferably, about 2.5: 1.
  • the ratio of the (i) plasmid containing the gene of interest and the Cap gene to the (ii) helper plasmid containing the Rep gene is at least 1: 1, at least 1.5: 1, at least 2: 1, at least 2.5: 1, at least 3: 1, at least 3.5: 1, or at least 4: 1; preferably, at least 3: 1.
  • the ratio of the (i) plasmid containing the gene of interest and the Cap gene to the (ii) helper plasmid containing the Rep gene is about 1: 1, about 1.5: 1, about 2: 1, about 2.5: 1, about 3: 1, about 3.5: 1, or about 4: 1; preferably, about 3: 1.
  • the host cells are cultured in an adherent culture or a suspension culture.
  • a host cell in one aspect, characterized in that the host cell contains all plasmids in a plasmid system as described herein. In one aspect, a host cell is provided, characterized in that the host cell contains plasmids as described herein.
  • the host cell is selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.
  • the production method described herein has a higher solid rate of recombinant adeno-associated virus than the production method based on the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the solid rate value is at least 0.05, at least 0.10, at least 0.15, at least 0.20, at least 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, or at least 0.80.
  • the production method described herein produces a higher genomic titer of the recombinant adeno-associated virus than the production method based on the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the genomic titer is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times.
  • the production method described herein produces a recombinant adeno-associated virus with a Cap coding sequence with lower impurity residues than the production method based on the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the Cap coding sequence has a lower residual impurity than the traditional method by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99%.
  • the production method described herein is lower than the AAV (rcAAV) residue with replication ability of the recombinant adeno-associated virus produced by the production method based on the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the rcAAV residue is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to the traditional method.
  • the adeno-associated virus AAV genome is about 4.7kb long and is a linear single-stranded DNA.
  • the wild-type AAV genome includes two open reading frames (ORFs), namely Rep and Cap, with inverted repeat sequences (ITRs) at both ends, with a length of about 100-200bp (for example, 145bp).
  • ITR consists of a palindromic sequence of about 125bp, which forms a T-hairpin structure through complementary base pairing. The remaining approximately 20 bases are unpaired and are called D sequences.
  • ITR generally also has a Rep protein binding site (RBS) and a terminal split site (TRS), both of which are the starting points of replication.
  • Wild-type AAV2 (wtAAV2, SEQ ID NO: 1) is a replication-defective parvovirus that requires a helper virus to help it replicate and amplify in vivo.
  • a recombinant adeno-associated virus is provided.
  • the recombinant adeno-associated virus is obtained according to the production method described herein.
  • the recombinant adeno-associated virus is produced based on the plasmid system described herein.
  • the plasmid described herein is used in the production of the recombinant adeno-associated virus.
  • the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or AAV-DJ.
  • the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV-DJ.
  • the AAV serotype is AAV2, AAV5, AAV8, or AAV9.
  • the AAV serotype is AAV2.
  • the recombinant adeno-associated virus provided herein has a higher solid rate than the recombinant adeno-associated virus produced by the three-plasmid system of the traditional helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the solid rate value is at least 0.05, at least 0.10, at least 0.15, at least 0.20, at least 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, or at least 0.80.
  • the recombinant adeno-associated virus provided herein has a higher genomic titer than the recombinant adeno-associated virus produced by the three-plasmid system of the traditional helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the genomic titer is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times.
  • the recombinant adeno-associated virus provided herein has lower impurity residues related to the Cap coding sequence of the recombinant adeno-associated virus produced by the three-plasmid system of the traditional helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the impurity residues related to the Cap coding sequence are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to the recombinant adeno-associated virus produced traditionally.
  • the recombinant adeno-associated virus provided herein has a lower AAV (rcAAV) residue with replication ability than the recombinant adeno-associated virus produced by the three-plasmid system of the traditional helper plasmid + Rep/Cap plasmid + target gene plasmid.
  • the rcAAV residue is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to the recombinant adeno-associated virus produced traditionally.
  • the Rep gene encodes four proteins required for AAV viral DNA replication and packaging, which are named according to their molecular weight, namely Rep78, Rep68, Rep52 and Rep40.
  • Rep78/68 expression is controlled by the P5 promoter, while the P19 promoter controls the expression of the smaller Rep protein Rep52/40.
  • Rep68 and Rep40 are produced as splicing isoforms, respectively, and the C-termini of Rep52 and Rep40 are identical to Rep78 and Rep68, respectively.
  • Rep78 and Rep68 promote AAV DNA rescue and subsequent replication, and participate in the positive and negative regulation of AAV gene expression.
  • Rep52 and Rep40 participate in the interaction between single-stranded DNA and preformed capsids, promoting the packaging of viral DNA into capsids.
  • the Rep gene comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2.
  • the Rep genes described herein express Rep78, Rep68, Rep52, and Rep40 proteins.
  • the Rep68 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:89.
  • the Rep78 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:90.
  • the Rep40 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:91.
  • the Rep52 protein comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:92.
  • the sequence of the Rep gene comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO: 1.
  • the sequence of the Rep gene comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.
  • the Rep gene expresses Rep78 and Rep52 proteins, but does not express Rep68 and Rep40 proteins.
  • the gene expressing Rep78 and Rep52 proteins (Rep78/52) comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • the Rep gene expresses Rep68 and Rep40 proteins, but does not express Rep78 and Rep52 proteins.
  • the gene expressing Rep68 and Rep40 proteins comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69.
  • the gene expressing Rep68 and Rep40 proteins comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:103.
  • the Rep gene expresses Rep52 and Rep40 proteins, but does not express Rep78 and Rep68 proteins.
  • the gene expressing Rep52 and Rep40 proteins comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:70.
  • the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably linked to the Rep gene.
  • the upstream of the Rep gene contains a P5 promoter.
  • the insertion position of the P5 promoter is immediately after the 120bp nucleotide site of the adeno-associated virus genome corresponding to SEQ ID NO:7, and is operably connected to the Rep gene.
  • the P5 promoter is spaced from the Rep gene by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides, or there are no intervening nucleotides. In some embodiments, there are no intervening nucleotides between the P5 promoter and the Rep gene.
  • the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.
  • the donor site of the original intron of the Rep gene comprises a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO:1.
  • the mutation of the donor site comprises mutating the CA equivalent to the nucleotides 1721-1722 of SEQ ID NO:7 to GT.
  • the mutation of the donor site comprises mutating the donor site sequence equivalent to the nucleotides 1717-1722 of SEQ ID NO:7 to GTACGT.
  • the mutated intron sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:11.
  • the upstream of the Rep gene contains an hGH intron.
  • the intron is located before the start codon of the Rep gene.
  • the upstream of the hGH intron contains a P5 promoter.
  • the P5 promoter is spaced from the hGH intron by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the P5 promoter and the hGH intron.
  • the hGH intron is spaced from the start codon of the Rep gene by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the hGH intron and the start codon of the Rep gene. In some embodiments, the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.
  • an intron of an adeno-associated virus genome is contained before the start codon of the Rep gene.
  • the sequence of the intron of the adeno-associated virus genome is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.
  • the intron of the adeno-associated virus genome is located downstream of the P5 promoter and upstream of the Rep gene.
  • the intron of the adeno-associated virus genome and the P5 promoter are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the intron of the adeno-associated virus genome and the P5 promoter.
  • the intron of the adeno-associated virus genome and the start codon of the Rep gene are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the intron of the adeno-associated virus genome and the Rep gene start codon.
  • the Rep gene contains a non-parental serotype promoter upstream, which is operably linked to the Rep gene.
  • the sequence of the non-parental serotype promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15.
  • the Rep gene contains an hGH intron upstream, and the non-parental serotype promoter is located upstream of the hGH intron.
  • the downstream of the Rep gene contains a polyA sequence.
  • the Rep gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the Rep gene and the polyA sequence.
  • the polyA sequence has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with nucleotides 4411-4460 of SEQ ID NO:1.
  • the Cap gene is operably linked to at least one promoter selected from the group consisting of a P5 promoter, a P19 promoter, and a P40 promoter. In some embodiments, the Cap gene is operably linked to a P5 promoter, a P19 promoter, and a P40 promoter. In some embodiments, the nucleic acid sequence encoding the Cap gene comprises, from the 5' end to the 3' end, a sequence encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • the promoters are spaced by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides. In some embodiments, there are no nucleotides spaced between the promoters. In some embodiments, the promoter and the Cap gene are spaced by no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides. In some embodiments, there are no nucleotides spaced between the promoters.
  • sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO: 1. In some embodiments, the sequence of the P19 promoter is T at a position relative to nucleotide 768 of SEQ ID NO: 1. In some embodiments, the sequence of the P19 promoter is 100% identical to nucleotides 741-890 of SEQ ID NO: 1 (except nucleotide 768).
  • the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO: 1. In some embodiments, the sequence of the P40 promoter is GT at a position relative to nucleotides 1911-1912 of SEQ ID NO: 1. In some embodiments, the sequence of the P40 promoter is 100% identical to nucleotides 1701-2202 of SEQ ID NO: 1 (except nucleotides 1911-1912).
  • the downstream of the Cap gene described herein contains a P5 promoter.
  • the P5 promoter is operably linked to the Cap gene.
  • the P5 promoter is spaced no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides, or no intervening nucleotides, between the P5 promoter and the Cap gene. In some embodiments, there are no intervening nucleotides between the P5 promoter and the Cap gene.
  • the P5 promoter is spaced no more than 30bp, no more than 25bp, no more than 20bp, no more than 15bp, no more than 10bp, or no more than 5bp of nucleotides, or no intervening nucleotides between the polyA downstream of the Cap gene.
  • the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4.
  • the TATA box mutation in the original P5 promoter located downstream of the Cap gene is corrected.
  • the correction of the TATA box mutation of the original P5 promoter comprises mutating the T equivalent to the 4469th nucleotide of SEQ ID NO:7 to A.
  • the correction of the TATA box mutation of the original P5 promoter comprises mutating the GGGGGGG equivalent to the 4477-4483rd nucleotides of SEQ ID NO:7 to TATTTAA.
  • the corrected sequence of the original P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:9.
  • the original liver-specific enhancer-promoter sequence downstream of the Cap gene is deleted.
  • the liver-specific enhancer-promoter sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12.
  • the liver-specific enhancer-promoter sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35.
  • the Cap gene contains a non-parental serotype promoter downstream, which is operably linked to the Cap gene.
  • the sequence of the non-parental serotype promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15.
  • the non-parental serotype promoter is located at a position corresponding to nucleotides 4270-4271 of SEQ ID NO: 7.
  • the Cap gene comprises a polyA sequence downstream. In some embodiments, the Cap gene and the polyA sequence are separated by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides. In some embodiments, there are no intervening nucleotides between the Cap gene and the polyA sequence. In some embodiments, the polyA sequence has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with nucleotides 4411-4460 of SEQ ID NO:1.
  • the Cap gene encodes a functional Cap protein.
  • the Cap gene encodes three capsid proteins VP1, VP2, VP3 and assembly activation protein AAP through alternative splicing and translation from different start codons, and is regulated by the P40 promoter. These three structural proteins are assembled with the assistance of AAP, and about 60 copies of VP1, VP2 and VP3 are assembled into an icosahedral particle with a diameter of about 26nm, with a molar ratio of 1:1:10. Different capsid proteins determine the serotype of AAV, and these serotypes constitute different tissue preferences.
  • the capsid protein encoded by the Cap gene is of the serotype AAV1 (SEQ ID NO: 48), AAV2 (SEQ ID NO: 49), AAV3 (SEQ ID NO: 50), AAV4 (SEQ ID NO: 51), AAV5 (SEQ ID NO: 52), AAV6 (SEQ ID NO: 53), AAV7 (SEQ ID NO: 54), AAV8 (SEQ ID NO: 55), AAV9 (SEQ ID NO: 56), AAV10 (SEQ ID NO: 57), AAVrh.10, or a derivative capsid protein based on one of the serotypes.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 48-57.
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:58-67.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:48 (AAV1).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:58.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:49 (AAV2).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:59.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:50 (AAV3).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:60.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:51 (AAV4).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:61.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:52 (AAV5).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:62.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:53 (AAV6).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:63.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:54 (AAV7).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:64.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55 (AAV8).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:65.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:56 (AAV9).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:66.
  • the capsid protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:57 (AAV10).
  • the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:67.
  • Capsid proteins include those produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be done, for example, by a marker rescue approach, in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and directed selection is used to select a capsid sequence with desired properties. Capsid sequences of different serotypes can be altered by homologous recombination in cells to produce novel capsid proteins.
  • Capsid proteins also include those produced by engineering the capsid protein sequence to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins (eg, between two or more capsid proteins of different serotypes).
  • Hybrid AAV capsid genes can be produced by randomly fragmenting the sequence of related AAV genes (e.g., sequences encoding capsid proteins of multiple different serotypes), and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology. Libraries of hybrid AAV genes produced by shuffling the capsid genes of several serotypes in this way can be screened to identify viral clones with desired functions. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to produce a diverse library of variants, which can then be selected for desired properties.
  • the sequence of the capsid gene can also be genetically modified to introduce specific deletions, substitutions or insertions relative to the natural wild-type sequence.
  • the capsid gene can be modified by inserting an unrelated protein or peptide sequence within the open reading frame of the capsid coding sequence or at the N and/or C-terminal of the capsid coding sequence.
  • the unrelated protein or peptide may advantageously be a protein or peptide that acts as a ligand for a specific cell type, thereby improving binding to the target cell, or increasing the specificity of the vector targeting a specific cell population.
  • An example may include using an RGD peptide to prevent uptake in retinal pigment epithelial cells, thereby enhancing transduction of surrounding retinal tissue.
  • the unrelated protein may also be a protein, i.e., an epitope or affinity tag, that helps purify viral particles as part of the production process.
  • the insertion site is usually selected so as not to interfere with other functions of the viral particles, such as internalization and transport of the viral particles. Technicians can identify suitable insertion sites based on their common knowledge.
  • This article also encompasses providing sequences of the AAV genome in an order and configuration different from the natural AAV genome.
  • This article also encompasses replacing one or more AAV sequences or genes with a sequence from another virus or with a chimeric gene composed of a sequence from more than one virus.
  • This type of chimeric gene may be composed of sequences of two or more related viral proteins from different viral species.
  • a helper virus e.g., adenovirus or herpes virus
  • a helper virus can provide helper genes that allow AAV virus to be produced in infected cells.
  • genes E1A, E1B, E2A, E4, and VA can provide helper functions.
  • the auxiliary genes described herein include one or more genes selected from E1A, E1B, E2A, E4, and VA RNA genes. In some embodiments, the auxiliary genes described herein include one or more genes selected from E2A, E4, and VA RNA genes.
  • all helper genes of the plasmid systems described herein are located on helper plasmids. In some embodiments, at least one helper gene of the plasmid systems described herein is not on a helper plasmid.
  • helper genes of the plasmid or plasmid system described herein are derived from an adenovirus.
  • the helper gene of the plasmid or plasmid system described herein is derived from adenovirus type 2 (Ad2).
  • Ad2A gene contained in the plasmid or plasmid system described herein comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:93.
  • the E4 gene contained in the plasmid or plasmid system described herein comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:94.
  • the VA RNA gene contained in the plasmid or plasmid system described herein comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:95.
  • helper genes of the plasmid systems described herein are derived from adenovirus type 2 (Ad2).
  • helper genes of the plasmid systems described herein are derived from herpes viruses.
  • the helper gene is the same as a gene in a corresponding virus. In some embodiments, the helper gene is a derivative sequence of a gene in a corresponding virus.
  • the plasmids and plasmid systems described herein have beneficial effects including but not limited to:
  • This article provides several methods for transforming the pRC2 plasmid in the traditional three-plasmid system.
  • the new plasmid system composed of the modified plasmid can not only improve the viral genome titer and solid rate, but also reduce the production of rcAAV.
  • It can be used for transfecting adherent culture HEK293 cells, and can also be used for the production of rAAV in a scalable suspension system; it is not only suitable for the production of rAAV2 vectors, but also for the production of other serotypes of rAAV; it meets the current clinical trial and market supply needs for rAAV vector materials, and can be quickly and easily applied to the production system, greatly reducing production costs, reducing a lot of manpower and material input, and improving production efficiency.
  • Another aspect of the present invention relates to pharmaceutical preparations, administration routes and effective doses.
  • Such pharmaceutical preparations can be used to treat diseases (such as eye diseases).
  • a pharmaceutical preparation comprising the recombinant adeno-associated virus described herein, and a pharmaceutically acceptable carrier and excipient.
  • Administration of the pharmaceutical formulations herein includes pharmaceutical formulations suitable for oral (including buccal and sublingual), rectal, nasal, topical, transdermal, pulmonary, vaginal, suppository or parenteral (including intraocular, intravitreal, intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) administration or in a form suitable for administration by nebulization, inhalation or insufflation.
  • parenteral including intraocular, intravitreal, intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous
  • General information on drug delivery systems can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999)).
  • pharmaceutical preparations include carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, polypeptides, amino acids, antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and/or preservatives), water, oils (including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), saline solutions, aqueous glucose solutions and glycerol solutions, flavoring agents, coloring agents, anti-adhesives and other acceptable additives, adjuvants or binders, other pharmaceutically acceptable auxiliary substances such as pH buffers, tension regulators, emulsifiers, wetting agents, etc., in order to approach physiological conditions.
  • carriers and excipients including but not limited to buffers, carbohydrates, mannitol, polypeptides, amino acids, antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and/or preservatives
  • oils including oils from petroleum, animal, plant or
  • excipients examples include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, glycerol, propylene, glycol, water, ethanol, etc.
  • pharmaceutical preparations are substantially free of preservatives.
  • pharmaceutical preparations may contain at least one preservative.
  • General methods for pharmaceutical dosage forms are found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott, Williams, & Wilkins, Baltimore Md. (1999)). It will be appreciated that while any suitable carrier known to those of ordinary skill in the art may be used to administer the compositions or formulations of the present disclosure, the type of carrier may vary depending on the mode of administration.
  • Biodegradable microspheres can also be used as carriers for the pharmaceutical formulations disclosed herein. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344, and 5,942,252.
  • the drug can be administered in the form of liposomes or microspheres (or microparticles).
  • Methods for preparing liposomes and microspheres for administration to a subject are well known to those skilled in the art.
  • U.S. Patent No. 4,789,734 (the contents of which are hereby incorporated by reference) describes a method for encapsulating biological substances in liposomes. Basically, the substance is dissolved in an aqueous solution, appropriate phospholipids and lipids are added, along with surfactants if necessary, and the substance is dialyzed or sonicated if necessary.
  • G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers in Biology and Medicine, 2, Supplement 87-341 (Academic Press, 1979) provides a review of known methods.
  • Microspheres formed from polymers or polypeptides are well known to those skilled in the art and can be designed to enter the bloodstream directly through the gastrointestinal tract. Alternatively, compounds can be incorporated and microspheres or microsphere complexes implanted for slow release over a period of days to months. See, e.g., U.S. Patents Nos. 4,906,474, 4,925,673, and 3,625,214 and Jein, TIPS 19:155-157 (1998), the contents of which are hereby incorporated by reference.
  • the concentration of the drug may be adjusted, the pH of the solution buffered, and the isotonicity adjusted to be compatible with intraocular or intravitreal injection.
  • the drugs disclosed herein can be formulated as sterile solutions or suspensions in suitable vehicles.
  • the pharmaceutical preparations can be sterilized by conventional well-known sterilization techniques, or can be sterile filtered.
  • the resulting aqueous solution can be packaged for use as is or lyophilized, and the lyophilized preparation is combined with a sterile solution before administration.
  • Suitable preparations and additional carriers are described in Remington "The Science and Practice of Pharmacy" (20th edition, Lippincott Williams & Wilkins, Baltimore MD).
  • the agent or its pharmaceutically acceptable salt can be provided alone or in combination with one or more other agents, or provided in one or more other forms.
  • the preparation may include one or more agents in a specific ratio, depending on the relative efficacy of each agent and the expected indication. For example, in a composition or preparation for targeting two different host targets, and in the case of similar efficacy, an agent of about 1:1 ratio can be used.
  • the two forms can be formulated together in the same dosage unit, such as in a cream, suppository, tablet, capsule, aerosol spray or a powder package to be dissolved in a beverage; or each form can be formulated in a separate unit, such as two creams, two suppositories, two tablets, two capsules, tablets and liquids for dissolving tablets, two aerosol sprays or powder packages and liquids for dissolving powders, etc.
  • pharmaceutically acceptable salts refers to those salts that retain the biological effectiveness and properties of the agents used in this disclosure and which are not biologically or otherwise undesirable.
  • Typical salts are salts of inorganic ions such as sodium, potassium, calcium, magnesium ions, etc.
  • Such salts include salts formed with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid.
  • the agent if the agent contains a carboxyl or other acidic groups, it can be converted into a pharmaceutically acceptable addition salt with an inorganic or organic base.
  • suitable alkalis include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, etc.
  • esters or amides refer to those esters and amides that retain the biological effectiveness and properties of the agents used in the present disclosure and are not biologically or otherwise undesirable.
  • Typical esters include ethyl esters, methyl esters, isobutyl esters, ethylene glycol esters, etc.
  • Typical amides include unsubstituted amides, alkyl amides, dialkyl amides, etc.
  • the drug can be administered in combination with one or more other drugs, forms and/or agents, such as those described above.
  • the pharmaceutical preparation containing one or more other active agents can be formulated to include a certain molar ratio.
  • the molar ratio of the first active agent to the other active agents can be about 99:1 to about 1:99.
  • the first active agent: the molar ratio range of the other active agents is selected from about 80:20 to about 20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90.
  • the first active agent the molar ratio of other active agents can be about 1:9, and in some embodiments can be about 1:1.
  • the two doses, forms and/or drugs can be formulated together in the same dosage unit, such as in one cream, suppository, tablet, capsule, or powder packet to be dissolved in a beverage; or each dose, form and/or drug can be formulated in a separate unit, such as two creams, suppositories, tablets, two capsules, a tablet and a liquid for dissolving the tablet, an aerosol spray, a powder packet and a liquid for dissolving the powder, etc.
  • agents and/or combinations of agents may then be administered with other agents if necessary or desired.
  • the choice of agents that may be co-administered with the agents and/or combinations of agents of the present disclosure may depend, at least in part, on the condition being treated.
  • the agent (or its pharmaceutically acceptable salt, ester or amide) can be administered alone or in the form of a pharmaceutical preparation, wherein the active agent is blended or mixed with one or more pharmaceutically acceptable carriers.
  • the pharmaceutical preparation can be any composition or preparation prepared for administration to a subject.
  • the pharmaceutical preparation used according to the present disclosure can be prepared in a conventional manner using one or more physiologically acceptable carriers, including excipients, diluents and/or adjuvants, which, for example, help to process the active agent into an administrable preparation.
  • the appropriate formulation may depend at least in part on the selected route of administration.
  • a variety of routes or modes of administration can be used to deliver the agent or its pharmaceutically acceptable salt, ester or amide for the present disclosure to a subject, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, intraocular, intravitreal and intramuscular application and by inhalation.
  • an oil or non-aqueous solvent may be used to bring the agent into solution.
  • an emulsion, suspension, or other formulation such as a liposomal formulation
  • any known method may be used to prepare liposomes for treating a disorder. See, for example, Bangham et al., J. Mol. Biol. 23:238-252 (1965) and Szoka et al., Proc. Natl Acad. Sci. USA 75:4194-4198 (1978), which are incorporated herein by reference.
  • Ligands may also be attached to liposomes to direct these compositions to specific sites of action.
  • the agents of the present disclosure may also be incorporated into foods such as cream cheese, butter, salad dressing, or ice cream to promote solubilization, administration, and/or compliance in certain subject populations.
  • the compounds of the present disclosure can be formulated for parenteral administration (e.g., by injection, such as intraocular injection or intravitreal injection), and can be present in unit dosage form in ampoules, prefilled syringes, small volume infusions, or in multi-dose containers with added preservatives.
  • the composition or formulation can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, such as a solution in aqueous polyethylene glycol.
  • the vehicle can be selected from those known in the art as suitable, including aqueous or oil suspensions or emulsions, and sesame oil, corn oil, cottonseed oil or peanut oil, and elixirs, mannitol, glucose, or sterile aqueous solutions and similar pharmaceutical vehicles.
  • the preparation can also include biocompatible biodegradable polymer compositions, such as poly (lactic acid-co-glycolic acid). These materials can be made into microspheres or nanospheres, loaded with drugs and further coated or derivatized, to provide excellent sustained release performance.
  • Vehicles suitable for periocular or intraocular injections include, for example, suspensions of therapeutic agents in injection-grade water, liposomes, and vehicles suitable for lipophilic substances. Other vehicles used for periocular or intraocular injections are well known in the art.
  • the composition is formulated into a pharmaceutical preparation suitable for intravenous administration to a human being according to conventional procedures.
  • the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer.
  • the composition or preparation may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site.
  • each component is provided in a unit dosage form alone or mixed together, such as a dry lyophilized powder or anhydrous concentrate in a sealed container, such as an ampoule or a sachet indicating the amount of an active agent.
  • composition or preparation in the case where the composition or preparation is to be administered by infusion, it may be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline may be provided so that each component may be mixed before administration.
  • the active compound When administered by injection, the active compound can be formulated in an aqueous solution, particularly in a physiologically compatible buffer, such as Hanks solution, Ringer's solution or physiological saline buffer.
  • the solution may contain formulating agents, such as suspending agents, stabilizers and/or dispersing agents.
  • the active compound may be in powder form and reconstituted with a suitable vehicle, such as sterile pyrogen-free water, before use.
  • the pharmaceutical preparation does not contain an adjuvant or any other substance added to enhance the immune response stimulated by the peptide.
  • the pharmaceutical preparation contains a substance that suppresses the immune response to the peptide. Formulation methods are known in the art, for example, as disclosed in Remington’s Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton P.
  • ophthalmic solution, suspension, ointment or insert comprising the disclosed agent or the agent combination can be used to effectively treat eye diseases.
  • Eye drops can be prepared by dissolving active ingredient in sterile aqueous solution such as physiological saline, buffer solution etc. or by merging the powder composition to be dissolved before use.
  • Other vehicles as known in the art can be selected, including but not limited to: balanced salt solution, saline solution, water-soluble polyethers such as polyethylene glycol, polyethylene compounds such as polyvinyl alcohol and polyvidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, petroleum derivatives such as mineral oil and white vaseline, animal fats such as lanolin, acrylic acid polymers such as carboxyl polymethylene gel, vegetable fats such as peanut oil and polysaccharides such as dextran and glycosaminoglycans such as sodium hyaluronate. If necessary, additives commonly used in eye drops can be added.
  • water-soluble polyethers such as polyethylene glycol, polyethylene compounds such as polyvinyl alcohol and polyvidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, petroleum derivatives such as mineral oil and white vaseline, animal fats such as lanolin, acrylic acid polymers such as carboxyl polymethylene gel, vegetable fats such as peanut oil and
  • Such additives include isotonic agents (e.g., sodium chloride, etc.), buffers (e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), thickeners (e.g., sugars such as lactose, mannitol, maltose, etc.; for example, hyaluronic acid or its salts such as sodium hyaluronate, potassium hyaluronate, etc.; for example, mucopolysaccharides such as chondroitin sulfate, etc.; for example, sodium polyacrylate, carboxyvinyl polymer, cross-linked polyacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, methylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxyprop
  • compositions or formulations herein may be enhanced by surfactants or other suitable co-solvents in the compositions or formulations.
  • suitable co-solvents include polysorbates 20, 60, and 80; Pluronic F68, F-84, and P-103; cyclodextrins, or other agents known to those skilled in the art.
  • co-solvents may be used at a level of about 0.01% to 2% by weight.
  • compositions or preparations of the present disclosure may be packaged in multiple doses.
  • Preservatives may preferably prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, Onamer M, or other agents known to those skilled in the art. In some embodiments, the use level of such preservatives may be 0.004% to 0.02%.
  • preservatives preferably benzalkonium chloride
  • benzalkonium chloride may be used at a level of 0.001% by weight to less than 0.01% by weight (e.g., 0.001% by weight to 0.008% by weight, preferably about 0.005% by weight). It has been found that benzalkonium chloride at a concentration of 0.005% may be sufficient to protect the compositions or preparations of the present disclosure from microbial attack.
  • the agents of the present disclosure are delivered in soluble rather than suspension form, which allows for more rapid and quantitative absorption to the site of action.
  • formulations such as gels, creams, lotions, suppositories, and ointments provide longer exposure to an area of the agents of the present disclosure, while formulations in solution form, such as sprays, provide more immediate, short-term exposure.
  • the compounds of the present disclosure can be attached to biocompatible polymers in a releasable manner for use in sustained release preparations, which are on, in or attached to inserts for topical, intraocular, periocular or systemic administration.
  • Controlled release from biocompatible polymers can also be utilized with water-soluble polymers to form drippable preparations.
  • Controlled release from biocompatible polymers such as PLGA microspheres or nanospheres can be used for preparations suitable for intraocular implantation or injection for sustained release administration, and any suitable biodegradable and biocompatible polymers can also be used.
  • the present invention provides the use of the pharmaceutical preparation described herein in the treatment of diseases.
  • the present invention provides the use of the plasmid described herein in the preparation of drugs.
  • the present invention provides the use of the plasmid system described herein in the preparation of drugs.
  • the present invention provides the use of the production method described herein in the preparation of drugs.
  • the present invention provides the use of the recombinant adeno-associated virus described herein in the preparation of drugs.
  • the present invention provides the use of the host cell described herein in the preparation of drugs.
  • provided herein is a method for treating a disease, comprising administering the recombinant adeno-associated virus or the pharmaceutical preparation described herein to a patient in need thereof.
  • the disease is caused by a gene mutation and/or gene deletion in a target gene in an organism.
  • the individual suffering from the disease is a human.
  • a kit comprising a plasmid as described herein. In one aspect, a kit is provided, comprising a plasmid system as described herein. In one aspect, a kit is provided, comprising a recombinant adeno-associated virus as described herein. In one aspect, a kit is provided, comprising a host cell as described herein.
  • the host cell is selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.
  • the term "about” and its grammatical equivalents associated with reference values and its grammatical equivalents as used herein can include a range of values plus or minus 10% of the value, such as a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.
  • the amount "about 10" includes amounts of 9 to 11.
  • subject refers to a mammal that has been or will be the object of treatment, observation or experiment.
  • mammal is intended to have its standard meaning and includes, for example, humans, dogs, cats, sheep and cattle.
  • the methods described herein can be used for human treatment and veterinary applications.
  • the subject is a human.
  • treat or “treatment” includes administering at least one compound disclosed herein or a pharmaceutically acceptable salt thereof to a mammalian subject, particularly a human subject, and includes prophylactic treatment to (i) arrest the development of clinical symptoms of a disease, (ii) cause regression of clinical symptoms of the disease and/or (iii) prevent the onset of the disease.
  • terapéuticaally effective amount refers to an effective amount that, when administered to a human or non-human subject, provides a therapeutic benefit, such as improvement of symptoms, slowing of disease progression, or prevention of disease.
  • nucleic acid and “polynucleotide” are used interchangeably.
  • upstream refers to the 5' end of the gene or nucleic acid site
  • downstream refers to the 3' end of the gene or nucleic acid site
  • promoter refers to a DNA sequence located in the 5'-upstream region of a structural gene, which can activate RNA polymerase, allowing it to accurately bind to the template DNA and have the specificity to initiate transcription.
  • liver specific enhancer-promoter refers to a promoter that can enhance the expression of foreign genes in the liver and is located between the stop codon and the right terminal inverted repeat sequence of the cap gene (see Logan et al., Identification of liver-specific enhancer–promoter activity in the 3’untranslated region of the wild-type AAV2 genome).
  • AAV expression systems containing liver-specific enhancer-promoter are prone to cause AAV accumulation in the liver, which may affect the safety of AAV vectors.
  • operably connected refers to a first polynucleotide component, such as a promoter, and a second transcribable polynucleotide component, such as a reading frame of a gene, arranged so that the first polynucleotide component affects the function of the second polynucleotide component.
  • Two polynucleotide components can be part of a single continuous polynucleotide molecule and can be adjacent. However, discontinuous or non-adjacent first and second polynucleotide components can also be arranged to be operably connected.
  • operably connected also refers to two polynucleotide components that are operably connected after recombination (e.g., mediated by a recombinase, or after introns are removed), rather than in the initial arrangement. Unless otherwise stated, when a promoter and a corresponding gene are described herein, the two are operably connected.
  • the term "host cell” refers to a cell in which the target gene (GOI) is cloned.
  • the plasmid can produce a replica substantially identical to its own sequence under the conditions provided by the host cell.
  • the host cell provides energy, materials, enzymes and other conditions.
  • the host cell can be selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.
  • sequence identity refers to the percentage of bases or amino acids that are identical and in the same relative position between two polynucleotide or polypeptide sequences. Thus, one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. The term “reference sequence” refers to the molecule to which the test sequence is compared. Unless otherwise indicated, the term “sequence identity” in the claims refers to the identity of the sequences as determined by Clustal Sequence identities were calculated using version 1.2.4 with default parameters.
  • Figure 1 is a schematic diagram of the structure of a traditional three-plasmid system.
  • FIG. 2A is a schematic diagram of the structure of the novel three-plasmid system I.
  • FIG. 2B is a schematic diagram of the structure of the novel three-plasmid system II.
  • Figure 3 is a schematic diagram of the structure of the two-plasmid system.
  • FIG. 4 is a schematic diagram of the wtAAV2 gene structure.
  • FIG. 5 is a schematic diagram of the pHelper-R2 plasmid structure.
  • FIG. 6 is a schematic diagram of the pHelper-R10 plasmid structure.
  • FIG. 7 is a schematic diagram of the structure of the pRC-2A plasmid.
  • FIG. 8 is a schematic diagram of the structure of the pRC-9A plasmid.
  • FIG. 9 is a schematic diagram of the structure of the pRep1 plasmid.
  • FIG. 10 is a schematic diagram of the structure of the pRep3 plasmid.
  • FIG. 11 is a schematic diagram of the structure of pAAV-GOI1-C1.
  • Figure 12 is a graph showing the fold change in genome yield of rAAV2-ND4 vectors packaged with pRC2, pRC2-1, pRC2-4, and pRC2-5.
  • FIG. 13 is a graph showing the fold change in genome yield of rAAV2-ND4 vectors packaged with pRC2, pRC2-4, and pRC2-8.
  • FIG. 14 shows the solidity ratio of rAAV2-ND4 vectors packaged by pRC2, pRC2-4, and pRC2-8.
  • FIG. 15 is a graph showing the fold change in genome yield of rAAV2-OPA1 vectors packaged with pRC2, pRC2-4, and pRC2-8.
  • FIG. 16 shows the solidity ratio of rAAV2-OPA1 vectors packaged with pRC2, pRC2-4, and pRC2-8.
  • FIG. 17 is a graph showing the fold change in genome yield of rAAV2-GOI1 vectors packaged with pRC2, pRC2-4, and pRC2-8.
  • FIG. 18 is a graph showing the fold change in genome yield of rAAV2-GOI1 vectors packaged with pRC2, pRC2-53, and pRC2-58.
  • FIG. 19 is a graph showing the fold change in genome yield of rAAV2-GOI1 vectors packaged with pRC2, pJSD33, and pJSD34.
  • FIG. 20 is a graph showing the fold change in genome yield of rAAV2-GOI1 vectors packaged with pRC2, pRC2-64, and pRC2-69.
  • Figure 21 is a graph showing the fold change in genome yield of rAAV2-EGFP vectors packaged with pHelper:pRC2:pAAV-EGFP and pHelper-R10:pRC-2A:pAAV-EGFP.
  • Figure 22 shows the viral genome titer of the rAAV2-hNRF2 vector packaged with an optimized ratio of pHelper-R10: pRC-2A: pAAV-hNRF2 three plasmids.
  • Figure 23 is a graph showing the fold change in genome yield of rAAV2-GOI1 vectors packaged with pHelper:pRC2:pAAV-GOI1, pHelper:pRep1:pAAV-GOI1-C1, and pHelper:pRep3:pAAV-GOI1-C1.
  • Figure 24 is a schematic diagram of the structure of the three-plasmid system of pHelper-Cap, pRep and pAAV-GOI.
  • FIG. 25 is a schematic diagram of the wtAAV2 genome structure.
  • Figure 26 is a schematic diagram of the pHelper-Cap2 plasmid structure.
  • Figure 27 is a schematic diagram of the pRep5 plasmid structure.
  • Figure 28 is a schematic diagram of the pRep6 plasmid structure.
  • Figure 29 is a graph showing the titer of rAAV2-GOI1 vector genomes packaged with pHelper-Cap2 plasmid and pRep1, pRep3, pRep5, and pRep6 plasmids, respectively.
  • Figure 30 is a graph showing viral genome titer of the optimized process of packaging rAAV2-GOI1 using pHelper-Cap2 plasmid and pRep5.
  • FIG. 31 is a graph showing the solidity rate of the new three-plasmid system III and the traditional three-plasmid system for packaging rAAV2-GOI1 vector.
  • Figure 32A is a graph showing the relative ratio of vector yields of multiple two-plasmid systems.
  • Figure 32B is a graph showing the relative ratio of Cap residues of multiple two-plasmid systems.
  • Figure 32C is a graph showing the relative ratio of plasmid residues of multiple two-plasmid systems.
  • Figure 33 is a graph showing the viral genome titers produced by the corresponding plasmids of two two-plasmid systems at different ratios.
  • Figure 34A is a graph showing the relative ratio of vector yields of multiple three-plasmid systems I.
  • Figure 34B is a graph showing the virus solidity rate produced by multiple three-plasmid systems I.
  • Figure 34C is a graph showing the comparison of genome titers of different serotypes of AAV viruses produced by the traditional three-plasmid system and the three-plasmid system I.
  • Figure 35A is a graph showing the relative ratio of vector yields of two three-plasmid systems II.
  • Figure 35B is a graph showing the viral genome titers produced by corresponding plasmids of multiple three-plasmid systems II at different ratios.
  • Figure 35C is a graph showing the genome titers of different serotypes of AAV viruses produced by the traditional three-plasmid system and the three-plasmid system II.
  • Figure 36A is a comparative diagram of the genome titers of different serotypes of AAV viruses produced by the traditional three-plasmid system and the three-plasmid system III.
  • Figure 36B is a comparative diagram of the solidity rates of different serotypes of AAV viruses produced by the traditional three-plasmid system and the three-plasmid system III.
  • Figure 37 is a graph showing the viral genome titers produced by the corresponding plasmids of the three-plasmid system III at different ratios.
  • Figure 38A shows the viral genome titer of the novel plasmid system after the Rep gene of the three-plasmid system III was modified and the Rep78/52 encoding gene was placed in the pHelper-Cap plasmid or the AAV plasmid encoding the heterologous gene sequence.
  • Figure 38B is the solidity rate result of the novel three-plasmid system III expansion system packaging rAAV2-EGFP virus after the modification.
  • Figures 39A and 39B show the viral genome titer ( Figure 39A) and solid rate ( Figure 39B) of the novel plasmid system formed by moving one or two of the three auxiliary genes on the pHelper-Cap plasmid of the three-plasmid system III to the pRep6 plasmid.
  • Figure 40A is a comparative diagram of the genome titers of rAAV-scEGFP viruses of different serotypes produced by the traditional three-plasmid system and the three-plasmid system III.
  • Figure 40B is a comparative diagram of the relative solidity of rAAV-scEGFP viruses of different serotypes packaged by the novel three-plasmid system III and the traditional three-plasmid system (the relative solidity of rAAV-scEGFP of various serotypes produced by the traditional three-plasmid system is set as 1).
  • Figure 41 is a schematic diagram of the pHelper-R5 plasmid structure.
  • Figure 42 is a schematic diagram of the structure of pRC-5A plasmid.
  • Figure 43 is a schematic diagram of the structure of pRC-8A plasmid.
  • Figure 44 is a schematic diagram of the pHelper-Cap5 plasmid structure.
  • Figure 45 is a schematic diagram of the pHelper-Cap8 plasmid structure.
  • Figure 46 is a schematic diagram of the pHelper-Cap9 plasmid structure.
  • Figure 47 is a schematic diagram of the pHelper-C2R4 plasmid structure.
  • Figure 48 is a schematic diagram of the VA-Cap2 plasmid structure.
  • Figure 49 is a schematic diagram of the VAE4-Cap2 plasmid structure.
  • Figure 50 is a schematic diagram of the pRep6-E2A plasmid structure.
  • Figure 51 is a schematic diagram of the pRep6-E4E2A plasmid structure.
  • Figure 52 is a schematic diagram of the pRep4 plasmid structure.
  • Figure 53 is a schematic diagram of the pEGFP-Cap2 plasmid structure.
  • Figure 54 is a schematic diagram of the pEGFP-Cap5 plasmid structure.
  • Figure 55 is a schematic diagram of the pEGFP-Cap8 plasmid structure.
  • Figure 56 is a schematic diagram of the pEGFP-Cap9 plasmid structure.
  • Embodiment 1 A recombinant nucleic acid comprising a Rep gene and/or a Cap gene of an adeno-associated virus (AAV) and having one or more of the following characteristics:
  • the donor site of the original intron of the viral genome of the adeno-associated virus contains a mutation, and the donor site is equivalent to the nucleotide sites 1717-1722 of SEQ ID NO:7;
  • a non-parental serotype promoter is contained upstream of the Rep gene or downstream of the Cap gene, and is operably linked to the Rep gene and/or the Cap gene.
  • Embodiment 2 The recombinant nucleic acid according to Embodiment 1 is characterized in that it comprises feature (1), and the insertion position of the P5 promoter located upstream of the Rep gene is immediately after the 120bp nucleotide site of the adeno-associated virus genome corresponding to SEQ ID NO:7, and is operably connected to the Rep gene.
  • Embodiment 3 The recombinant nucleic acid according to any one of Embodiments 1-2, characterized in that a P5 promoter is inserted downstream of the Cap gene and is operably linked to the Cap gene.
  • Embodiment 4 The recombinant nucleic acid according to any one of Embodiments 2-3 is characterized in that the interval between the P5 promoter and the Rep gene or the Cap gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • Embodiment 5 The recombinant nucleic acid according to any one of embodiments 1-4 is characterized in that the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.
  • Embodiment 6 The recombinant nucleic acid according to any one of embodiments 1-5 is characterized in that it comprises feature (2), and the correction of the TATA box mutation of the original P5 promoter comprises mutating T corresponding to nucleotide position 4469 of SEQ ID NO:7 to A, and/or mutating GGGGGGG corresponding to nucleotide positions 4477-4483 of SEQ ID NO:7 to TATTTAA.
  • Embodiment 7 The recombinant nucleic acid according to Embodiment 6 is characterized in that the corrected sequence of the original P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:9.
  • Embodiment 8 The recombinant nucleic acid according to any one of embodiments 1-7 is characterized in that it comprises feature (3), and the mutation of the donor site comprises mutating the CA equivalent to the nucleotides at positions 1721-1722 of SEQ ID NO:7 to GT, or mutating the donor site sequence equivalent to the nucleotides at positions 1717-1722 of SEQ ID NO:7 to GTACGT.
  • Embodiment 9 The recombinant nucleic acid according to embodiment 8 is characterized in that the mutated intron sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:11.
  • Embodiment 10 The recombinant nucleic acid according to any one of embodiments 1 to 9, characterized in that it comprises feature (4), and the liver-specific enhancer-promoter sequence is deleted.
  • Embodiment 11 The recombinant nucleic acid according to any one of embodiments 1-10 is characterized in that the liver-specific enhancer-promoter sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:12 or SEQ ID NO:35.
  • Embodiment 12 The recombinant nucleic acid according to any one of embodiments 1-11 is characterized in that it comprises feature (5), and the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13.
  • Embodiment 13 The recombinant nucleic acid according to any one of Embodiment 12, characterized in that the hGH intron is located downstream of the P5 promoter described in feature (1) and upstream of the Rep gene described in feature (1).
  • Embodiment 14 The recombinant nucleic acid according to embodiment 13 is characterized in that the interval between the hGH intron and the P5 promoter of feature (1) is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there is no intervening nucleotides.
  • Embodiment 15 The recombinant nucleic acid according to any one of embodiments 1-14, characterized in that the interval between the hGH intron and the start codon of the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • Embodiment 16 The recombinant nucleic acid according to Embodiment 15, characterized in that there are no intervening nucleotides between the hGH intron and the start codon of the Rep gene.
  • Embodiment 17 The recombinant nucleic acid according to any one of embodiments 1-16 is characterized in that it comprises feature (6), and the sequence of the intron of the adeno-associated virus genome is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:14.
  • Embodiment 18 The recombinant nucleic acid according to any one of Embodiments 1 to 17, characterized in that the intron of the adeno-associated virus genome is located downstream of the P5 promoter of feature (1) and upstream of the Rep gene of feature (1).
  • Embodiment 19 The recombinant nucleic acid according to Embodiment 18 is characterized in that the introns of the adeno-associated virus genome and the P5 promoter of characteristic (1) are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no spacers of nucleotides.
  • Embodiment 20 The recombinant nucleic acid according to any one of embodiments 1-19 is characterized in that the spacing between the intron of the adeno-associated virus genome and the start codon of the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • Embodiment 21 The recombinant nucleic acid according to any one of embodiments 1-20 is characterized in that it comprises feature (7), and the sequence of the non-parent serotype promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:15.
  • Embodiment 22 The recombinant nucleic acid according to any one of Embodiments 1-21, characterized in that (i) the non-parental serotype promoter is located downstream of the Cap gene, or (ii) the non-parental serotype promoter is located upstream of the Rep gene, and the non-parental serotype promoter is located upstream of the hGH intron.
  • Embodiment 23 The recombinant nucleic acid according to Embodiment 22 is characterized in that the non-parent serotype promoter is located at a position corresponding to nucleotides 4270-4271 of SEQ ID NO:7.
  • Embodiment 24 The recombinant nucleic acid according to any one of embodiments 1-23 is characterized in that the recombinant nucleic acid does not contain a sequence equivalent to nucleotides 4368-4532 of SEQ ID NO:7, or does not contain a sequence equivalent to nucleotides 4373-4532 of SEQ ID NO:7.
  • Embodiment 25 The recombinant nucleic acid according to any one of embodiments 1-24 is characterized in that the recombinant nucleic acid does not contain a sequence corresponding to nucleotides 121-130 of SEQ ID NO:7.
  • Embodiment 26 The recombinant nucleic acid according to any one of embodiments 1-25, characterized in that:
  • the serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or AAV-DJ; preferably, the serotype of the AAV is AAV2, AAV5, AAV8, AAV9; more preferably, the serotype of the AAV is AAV2;
  • the sequence of the Rep gene is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2;
  • the sequence of the Cap gene is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3.
  • Embodiment 27 The recombinant nucleic acid according to any one of Embodiments 1-26 is characterized in that the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:48-57.
  • Embodiment 28 The recombinant nucleic acid according to any one of embodiments 1-27 is characterized in that the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:58-67.
  • Embodiment 29 The recombinant nucleic acid according to any one of embodiments 1-28, characterized in that it includes the characteristics:
  • Embodiment 30 The recombinant nucleic acid according to any one of Embodiments 1-29, characterized in that it comprises the Rep gene and the Cap gene.
  • Embodiment 31 The recombinant nucleic acid according to any one of embodiments 1, 3-7, 10-11, 21-29, characterized in that it comprises the Cap gene but does not comprise a nucleic acid sequence encoding the Rep gene.
  • Embodiment 32 The recombinant nucleic acid according to any one of embodiments 1-2, 4-5, 8-9, 12-29, characterized in that it comprises the Rep gene but does not comprise a nucleic acid sequence encoding the Cap gene.
  • Embodiment 33 A plasmid comprising the recombinant nucleic acid according to any one of embodiments 1-32.
  • Embodiment 34 A plasmid according to Embodiment 33, characterized in that the plasmid is a packaging plasmid (RC plasmid) in a recombinant adeno-associated virus helper virus-free system, or a helper plasmid (pHelper plasmid), or a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.
  • RC plasmid packaging plasmid
  • pHelper plasmid helper plasmid
  • Embodiment 35 A plasmid according to embodiments 33-34, characterized in that the plasmid contains the Cap gene, but does not contain a nucleic acid sequence encoding a Rep gene, nor does it contain a nucleic acid sequence encoding an auxiliary gene for AAV production, nor does it contain an AAV inverted repeat sequence (ITR).
  • ITR AAV inverted repeat sequence
  • Embodiment 36 A plasmid characterized by comprising a Cap gene but not comprising a nucleic acid sequence encoding a Rep gene, nor a nucleic acid sequence encoding an auxiliary gene for AAV production, nor an AAV inverted repeat sequence (ITR).
  • ITR AAV inverted repeat sequence
  • Embodiment 37 A plasmid according to any one of Embodiments 33-34, characterized in that the plasmid is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence, and contains the Cap gene.
  • Embodiment 38 A plasmid according to Embodiment 37, characterized in that the target gene plasmid contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment 39 A helper plasmid, characterized in that it contains the recombinant nucleic acid containing the Cap gene according to any one of Embodiments 1-31, and the helper plasmid contains a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production.
  • AAV adeno-associated virus
  • Embodiment 40 A helper plasmid, characterized in that it contains a nucleic acid sequence encoding a Cap gene and a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production, wherein the Cap gene encodes a functional Cap protein.
  • AAV adeno-associated virus
  • the auxiliary plasmid according to any one of Embodiments 39-40 is characterized in that the auxiliary plasmid contains a Rep gene; optionally, the Rep gene expresses Rep78 and Rep52 proteins; optionally, the Rep gene does not express Rep68 or Rep40 proteins; optionally, the Rep gene contains a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • Embodiment 42 The auxiliary plasmid according to any one of Embodiments 39-41 is characterized in that the auxiliary genes for AAV production include one or more genes selected from E2A, E4, and VA RNA.
  • Embodiment 43 The auxiliary plasmid according to Embodiment 42 is characterized in that the auxiliary genes for AAV production include two genes selected from E2A, E4, and VA RNA; optionally, the auxiliary plasmid contains the E4 gene and the VA RNA gene.
  • Embodiment 44 The auxiliary plasmid according to Embodiment 42 is characterized in that the auxiliary genes for AAV production contain E2A, E4, and VA RNA.
  • Embodiment 45 The helper plasmid according to any one of Embodiments 42-44, characterized in that the E2A, E4, and/or VA RNA are derived from adenovirus type 2 (Ad2); optionally, the E2A gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:93; optionally, the E4 gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:94; optionally, the VA RNA gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least
  • Embodiment 46 The auxiliary plasmid according to any one of Embodiments 42-44 is characterized in that the E2A, E4, and/or VA RNA is derived from adenovirus type 2 (Ad2).
  • Ad2 adenovirus type 2
  • Embodiment 47 The recombinant nucleic acid according to any one of embodiments 1-31, or the plasmid according to any one of embodiments 31-41, characterized in that it comprises at least one promoter selected from the group consisting of a P5 promoter, a P19 promoter, and a P40 promoter, and the promoter is operably linked to the Cap gene.
  • Embodiment 48 The recombinant nucleic acid or plasmid according to Embodiment 47 is characterized in that it contains a P5 promoter, a P19 promoter, and a P40 promoter; preferably, the nucleic acid sequence encoding the Cap gene contains, from the 5' end to the 3' end, a sequence encoding the P5 promoter, the P19 promoter, the P40 promoter, and the Cap gene.
  • Embodiment 49 The recombinant nucleic acid or plasmid according to any one of Embodiments 47-48, characterized in that the spacing between the promoters and/or between the promoter and the Cap gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment 50 The recombinant nucleic acid or plasmid according to any one of Embodiments 47-49 is characterized in that the sequence of the P5 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 191-320 of SEQ ID NO:1.
  • Embodiment 51 The recombinant nucleic acid or plasmid according to any one of Embodiments 47-50 is characterized in that the sequence of the P19 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 741-890 of SEQ ID NO:1; preferably, the sequence of the P19 promoter is T at the position relative to nucleotide 768 of SEQ ID NO:1.
  • Embodiment 52 The recombinant nucleic acid or plasmid according to any one of Embodiments 47-51 is characterized in that the sequence of the P40 promoter is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 1701-2202 of SEQ ID NO:1; preferably, the sequence of the P40 promoter is GT relative to nucleotides 1911-1912 of SEQ ID NO:1.
  • Embodiment 53 The recombinant nucleic acid according to any one of embodiments 1-31, the plasmid according to any one of embodiments 33-46, or the recombinant nucleic acid or plasmid according to any one of embodiments 47-52, characterized in that the sequence of the Cap gene comprises a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 2203-4410 of SEQ ID NO:1.
  • Embodiment 54 The recombinant nucleic acid according to any one of embodiments 1-31, the plasmid according to any one of embodiments 33-46, or the recombinant nucleic acid or plasmid according to any one of embodiments 47-52, characterized in that the Cap protein encoded by the Cap gene comprises a protein sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:48-57.
  • Embodiment 55 The recombinant nucleic acid or plasmid according to Embodiment 54 is characterized in that the Cap gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO:58-67.
  • Embodiment 56 The recombinant nucleic acid according to any one of embodiments 1-31, the plasmid according to any one of embodiments 33-46, or the recombinant nucleic acid or plasmid according to any one of embodiments 47-55, characterized in that the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.
  • Embodiment 57 The recombinant nucleic acid or plasmid according to Embodiment 56 is characterized in that the interval between the Cap gene and the polyA sequence is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment 58 The recombinant nucleic acid or plasmid according to any one of Embodiments 56-57 is characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment 59 The recombinant nucleic acid according to any one of embodiments 1-31, the plasmid according to any one of embodiments 33-46, or the recombinant nucleic acid or plasmid according to any one of embodiments 47-58, characterized in that a P5 promoter is further included downstream of the Cap gene, which is operably connected to the Cap gene; optionally, the P5 promoter is located downstream of the polyA sequence downstream of the Cap gene; optionally, the P5 promoter and the Cap gene, or the P5 promoter and the polyA sequence, are spaced by no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides, or there are no intervening nucleotides.
  • Embodiment 60 The recombinant nucleic acid according to any one of embodiments 1-31, the plasmid according to any one of embodiments 33-46, or the recombinant nucleic acid or plasmid according to any one of embodiments 47-59, characterized in that the Cap gene encodes a functional Cap protein.
  • Embodiment 61 A plasmid according to any one of Embodiments 33-60, characterized in that the plasmid cannot express functional Rep protein.
  • Embodiment 62 A plasmid according to any one of Embodiments 33-61, characterized in that the plasmid does not contain a nucleic acid sequence encoding the Rep gene.
  • Embodiment 63 A helper plasmid, characterized in that it contains the recombinant nucleic acid containing the Rep gene according to any one of embodiments 1-30, 32, and the helper plasmid contains a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production.
  • AAV adeno-associated virus
  • Embodiment 64 A helper plasmid, characterized in that it comprises a nucleic acid sequence encoding a Rep gene and a nucleic acid sequence encoding at least one helper gene for adeno-associated virus (AAV) production,
  • AAV adeno-associated virus
  • Embodiment 65 The auxiliary plasmid according to any one of Embodiments 63-64 is characterized in that the auxiliary genes for AAV production include one or more genes selected from E2A, E4, and VA RNA.
  • Embodiment 66 The auxiliary plasmid according to Embodiment 65 is characterized in that the auxiliary genes for AAV production include two genes selected from E2A, E4, and VA RNA; optionally, the auxiliary plasmid contains the E4 gene and the VA RNA gene.
  • Embodiment 67 The auxiliary plasmid according to Embodiment 65 is characterized in that the auxiliary genes for AAV production contain E2A, E4, and VA RNA.
  • Embodiment 68 The helper plasmid according to any one of Embodiments 65-67 is characterized in that the E2A, E4, and/or VA RNA are derived from adenovirus type 2 (Ad2); optionally, the E2A gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:93; optionally, the E4 gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:94; optionally, the VA RNA gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least
  • Embodiment 69 The auxiliary plasmid according to any one of Embodiments 65-67 is characterized in that the E2A, E4, and/or VA RNA is derived from adenovirus type 2 (Ad2).
  • Ad2 adenovirus type 2
  • Embodiment 70 The helper plasmid according to any one of Embodiments 63-69 is characterized in that it contains a nucleic acid sequence encoding the Rep gene, and the plasmid (a) cannot express a functional Cap protein, and/or (b) does not contain a nucleic acid sequence encoding the Cap gene.
  • Embodiment 71 A plasmid according to any one of Embodiments 33-34, characterized in that it contains a nucleic acid sequence encoding the Rep gene, and the plasmid (a) cannot express a functional Cap protein, and/or (b) does not contain a nucleic acid sequence encoding the Cap gene.
  • Embodiment 73 A plasmid according to Embodiment 72, characterized in that the plasmid does not contain a nucleic acid sequence encoding a Cap gene.
  • Embodiment 74 A plasmid according to embodiment 72 or 73, characterized in that the plasmid is unable to express functional expression products of auxiliary genes for AAV production.
  • Embodiment 75 A plasmid according to any one of Embodiments 72-74, characterized in that the plasmid does not contain a nucleic acid sequence encoding an auxiliary gene for AAV production.
  • Embodiment 76 A recombinant nucleic acid comprising the Rep gene according to any one of embodiments 1-30, 32, or a plasmid according to any one of embodiments 33-34, 63-75, characterized in that the Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.
  • Embodiment 77 The recombinant nucleic acid or plasmid according to Embodiment 76 is characterized in that the sequence of the Rep gene comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2300 of SEQ ID NO:1.
  • Embodiment 78 The recombinant nucleic acid comprising the Rep gene according to any one of embodiments 1-30, 32, or the plasmid according to any one of embodiments 33-34, 63-75, characterized in that the Rep gene expresses Rep78 and Rep52 proteins; optionally, the Rep gene does not express Rep68 or Rep40 proteins; optionally, the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:71.
  • Embodiment 79 The recombinant nucleic acid or plasmid according to Embodiment 78 is characterized in that the sequence of the Rep gene contains a sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 321-2196 of SEQ ID NO:1.
  • Embodiment 81 A recombinant nucleic acid or plasmid according to Embodiment 80, characterized in that the Rep gene comprises a polynucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:103.
  • Embodiment 82 The recombinant nucleic acid or plasmid according to any one of Embodiments 80-81, characterized in that the recombinant nucleic acid or plasmid does not contain genes that only express Rep52 and Rep40 proteins.
  • Embodiment 83 The recombinant nucleic acid comprising the Rep gene according to any one of embodiments 1-30, 32, the plasmid according to any one of embodiments 33-34, 63-75, or the recombinant nucleic acid or plasmid according to any one of embodiments 76-82, characterized in that it comprises a P5 promoter located upstream of the Rep gene and/or a P5 promoter located downstream of the Rep gene, and the P5 promoter is operably connected to the Rep gene.
  • Embodiment 84 The recombinant nucleic acid or plasmid according to Embodiment 83 is characterized in that the spacing between the promoter and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment 86 A recombinant nucleic acid comprising the Rep gene according to any one of embodiments 1-30, 32, a plasmid according to any one of embodiments 33-34, 63-75, or a recombinant nucleic acid or plasmid according to any one of embodiments 76-85, characterized in that the Rep gene encodes a functional Rep protein.
  • Embodiment 87 The recombinant nucleic acid comprising the Rep gene according to any one of embodiments 1-30, 32, the plasmid according to any one of embodiments 33-34, 63-75, or the recombinant nucleic acid or plasmid according to any one of embodiments 76-86, characterized in that the nucleic acid sequence encoding the Rep gene encodes a polyA sequence, and the polyA sequence is located downstream of the Rep gene.
  • Embodiment 88 The recombinant nucleic acid or plasmid according to Embodiment 87 is characterized in that the interval between the Rep gene and the polyA sequence is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment 89 The recombinant nucleic acid or plasmid according to any one of Embodiments 87-88 is characterized in that the polyA sequence is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to nucleotides 4411-4460 of SEQ ID NO:1.
  • Embodiment 90 A plasmid according to any one of embodiments 33-34, 63-89, characterized in that the nucleic acid sequence encoding the Rep gene contains an hGH intron; preferably, the intron is located before the start codon of the Rep gene.
  • Embodiment 91 A plasmid according to Embodiment 90, characterized in that the spacing between the P5 promoter and the hGH intron, and/or between the hGH intron and the Rep gene is no more than 30 bp, no more than 25 bp, no more than 20 bp, no more than 15 bp, no more than 10 bp, or no more than 5 bp of nucleotides; preferably, there are no intervening nucleotides.
  • Embodiment 92 A plasmid according to any one of Embodiments 90-91, characterized in that the sequence of the hGH intron is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13.
  • Embodiment 93 A plasmid according to any one of embodiments 33-34, 63-92, characterized in that the donor site of the original intron of the Rep gene contains a mutation, and the donor site is equivalent to the nucleotide sites 1907-1912 of SEQ ID NO:1.
  • Embodiment 94 A plasmid according to Embodiment 93, characterized in that the mutation of the donor site comprises mutating CA corresponding to nucleotides at positions 1911-1912 of SEQ ID NO:1 to GT, or mutating the donor site sequence corresponding to nucleotides at positions 1907-1912 of SEQ ID NO:1 to GTACGT.
  • Embodiment 95 The plasmid according to any one of Embodiments 33-94 is characterized in that the plasmid also contains an f1 origin site.
  • Embodiment 96 A three-plasmid system, characterized in that it comprises:
  • a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, further comprising at least two AAV inverted repeat sequences (ITR), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence,
  • the plasmid described in (i) and/or (ii) comprises the recombinant nucleic acid encoding the Rep gene and/or the Cap gene according to any one of embodiments 1-32, 47-60, 76-89.
  • Embodiment 97 A three-plasmid system according to Embodiment 96, characterized in that the plasmid in (ii) comprises the Rep gene and the Cap gene.
  • Embodiment 98 A three-plasmid system according to Embodiment 96, characterized in that the Rep gene and the Cap gene are on different plasmids.
  • Embodiment 99 A three-plasmid system according to Embodiment 98, characterized in that the helper plasmid in (i) contains the Rep gene, and the plasmid in (ii) contains the Cap gene.
  • Embodiment 100 A three-plasmid system according to Embodiment 98, characterized in that the plasmid in (ii) contains the Rep gene, and the plasmid of the recombinant adeno-associated virus in (iii) contains the Cap gene.
  • Embodiment 101 A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that one plasmid contains the Rep gene of AAV but does not contain the Cap gene of AAV, and another different plasmid contains the Cap gene of AAV but does not contain the Rep gene of AAV.
  • AAV adeno-associated virus
  • Embodiment 102 According to the three-plasmid system described in Embodiment 101, it is characterized in that it comprises an auxiliary plasmid (pHelper plasmid) comprising the Rep gene, a second plasmid comprising the Cap gene, and a third plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, the third plasmid also comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • pHelper plasmid auxiliary plasmid
  • ITR AAV inverted repeat sequences
  • Embodiment 103 According to the three-plasmid system described in Embodiment 101, it is characterized in that it comprises an auxiliary plasmid (pHelper plasmid), a second plasmid comprising the Rep gene, and a third plasmid comprising the Cap gene and encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, the third plasmid also comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • pHelper plasmid auxiliary plasmid
  • a second plasmid comprising the Rep gene
  • a third plasmid comprising the Cap gene and encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence
  • Embodiment 104 A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that it comprises:
  • helper plasmid according to any one of embodiments 63-70, 76-95, which comprises the Rep gene, and the helper plasmid (a) cannot express a functional Cap protein, and/or (b) does not comprise a nucleic acid sequence encoding the Cap gene;
  • plasmid according to any one of embodiments 35-36, 47-62, comprising the Cap gene, and the plasmid (a) cannot express a functional Rep protein, and/or (b) does not comprise a nucleic acid sequence encoding a Rep gene;
  • a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, it also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment 105 A three-plasmid system for adeno-associated virus (AAV) expression, comprising:
  • helper plasmid a helper plasmid
  • the helper plasmid comprises a recombinant nucleic acid encoding a Rep gene
  • the helper plasmid (a) cannot express a functional Cap protein, and/or (b) does not comprise a nucleic acid sequence encoding a Cap gene
  • plasmid comprising a Cap gene; optionally, the plasmid (a) is unable to express a functional Rep protein, and/or (b) does not comprise a nucleic acid sequence encoding a Rep gene; and
  • a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, it also contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment 106 A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that it comprises:
  • a recombinant adeno-associated virus plasmid comprising the Cap gene and the heterologous nucleic acid sequence according to any one of embodiments 37-38, 47-62; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, further comprising at least two AAV inverted repeat sequences (ITR), wherein the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment 107 A three-plasmid system for adeno-associated virus (AAV) expression, comprising:
  • plasmid comprising a Rep gene
  • the plasmid is unable to express a functional Cap protein, and/or (b) does not comprise a nucleic acid sequence encoding a Cap gene
  • plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence and comprising a Cap gene; optionally, the plasmid (a) cannot express a functional Rep protein, and/or (b) does not contain a nucleic acid sequence encoding a Rep gene; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, it further comprises at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment 108 A three-plasmid system for adeno-associated virus (AAV) expression, comprising:
  • Embodiment 109 A three-plasmid system for adeno-associated virus (AAV) expression, comprising:
  • helper plasmid (i) a helper plasmid (pHelper plasmid), wherein the helper plasmid comprises a recombinant nucleic acid encoding a Cap gene;
  • Embodiment 110 A three-plasmid system according to any one of Embodiments 108-109, characterized in that the (i) auxiliary plasmid cannot express functional Rep protein, and/or the (ii) plasmid encoding the Rep gene cannot express functional Cap protein.
  • Embodiment 112 A three-plasmid system according to any one of Embodiments 108-111, characterized in that the (ii) plasmid encoding the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 or Rep52 proteins, and the (i) auxiliary plasmid contains a gene expressing Rep78 and/or Rep52 proteins; optionally, the gene does not express Rep68 or Rep40.
  • Embodiment 114 A three-plasmid system according to any one of Embodiments 108-113, characterized in that the (i) auxiliary plasmid contains the E4 gene and the VA RNA gene; and the (ii) plasmid encoding the Rep gene contains the E2A gene.
  • Embodiment 116 A three-plasmid system according to any one of Embodiments 108-115, characterized in that the heterologous nucleic acid sequence encodes a heterologous gene.
  • Embodiment 117 A two-plasmid system, characterized in that it comprises (i) the first plasmid containing the Rep gene according to any one of embodiments 63-70, 76-95 and (ii) the second plasmid containing the Cap gene according to any one of embodiments 37-38, 47-62.
  • Embodiment 118 A two-plasmid system, characterized in that it comprises a first plasmid containing the Rep gene of AAV and a second plasmid containing the Cap gene of AAV, wherein the Rep gene and the Cap gene are located on different plasmids respectively.
  • Embodiment 119 A two-plasmid system according to any one of Embodiments 117-118, characterized in that the Rep gene and the Cap gene are located on different plasmids.
  • Embodiment 121 A two-plasmid system according to any one of Embodiments 117-119, characterized in that the second plasmid containing the Cap gene is a helper plasmid (pHelper plasmid), and the first plasmid containing the Rep gene is a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence; optionally, the heterologous nucleic acid sequence encodes a heterologous gene; optionally, the first plasmid further contains at least two AAV inverted repeat sequences (ITR), and the two AAV inverted repeat sequences are respectively located upstream and downstream of the heterologous nucleic acid sequence.
  • ITR AAV inverted repeat sequences
  • Embodiment 122 A plasmid system according to any one of Embodiments 96-121, characterized in that the Rep gene expresses Rep78, Rep68, Rep52 and Rep40 proteins.
  • Embodiment 123 A plasmid system according to any one of Embodiments 96-122, characterized in that the Rep gene expresses Rep78 and Rep52 proteins, but does not express Rep68 and Rep40 proteins.
  • Embodiment 124 A plasmid system according to any one of Embodiments 96-122, characterized in that the Rep gene expresses Rep68 and Rep40 proteins but does not express Rep78 and Rep52 proteins.
  • Embodiment 125 A plasmid system according to any one of Embodiments 96-124, characterized in that the plasmid system does not contain genes for co-expressing Rep52 protein and Rep40 protein.
  • Embodiment 126 A plasmid system according to any one of Embodiments 96-125, characterized in that the upstream of the Rep gene contains an hGH intron.
  • Embodiment 127 A plasmid system according to Embodiment 126, characterized in that the upstream of the hGH intron contains a P5 promoter.
  • Embodiment 128 A plasmid system according to any one of Embodiments 96-127, characterized in that the downstream of the Rep gene contains a polyA sequence.
  • Embodiment 129 A plasmid system according to any one of Embodiments 96-128, characterized in that the upstream and/or downstream of the Rep gene contains a P5 promoter, which is operably linked to the Rep gene.
  • Embodiment 130 A plasmid system according to any one of Embodiments 96-129, characterized in that the upstream of the Cap gene contains three promoters, which from 5' to 3' direction are: P5 promoter, P19 promoter, and P40 promoter, all of which are operably linked to the Cap gene, and the downstream of the Cap gene contains a polyA sequence.
  • Embodiment 131 A plasmid system according to any one of Embodiments 96-130, characterized in that the plasmid system comprises at least one polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 16--28, 33-34, 40, 45-47, 68, 80-87, 96-98.
  • Embodiment 132 A method for modifying a plasmid system, characterized by comprising one or more of the following modification methods:
  • Embodiment 133 The method for modifying the plasmid system according to Embodiment 132, characterized in that:
  • the P5 promoter inserted upstream of the Rep gene as described in (1) has a sequence as shown in SEQ ID NO:4;
  • the mutated P5 promoter has the sequence shown in SEQ ID NO:8, and the P5 promoter inserted downstream of the Cap gene has the sequence of SEQ ID NO:4;
  • Mutating the donor site of the intron is to mutate GTACCA at the donor site of the intron to GTACGT, preferably, the intron before the mutation has a sequence as shown in SEQ ID NO: 10; and the intron after the mutation has a sequence as shown in SEQ ID NO: 11;
  • the hGH intron inserted in has the sequence shown in SEQ ID NO:13;
  • the modified wtAAV2 intron has a sequence as shown in SEQ ID NO:14; and/or
  • the non-parental serotype promoter is wtAAV7 P5 promoter, which has the sequence shown in SEQ ID NO:15.
  • Embodiment 134 A method for producing a recombinant adeno-associated virus, characterized in that the plasmid according to any one of embodiments 33-95 or all plasmids in the plasmid system according to any one of embodiments 96-131 are transfected into a host cell, cultured, and isolated to obtain the recombinant adeno-associated virus.
  • Embodiment 135. A production method according to Embodiment 134, characterized in that the ratio of the plasmid comprising the Cap gene to the plasmid comprising the Rep gene is at least 1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, or at least 4:1; preferably, at least 3:1.
  • Embodiment 136 A production method according to any one of Embodiments 134-135, characterized in that the ratio of the plasmid containing the Cap gene to the plasmid containing the Rep gene is about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, or about 4:1; preferably, about 3:1.
  • Embodiment 137 A production method according to any one of embodiments 134-136, characterized in that the ratio of the plasmid containing the Rep gene to the plasmid containing the recombinant adeno-associated virus encoding the heterologous nucleic acid sequence is at least 1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, or at least 4:1; preferably, at least 2.5:1.
  • Embodiment 138 A production method according to any one of embodiments 134-137, characterized in that the ratio of the plasmid containing the Rep gene to the plasmid containing the recombinant adeno-associated virus encoding the heterologous nucleic acid sequence is about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, or about 4:1; preferably, about 2.5:1.
  • Embodiment 139 The production method according to any one of Embodiments 134-138, characterized in that, compared with the production method based on the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid, the recombinant adeno-associated virus produced by the method is:
  • the solidity is higher; optionally, the solidity value is at least 0.05, at least 0.10, at least 0.15, at least 0.20, at least 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, or at least 0.80 higher;
  • the genomic titer is higher; optionally, the genomic titer is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher;
  • the residual impurities associated with the Cap encoding sequence are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventional methods; and/or
  • the residual rcAAV is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventional methods.
  • Embodiment 140 A recombinant adeno-associated virus, characterized in that it is obtained using the production method described in any one of Embodiments 134-139.
  • Embodiment 141 The recombinant adeno-associated virus according to Embodiment 140 is characterized in that the culture method of the host cells includes adherent culture or suspension culture.
  • Embodiment 142 The recombinant adeno-associated virus according to any one of Embodiments 140-141, characterized in that compared with the recombinant adeno-associated virus produced by the traditional three-plasmid system of helper plasmid + Rep/Cap plasmid + target gene plasmid:
  • the solidity is higher; optionally, the solidity value is at least 0.05, at least 0.10, at least 0.15, at least 0.20, at least 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, or at least 0.80 higher;
  • the genomic titer is higher; optionally, the genomic titer is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher;
  • the residual impurities associated with the Cap coding sequence are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventionally produced recombinant adeno-associated viruses; and/or
  • the residual rcAAV is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% compared to conventionally produced recombinant adeno-associated viruses.
  • Embodiment 143 A host cell, characterized in that the host cell contains the plasmid according to any one of embodiments 33-95 or all plasmids in the plasmid system according to any one of embodiments 96-131.
  • Embodiment 144 A production method according to any one of Embodiments 134-139, or a host cell according to Embodiment 143, characterized in that the host cell is selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.
  • Embodiment 145 Use of the recombinant nucleic acid described in any one of embodiments 1-32, the plasmid described in any one of embodiments 33-95, the plasmid system described in any one of embodiments 96-141, the recombinant adeno-associated virus described in any one of embodiments 140-142, or the host cell described in any one of embodiments 143-144 in the preparation of a drug.
  • Embodiment 146 A pharmaceutical preparation, characterized in that the drug comprises the recombinant adeno-associated virus according to any one of Embodiments 140-142, and a pharmaceutically acceptable carrier and excipient.
  • Embodiment 147 Use of the pharmaceutical preparation described in Embodiment 146 in treating a disease.
  • Embodiment 148 A method for treating a disease, comprising administering the recombinant adeno-associated virus according to any one of Embodiments 140-142, or the pharmaceutical formulation according to Embodiment 146 to a patient in need thereof.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明涉及生物技术领域，特别涉及质粒系统及其应用。具体是用于生产重组腺相关病毒(Recombinant adeno-associated virus，rAAV)载体的新型质粒包装系统，通过对传统三质粒系统的改造(例如将包装质粒pRC2进行拆分和修饰，将Cap基因与辅助质粒pHelper组合，而Rep基因单独置于一个质粒，由此获得新型的辅助包装质粒)，由此获得不同结构的辅助质粒、包装质粒和/或载体质粒。这些质粒系统不仅可以提高rAAV产量，节约高昂的生产成本，还能降低载体中的污染杂质-具有复制能力的AAV(Replication-competent AAV，rcAAV)含量，控制纯度，增加AAV载体制剂的经济性和安全性。

Description

质粒包装系统及其应用
相关申请的交叉引用
本申请要求2023年2月1日提交的中国专利申请号202310049101.7，和2023年6月2日提交的中国专利申请号202310651836.7的权益，将每个文献的内容通过引用以其整体并入本文。
通过引用并入序列表
将与此说明书一起以电子方式提交的序列文件的内容通过引用以其整体并入本文：序列文件的计算机可读格式拷贝(文件名：F24W1513PCT-new-sequence listing.xml，创建日期：2024年1月30日，文件大小：630kb)。
技术领域
本发明涉及生物技术领域，特别涉及质粒系统及其应用。
背景内容
重组腺相关病毒(Recombinant adeno-associated virus，rAAV)可以在体内长期稳定表达外源基因，是一种低免疫原性病毒载体，所以成为基因治疗领域中最有希望的递送工具，被广泛应用于基础和临床研究中，目前正在进行的临床试验就有几百项，且已有八款AAV基因治疗药物上市，商业前景十分广阔。但是在2017年第一种AAV基因治疗药物Glybera宣布退市，究其原因是有价无市，其一次疗法的费用高达100万美元，生产成本高是其定价高的根本原因。所以实现规模化生产，得到高滴度高纯度的rAAV载体一直是生物制药企业需要解决的重大难题。
尽管AAV在生物技术制品的相关评级中被认为安全性较高，但近年来，FDA咨询委员会对于AAV临床研究的结果也越发关注，在IND申请指南中对于复制型腺相关病毒(Replication-competent adeno-associated virus，rcAAV)的问题更是十分重视，他们建议检测rcAAV，且描述出检测方法和检测灵敏度。rcAAV是指含有wtAAV Rep和Cap基因的有复制能力的病毒颗粒，当辅助病毒存在时，这种AAV能够进行复制。中国《体内基因治疗产品药学研究与评价技术指导原则(试行)》也明确指出，对于病毒载体类产品的起始原材料，比如瞬时转染时用于病毒载体包装的质粒，应全面考虑病毒载体设计，严格控制潜在风险。为了降低病毒生产和使用过程中重组产生可复制型或假野生型病毒的风险，建议优先选择经验证具有较高安全等级的病毒包装系统，将病毒包装必需元件适当拆分至不同的质粒中，尽量去除非必需的病毒基因，降低质粒之间，质粒与包装细胞及野生病毒序列之间的同源性。特别说明AAV包装系统构建时，将AAV的结构基因分离于不同质粒的转录单元中，并尽可能降低Rep/Cap基因序列和ITR等序列之间的相似，降低重组产生新型感染性病毒或复制型病毒的风险，充分考虑生产和使用过程中通过同源或非同源重组产生复制型病毒的可能，载体应避免包含任何已知的、具有人体致瘤性风险的元件等。
由于复制蛋白Rep具有解旋酶和内切酶活性，可能会诱发DNA损伤引起细胞毒性，且衣壳蛋白Cap也被发现会导致严重的细胞免疫反应，而rcAAV就是这两种蛋白的重要来源之一，因此会严重影响药物安全性和质量。rcAAV作为AAV载体制剂中的一种杂质，在下游纯化过程中是无法被区分去除的，rcAAV残留检测也是法规要求的重点检验项目，所以必须降低rcAAV含量。
现用生产rAAV的主要方式是AAV无辅助病毒系统(AAV Helper-Free System)，该系统包含三个质粒：第一个是含有目的基因(gene-of-interest(GOI))的载体质粒，其两端保留了AAV的末端反向重复序列(Inverted terminal repeat，ITR)，是复制和包装必需的顺式作用元件；第二个是决定了血清型的RC质粒(pRC)，编码AAV自身的复制蛋白Rep和结构蛋白Cap；最后需要一个辅助质粒pHelper，提供从腺病毒Ad(adenovirus)中分离的辅助基因E2A、E4orf6和VA RNA。通过三质粒瞬转哺乳动物细胞，能够快速、简便生产出rAAV，但是此种方法生产用细胞多为贴壁细胞，需要胎牛血清培养，对生长条件和下游纯化要求较严格，且需要制备大量高质量的质粒，因此生产成本很高，难以扩大。虽然目前已有商业化的悬浮HEK293细胞，优点是使用无血清培养基进行培养，不仅可以减少下游纯化步骤，还可以实现规模放大，缺点是受转染复合物形成的动力学因素影响，转染效率低，投料量相等的情况下，rAAV的产量不如贴壁培养高。另外由于所用包装RC质粒的Rep和Cap基因在同一质粒骨架上，和wtAAV基因序列排布基本相同，所以生产过程中载体质粒(pAAV-GOI)的ITR及其附近序列和包装质粒(pRC)的Rep-Cap序列之间极易于发生同源或非同源重组形成rcAAV。目前已有多种抑制或避免包装wtAAV基因的方法，减少rcAAV形成的第一种方法是：消除载体质粒和RC质粒之间的同源序列，进一步可以将Rep和Cap置于异源启动子的控制下，代替AAV的天然启动子P5、P19或P40，甚至以相反的方向转录，有文献报道通过这种方法可以将rcAAV降低到检测水平以下，缺点是rAAV产量可能大幅降低。另一种方法是通过在Rep或Cap基因内部插入内含子来产生超大的RC质粒，从而使得最终的序列超过AAV的包装限度，但此种方法在包装过程中内含子很有可能被剪切掉，所以wtAAV基因还是会被包装进去，rcAAV残留量可能不符合临床级AAV载体制剂的监管要求。
由于存在以上产量和安全问题，所以提高产量降低rAAV载体的生产成本，尽量减少污染杂质rcAAV造成的安全影响仍然是一个需要解决的技术问题。
发明内容
本文的一方面，提供了一种重组核酸，包含腺相关病毒(AAV)的Rep基因和/或Cap基因，且有如下的一种或多种特征：
(1)在所述Rep基因的上游和/或所述Cap基因的下游含有一个P5启动子；
(2)位于所述Cap基因下游的原有P5启动子中的TATA box突变被修正，或在所述Cap基因下游插入一个所述P5启动子，与所述Cap基因可操作地连接；
(3)所述腺相关病毒的病毒基因组原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:7的第1717-1722核苷酸位点；
(4)所述Cap基因下游原有的肝脏特异性增强子-启动子序列被删除；
(5)在所述Rep基因起始密码子前含有一个hGH内含子；
(6)在所述Rep基因起始密码子前含有一个腺相关病毒基因组的内含子；和/或
(7)在所述Rep基因上游或在所述Cap基因下游含有一个非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
在一些实施例中，所述的重组核酸包含特征(1)，且位于所述Rep基因上游的所述P5启动子的插入位置紧接在相当于SEQ ID NO:7的腺相关病毒基因组的120bp的核苷酸位点后，与所述Rep基因可操作地连接。在一些实施例中，所述的重组核酸在所述Cap基因下游插入一个P5启动子，与所述Cap基因可操作地连接。在一些实施例中，所述P5启动子与所述Rep基因或所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述P5启动子的序列与SEQ ID NO:4有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的重组核酸包含特征(2)，且所述原有P5启动子的TATA box突变的修正包含将相当于SEQ ID NO:7第4469位核苷酸的T突变为A，和/或将相当于SEQ ID NO:7第4477-4483位核苷酸的GGGGGGG突变为TATTTAA。在一些实施例中，所述原有P5启动子修正后的序列与SEQ ID NO:9有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的重组核酸包含特征(3)，且所述donor位点的突变包含将相当于SEQ ID NO:7第1721-1722位核苷酸的CA突变为GT，或相当于SEQ ID NO:7第1717-1722位核苷酸的donor位点序列突变为GTACGT。在一些实施例中，突变后的内含子序列与SEQ ID NO:11有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的重组核酸包含特征(4)，且所述肝脏特异性增强子-启动子序列被删除。在一些实施例中，所述肝脏特异性增强子-启动子序列与SEQ ID NO:12或SEQ ID NO:35有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的重组核酸包含特征(5)，且所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述hGH内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。在一些实施例中，所述hGH内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述hGH内含子与所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述hGH内含子与所述Rep基因起始密码子之间没有间隔的核苷酸。
在一些实施例中，所述的重组核酸包含特征(6)，且所述腺相关病毒基因组的内含子的序列与SEQ ID NO:14有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述腺相关病毒基因组的内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。在一些实施例中，所述腺相关病毒基因组的内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述腺相关病毒基因组的内含子和所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
在一些实施例中，所述的重组核酸包含特征(7)，且所述非亲本血清型启动子的序列与SEQ ID NO:15有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述非亲本血清型启动子位于所述Cap基因的下游。在一些实施例中，所述非亲本血清型启动子位于所述Rep基因上游，且所述所述非亲本血清型启动子位于所述hGH内含子的上游。在一些实施例中，所述非亲本血清型启动子位于相当于SEQ ID NO:7第4270-4271位核苷酸之间的位置。
在一些实施例中，所述重组核酸不含有相当于SEQ ID NO:7第4368-4532位核苷酸的序列，或不含有相当于SEQ ID NO:7第4373-4532位核苷酸的序列。
在一些实施例中，所述重组核酸不含有相当于SEQ ID NO:7第121-130位核苷酸的序列。
在一些实施例中，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ。在一些实施例中，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12或AAV-DJ。在一些实施例中，所述AAV血清型是AAV2、AAV5、AAV8、或AAV9。在一些实施例中，所述AAV的血清型是AAV2。在一些实施例中，所述Rep基因的序列与SEQ ID NO:2有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述Cap基因的序列与SEQ ID NO:3有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述的重组核酸包括所述特征：
(1)；或
(1)和(2)；或
(3)；或
(1)(2)和(3)；或
(1)(2)(3)和(4)；或
(1)(2)(3)(4)和(5)；或
(1)和(6)；或
(1)(2)和(6)；或
(2)(3)(4)(5)和(7)；或
(1)(3)(4)(5)和(7)。
在一些实施例中，所述的重组核酸包含所述Rep基因和所述Cap基因。在一些实施例中，所述的重组核酸包含所述Cap基因，但不包含编码Rep基因的核酸序列。在一些实施例中，所述的重组核酸包含所述Rep基因，但不包含编码Cap基因的核酸序列。
本文的一方面，提供了一种质粒，包含本文所述的重组核酸。
在一些实施例中，所述的质粒为重组腺相关病毒无辅助病毒系统中的包装质粒(RC质粒)，或辅助质粒(pHelper质粒)，或编码包含异源核酸序列的重组腺相关病毒的质粒。
在一些实施例中，所述质粒包含所述Cap基因，但不包含编码Rep基因的核酸序列，也不包含编码用于AAV生产的辅助基因的核酸序列，也不包含AAV反向重复序列(ITR)。在一些实施例中，所述质粒为编码包含异源核酸序列的重组腺相关病毒的质粒，且包含所述Cap基因。
本文的一方面，提供了一种质粒，其特征在于，包含Cap基因，但不包含编码Rep基因的核酸序列，也不包含编码用于AAV生产的辅助基因的核酸序列，也不包含AAV反向重复序列(ITR)。
本文的一方面，提供了一种质粒，其特征在于，所述目的基因质粒包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
本文的一方面，提供了一种辅助质粒，其特征在于，包含本文所述的包含所述Cap基因的重组核酸，且所述辅助质粒包含编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。
本文的一方面，提供了一种辅助质粒，其特征在于，包含编码Cap基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列，所述Cap基因编码一个功能性的Cap蛋白。
在一些实施例中，所述辅助质粒包含Rep基因。在一些实施例中，所述Rep基因表达Rep78和Rep52蛋白。在一些实施例中，所述Rep基因不表达Rep68或Rep40蛋白。在一些实施例中，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。在一些实施例中，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的两个基因。在一些实施例中，所述辅助质粒包含E4基因和VA RNA基因。在一些实施例中，所述辅助基因包含E2A，E4，和VA RNA。
在一些实施例中，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)。在一些实施例中，所述E2A基因包含与SEQ ID NO:93有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。在一些实施例中，所述E4基因包含与SEQ ID NO:94有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。在一些实施例中，所述VA RNA基因包含与SEQ ID NO:95有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)。
在一些实施例中，所述的质粒，其特征在于，包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。在一些实施例中，包含P5启动子，P19启动子，和P40启动子。在一些实施例中，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。在一些实施例中，所述启动子之间，和/或所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，没有间隔的核苷酸。在一些实施例中，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。在一些实施例中，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。
在一些实施例中，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。在一些实施例中，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，在所述Cap基因下游还包含一个P5启动子，与所述Cap基因可操作地连接。在一些实施例中，所述P5启动子位于Cap基因下游的polyA序列的下游。在一些实施例中，所述P5启动子与所述Cap基因之间，或所述P5启动子与所述polyA序列之间，间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
在一些实施例中，所述Cap基因编码一个功能性的Cap蛋白。
在一些实施例中，所述质粒不能表达功能性的Rep蛋白。在一些实施例中，所述质粒不包含编码Rep基因的核酸序列。
本文的一方面，提供了一种辅助质粒，其特征在于，包含根据本文所述的包含所述Rep基因的重组核酸，且所述辅助质粒包含编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。本文的一方面，提供了一种辅助质粒，其特征在于，包含编码Rep基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列，
在一些实施例中，所述的辅助质粒，其特征在于，包含编码所述Rep基因的核酸序列，并且所述质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列。
本文的一方面，提供了一种质粒，其特征在于，包含编码所述Rep基因的核酸序列，并且所述质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列。本文的一方面，提供了一种质粒，其特征在于，包含编码Rep基因的核酸序列，并且所述质粒不能表达功能性的Cap蛋白。在一些实施例中，所述质粒不包含编码Cap基因的核酸序列。在一些实施例中，所述质粒不能表达功能性的用于AAV生产的辅助基因的表达产物。在一些实施例中，所述质粒不包含编码用于AAV生产的辅助基因的核酸序列。
在一些实施例中，所述Rep基因表达Rep78，Rep68，Rep52，和Rep40蛋白。
在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
在一些实施例中，所述Rep基因表达Rep78和Rep52蛋白。在一些实施例中，所述Rep基因不表达Rep68或Rep40蛋白。在一些实施例中，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:1第321-2196位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
在一些实施例中，所述Rep基因表达Rep68和Rep40蛋白。在一些实施例中，所述Rep基因不表达Rep78或Rep52蛋白。在一些实施例中，所述Rep基因包含与SEQ ID NO:69或SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述的重组核酸或质粒不包含仅表达Rep52和Rep40蛋白的基因。
在一些实施例中，包含所述Rep基因的重组核酸或质粒包含一个位于所述Rep基因的上游的P5启动子和/或一个位于所述Rep基因的下游的P5启动子，所述P5启动子与所述Rep基因可操作地连接。在一些实施例中，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，没有间隔的核苷酸。在一些实施例中，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸，或与SEQ ID NO:1第191-310位核苷酸，有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，包含所述Rep基因的重组核酸或质粒的Rep基因编码一个功能性的Rep蛋白。
在一些实施例中，所述的包含所述Rep基因的重组核酸或质粒，其特征在于，所述编码Rep基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Rep基因下游。在一些实施例中，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的质粒中的所述编码Rep基因的核酸序列包含一个hGH内含子。在一些实施例中，所述内含子位于所述Rep基因的起始密码子前。
在一些实施例中，所述P5启动子与所述hGH内含子之间，和/或所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，没有间隔的核苷酸。在一些实施例中，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。在一些实施例中，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT，或相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
在一些实施例中，所述质粒还包含f1 origin位点。
本文的一方面，提供了一种三质粒系统，其特征在于，包含:
(i)辅助质粒(pHelper质粒)，
(ii)一个包含腺相关病毒(AAV)的Rep基因和/或Cap基因的质粒，和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游，其中，(i)和/或(ii)中所述的质粒包含根据本文所述的编码所述Rep基因和/或所述Cap基因的重组核酸。
在一些实施例中，(ii)中所述质粒包含所述Rep基因和所述Cap基因。在一些实施例中，所述Rep基因和所述Cap基因在不同的质粒上。在一些实施例中，(i)所述的辅助质粒包含所述Rep基因，且(ii)中所述质粒包含所述Cap基因。在一些实施例中，(ii)中所述质粒包含所述Rep基因，且(iii)中所述重组腺相关病毒的质粒包含所述Cap基因。
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，其中一个质粒包含AAV的Rep基因但不包含AAV的Cap基因，另一个不同的质粒包含AAV的Cap基因但不包含AAV的Rep基因。在一些实施例中，所述的三质粒系统包含一个包含所述Rep基因的辅助质粒(pHelper质粒)，一个包含所述Cap基因的第二质粒，和一个编码包含异源核酸序列的重组腺相关病毒的第三质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，所述第三质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
在一些实施例中，所述的三质粒系统包含一个辅助质粒(pHelper质粒)，一个包含所述Rep基因的第二质粒，和一个包含所述Cap基因且编码包含异源核酸序列的重组腺相关病毒的第三质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，所述第三质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含:
(i)根据本文所述的辅助质粒，其包含所述Rep基因，且所述辅助质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；
(ii)根据本文所述的质粒，其包含所述Cap基因，且该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
在一些实施例中，所述的质粒系统用于生产血清型为AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、或AAV-DJ的重组载体。
优选地，在一些实施例中，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9。
更优选地，在一些实施例中，所述AAV血清型为AAV2。
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Rep基因的重组核酸。在一些实施例中，所述辅助质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；
(ii)一个包含Cap基因的质粒。在一些实施例中，该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含:
(i)辅助质粒(pHelper质粒)，
(ii)根据本文所述的质粒，其包含所述Rep基因，且该质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列，和
(iii)根据本文所述的包含所述Cap基因和所述异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)；
(ii)一个包含Rep基因的质粒。在一些实施例中，该质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒，且包含Cap基因的质粒。在一些实施例中，该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)根据本文所述的辅助质粒，其包含所述Cap基因；
(ii)根据本文所述的编码Rep基因的质粒；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Cap基因的重组核酸；
(ii)一个包含Rep基因的质粒；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
在一些实施例中，所述(i)辅助质粒不能表达功能性的Rep蛋白，和/或所述(ii)编码Rep基因的质粒不能表达功能性的Cap蛋白。在一些实施例中，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。在一些实施例中，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(i)辅助质粒包含一个表达Rep78和/或Rep52蛋白的基因。在一些实施例中，该基因不表达Rep68或Rep40。在一些实施例中，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒包含一个表达Rep78和/或Rep52蛋白的基因。在一些实施例中，该基因不表达Rep68或Rep40。
在一些实施例中，所述(i)辅助质粒包含E4基因和VA RNA基因；所述(ii)编码Rep基因的质粒包含E2A基因。
在一些实施例中，所述的质粒系统用于生产血清型为AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、或AAV-DJ的重组载体。
优选地，在一些实施例中，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9。
更优选地，在一些实施例中，所述AAV的血清型是AAV2。
在一些实施例中，所述异源核酸序列编码一个异源基因。
本文的一方面，提供了一种二质粒系统，其特征在于，包含(i)根据本文所述的含所述Rep基因的第一质粒和(ii)根据本文所述的含所述Cap基因的第二质粒。本文的一方面，提供了一种二质粒系统，其特征在于，包含一个含有AAV的Rep基因的第一质粒和一个含有AAV的Cap基因的第二质粒，所述Rep基因和所述Cap基因分别位于不同质粒上。在一些实施例中，所述Rep和所述Cap基因分别位于不同质粒上。
在一些实施例中，含有所述Rep基因的第一质粒为辅助质粒(pHelper质粒)，且含有所述Cap基因的第二质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，所述第二质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
在一些实施例中，含有所述Cap基因的第二质粒为辅助质粒(pHelper质粒)，且含有所述Rep基因的第一质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。在一些实施例中，所述第一质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
在一些实施例中，所述Rep基因表达Rep78、Rep68、Rep52和Rep40蛋白。在一些实施例中，所述Rep基因表达Rep78和Rep52蛋白，而不表达Rep68和Rep40蛋白。在一些实施例中，所述Rep基因表达Rep68和Rep40蛋白，而不表达Rep78和Rep52蛋白。在一些实施例中，所述质粒系统不包含共表达Rep52蛋白和Rep40蛋白的基因。
在一些实施例中，所述Rep基因的上游含有一个hGH内含子。在一些实施例中，所述hGH内含子的上游含有一个P5启动子。在一些实施例中，所述Rep基因的下游含有polyA序列。在一些实施例中，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
在一些实施例中，所述Cap基因的上游含有三个启动子，从5’至3’方向分别为：P5启动子，P19启动子，和P40启动子，均与所述Cap基因可操作地连接，所述Cap基因的下游含有polyA序列。
在一些实施例中，所述质粒系统包含至少一条与SEQ ID NO:16--28,33-34,40,45-47,68,80-87,96-98任一条有至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述的质粒系统用于生产血清型为AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、或AAV-DJ的重组载体。在一些实施例中，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9。在一些实施例中，所述AAV血清型为AAV2。
本文的一方面，提供了一种质粒系统的改造方法，其特征在于，具有根据下一种或多种修饰方法：
(1)在Rep基因的上游插入P5启动子，与所述Rep基因可操作地连接；
(2)校正位于Cap基因下游的TATA box突变的P5启动子或在Cap基因下游插入P5启动子，与所述Cap基因可操作地连接；
(3)对内含子的donor位点进行突变；
(4)删除掉Cap基因下游包括肝脏特异性增强子-启动子的一段序列；
(5)在Rep基因起始密码子AUG前插入hGH内含子；
(6)在Rep基因起始密码子AUG前插入wtAAV2内含子；
(7)在Rep基因上游或在Cap基因下游插入非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
在一些实施例中，所述改造方法，其特征在于：
(1)中所述在Rep基因的上游插入P5启动子具有如SEQ ID NO:4所示的序列；
(2)中所述突变的P5启动子具有如SEQ ID NO:8所示的序列，在Cap基因下游插入的所述P5启动子具有SEQ ID NO:4的序列；
(3)对内含子的donor位点进行突变是将内含子的donor位点处的GTACCA突变为GTACGT，优选地，突变前的内含子具有如SEQ ID NO:10所示的序列；突变donor后的内含子具有如SEQ ID NO:11所示的序列；
(4)中删除的序列如SEQ ID NO:12所示；
(5)中插入的hGH内含子具有如SEQ ID NO:13所示的序列；
(6)中在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子，优选地，经过改造的wtAAV2内含子具有如SEQ ID NO:14所示的序列；和/或
(7)中非亲本血清型启动子为wtAAV7 P5启动子，其有如SEQ ID NO:15所示的序列。
本文的一方面，提供了一种重组腺相关病毒的生产方法，其特征在于，将根据本文所述的质粒或所述质粒系统中的所有质粒转染至宿主细胞，培养，分离得到所述重组腺相关病毒。
在一些实施例中，所述的生产方法，其特征在于，包含所述Cap基因的质粒与包含所述Rep基因的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1。在一些实施例中，至少3:1。
在一些实施例中，所述的生产方法，其特征在于，包含所述Cap基因的质粒与包含所述Rep基因的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1。在一些实施例中，约3:1。
在一些实施例中，所述的生产方法，其特征在于，包含所述Rep基因的质粒与包含所述编码包含异源核酸序列的重组腺相关病毒的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1。在一些实施例中，至少2.5:1。
在一些实施例中，所述的生产方法，其特征在于，包含所述Rep基因的质粒与包含所述编码包含异源核酸序列的重组腺相关病毒的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1。在一些实施例中，约2.5:1。
在一些实施例中，所述的生产方法，其特征在于，相比基于传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统的生产方法，所述方法产生的重组腺相关病毒的：
(1)实心率更高；可选地，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少0.70,至少0.75,或至少0.80；
(2)基因组滴度更高；可选地，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍；
(3)Cap编码序列的相关杂质残留更低；可选地，Cap编码序列的相关杂质残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％；和/或
(4)具有复制能力的AAV(rcAAV)残留更低；可选地，rcAAV残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
本文的一方面，提供了一种重组腺相关病毒，其特征在于，采用根据本文所述的生产方法获得。在一些实施例中，所述宿主细胞的培养方式包括贴壁培养或悬浮培养。在一些实施例中，所述重组腺相关病毒相比由传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统生产的重组腺相关病毒的：
(1)实心率更高；可选地，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少0.70,至少0.75,或至少0.80；
(2)基因组滴度更高；可选地，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍；
(3)Cap编码序列的相关杂质残留更低；可选地，Cap编码序列的相关杂质残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％；和/或
(4)具有复制能力的AAV(rcAAV)残留更低；可选地，rcAAV残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
本文的一方面，提供了一种宿主细胞，其特征在于，所述宿主细胞含有根据本文所述的质粒或所述质粒系统中的所有质粒。
在一些实施例中，所述宿主细胞选自HEK293细胞、HEK293T细胞、HEK293F细胞、VPC2.0细胞、Hela细胞和A549细胞。
本文的一方面，提供了一种本文所述的重组核酸在制备药物中的应用。本文的一方面，提供了一种本文所述的质粒在制备药物中的应用。本文的一方面，提供了一种本文所述的质粒系统在制备药物中的应用。本文的一方面，提供了一种本文所述的重组腺相关病毒在制备药物中的应用。本文的一方面，提供了一种本文所述的宿主细胞在制备药物中的应用。
本文的一方面，提供了一种药物制剂，其特征在于，所述药物包括本文所述的重组腺相关病毒，以及药学上可接受的载体和赋形剂。
本文的一方面，提供了一种本文所述的药物制剂在治疗疾病中的应用。
本文的一方面，提供了一种治疗疾病的方法，其包括向有需要的患者施用本文所述的重组腺相关病毒或药物制剂。
在一些实施例中，所述疾病是由于人体中的靶基因发生基因突变和/或基因缺失导致的。
本文的一方面，提供了一种，其包含本文所述的重组核酸，质粒，质粒系统，重组腺相关病毒，和/或宿主细胞。
详述
本文的一方面，针对AAV表达系统的现有缺陷，为了实现产量的提升，节约高昂的制造成本，降低生产过程中的污染杂质(例如具有复制能力的AAV(rcAAV)等目的，提供了一系列技术方案，包括但不限于：
-拆分包装质粒中的Rep和Cap基因，置于不同的质粒上；和/或
-调整Cap蛋白和/或Rep蛋白相关基因表达元件的设计。
通过对这些技术方案的优化和组合，实现上述技术效果。
本文的一方面，提供了多种新型三质粒系统，其将Rep基因或Cap基因放在辅助质粒或目的基因质粒上，另外一个质粒单独表达Rep或Cap。在一些实施例中，该系统中没有任何一个质粒同时包含Rep基因和Cap基因。
本文的一方面，提供了多种新型二质粒系统，其将Rep基因和Cap基因拆分，一个置于辅助质粒上，另一个置于目的基因质粒上。
本文的一方面，通过改造AAV质粒(例如传统的包装质粒，或新型三质粒系统或新型二质粒系统中的质粒)，修改、增加、替换P5启动子，删除掉非必要序列，插入内含子或改变内含子donor等结构，通过调控Rep和Cap基因产物的表达水平和比例，从而达到提高rAAV病毒载体产量和实心率的目的。具体改造方法如下：
(1)在Rep基因的上游插入P5启动子；优选地，所述P5启动子具有如SEQ ID NO:4所示的序列。
(2)校正位于Cap基因下游的TATA box突变的P5启动子或在Cap基因下游插入P5启动子；优选地，所述突变的P5启动子具有如SEQ ID NO:8所示的序列；更优选地，校正后的P5启动子具有如SEQ ID NO:9所示的序列；在Cap基因下游插入具有SEQ ID NO:4的序列。
(3)对内含子的donor位点进行突变；优选地，将内含子的donor位点处的GTACCA突变为GTACGT；优选地，突变前的内含子具有如SEQ ID NO:10所示的序列；更优选地，突变donor后的内含子具有如SEQ ID NO:11所示的序列。
(4)删除掉Cap基因下游包括肝脏特异性增强子-启动子的一段序列(Liver specific enhancer promotor，LSP)；优选地，删除的序列如SEQ ID NO:12所示。
(5)在Rep基因起始密码子AUG前插入hGH内含子；优选地，所述hGH内含子具有如SEQ ID NO:13所示的序列。
(6)在Rep基因起始密码子AUG前插入wtAAV2内含子；优选地，在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子；更优选地，经过改造的wtAAV2内含子具有如SEQ ID NO:14所示的序列。
(7)在Rep基因上游或在Cap基因下游插入非亲本血清型启动子；优选地，在Rep基因上游或在Cap基因下游插入wtAAV7 P5启动子；更优选地，wtAAV7 P5启动子有如SEQ ID NO:15所示的序列。
优选地，所述质粒改造包括如下修饰(改造方法序号与前述具体改造方法序号保持一致)：
(1)；或
(1)和(2)；或
(3)；或
(1)(2)和(3)；或
(1)(2)(3)和(4)；或
(1)(2)(3)(4)和(5)；或
(1)和(6)；或
(1)(2)和(6)；或
(2)(3)(4)(5)和(7)；或
(1)(3)(4)(5)和(7)。
在一些实施例中，插入的AAV启动子(例如P5启动子)来源于和可操作地连接的相应Rep基因和/或Cap基因的来源相同的亲本AAV血清型(例如，来源于AAV2的P5启动子与相应的AAV2 Rep基因和/或AAV2 Cap基因可操作地连接)。在一些实施例中，插入的AAV启动子(例如P5启动子)来源于和可操作地连接的相应Rep基因和/或Cap基因的来源不同的非亲本AAV血清型(例如，来源于AAV7的P5启动子与AAV2 Rep基因和/或AAV2 Cap基因可操作地连接)。
本文的一方面，提供了多种质粒。在一些实施例中，所述质粒包含本文所述的重组核酸。在一些实施例中，所述质粒为重组腺相关病毒无辅助病毒系统中的包装质粒(RC质粒)。在一些实施例中，所述质粒为将包装质粒的Rep或Cap基因拆分后形成的质粒。在一些实施例中，所述质粒为辅助质粒(pHelper质粒)。在一些实施例中，所述质粒为编码包含异源核酸序列的重组腺相关病毒的质粒(目的基因质粒)。
pHelper-Cap辅助质粒
本文的一方面，提供了一种能够表达Cap蛋白的辅助质粒(pHelper-Cap)，其特征在于，包含编码Cap基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。
在一些实施例中，所述辅助质粒包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。在一些实施例中，所述辅助质粒包含P5启动子，P19启动子，和P40启动子。在一些实施例中，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。在一些实施例中，所述启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述启动子之间没有间隔的核苷酸。在一些实施例中，所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述启动子和所述Cap基因之间没有间隔的核苷酸。
在一些实施例中，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。在一些实施例中，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸(除第768位核苷酸以外)100％相同。
在一些实施例中，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。在一些实施例中，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸(除第1911-1912位核苷酸以外)100％相同。
在一些实施例中，所述Cap基因编码一个功能性的Cap蛋白。在一些实施例中，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
在一些实施例中，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
在一些实施例中，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述Cap基因与所述polyA序列之间没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述辅助质粒以本文所述的方式对Cap基因的表达进行了优化设计，包括但不限于以下优化方式的一种或多种：
(1)在所述Cap基因的下游含有一个P5启动子；
(2)修正位于所述Cap基因下游的原有P5启动子中的TATA box；
(3)删除所述Cap基因下游原有的肝脏特异性增强子-启动子序列；
(4)在所述Cap基因下游含有一个非亲本血清型启动子，与所述Cap基因可操作地连接。
在一些实施例中，所述辅助质粒不能表达功能性的Rep蛋白。在一些实施例中，所述辅助质粒不包含编码Rep基因的核酸序列。
在一些实施例中，所述辅助质粒仅能表达特定的Rep蛋白，但不能表达另外的Rep蛋白。例如，在有些实施例中，所述辅助质粒仅能表达Rep78和Rep52蛋白，但不能表达Rep68和Rep40蛋白。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个基因。在一些实施例中，所述辅助质粒含有E2A基因。在一些实施例中，所述辅助质粒含有E4基因。在一些实施例中，所述辅助质粒含有VA RNA基因。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的至少两个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4基因，但不包含VA RNA基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，VA RNA基因，但不包含E4基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E4，VA RNA基因，但不包含E2A基因。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于同一个AAV质粒系统里的另一个质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于同一个AAV质粒系统里的表达Rep蛋白的另一个质粒中。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4，和VA RNA基因。
pHelper-Rep辅助质粒
本文的一方面，提供了一种能够表达Rep蛋白的辅助质粒(pHelper-Rep)，其特征在于，包含编码Rep基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。
在一些实施例中，所述Rep基因编码一个功能性的Rep蛋白。在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:2有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
在一些实施例中，所述编码Rep基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Rep基因下游。在一些实施例中，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述Rep基因与所述polyA序列之间没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的Rep基因表达Rep78、Rep68、Rep52和Rep40蛋白。
在一些实施例中，所述Rep基因表达Rep78和Rep52蛋白，而不表达Rep68和Rep40蛋白。在一些实施例中，所述Rep基因表达Rep68和Rep40蛋白，而不表达Rep78和Rep52蛋白。
在一些实施例中，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
在一些实施例中，所述辅助质粒以本文所述的方式对Rep基因的表达进行了优化设计，包括但不限于以下优化方式的一种或多种：
(1)在所述Rep基因的上游含有一个P5启动子；
(2)所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:7的第1717-1722核苷酸位点；
(3)在所述Rep基因起始密码子前含有一个hGH内含子；
(6)在所述Rep基因起始密码子前含有一个腺相关病毒基因组的内含子；和/或
(7)在所述Rep基因上游含有一个非亲本血清型启动子，与所述Rep基因可操作地连接。
在一些实施例中，所述辅助质粒不能表达功能性的Cap蛋白。在一些实施例中，所述辅助质粒不包含编码Cap基因的核酸序列。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个基因。在一些实施例中，所述辅助质粒含有E2A基因。在一些实施例中，所述辅助质粒含有E4基因。在一些实施例中，所述辅助质粒含有VA RNA基因。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的至少两个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4基因，但不包含VA RNA基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，VA RNA基因，但不包含E4基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E4，VA RNA基因，但不包含E2A基因。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于同一个AAV质粒系统里的另一个质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于同一个AAV质粒系统里的表达Rep蛋白的另一个质粒中。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4，和VA RNA基因。
pHelper质粒
在一些实施例中，本文所述辅助质粒含有用于AAV生产的辅助基因。在一些实施例中，所述辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个基因。在一些实施例中，所述辅助质粒含有E2A基因。在一些实施例中，所述辅助质粒含有E4基因。在一些实施例中，所述辅助质粒含有VA RNA基因。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的至少两个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4基因，但不包含VA RNA基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，VA RNA基因，但不包含E4基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E4，VA RNA基因，但不包含E2A基因。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于同一个AAV质粒系统里的另一个质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于同一个AAV质粒系统里的表达Rep蛋白的另一个质粒中。
在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4，和VA RNA基因。
在一些实施例中，所述辅助质粒不能表达功能性的Cap蛋白。在一些实施例中，所述辅助质粒不包含编码Cap基因的核酸序列。
在一些实施例中，所述辅助质粒不能表达功能性的Rep蛋白。在一些实施例中，所述辅助质粒不包含编码Rep基因的核酸序列。
pRep质粒
本文的一方面，提供了一种用于表达Rep蛋白的质粒(pRep)，其特征在于，包含编码Rep基因的核酸序列。在一些实施例中，该质粒不能表达功能性的Cap蛋白。在一些实施例中，所述质粒不包含编码Cap基因的核酸序列。
在一些实施例中，所述用于表达Rep蛋白的质粒不能表达功能性的用于AAV生产的辅助基因的表达产物。在一些实施例中，所述质粒不包含编码用于AAV生产的辅助基因的核酸序列。
在一些实施例中，所述用于表达Rep蛋白的质粒包含不超过一个用于AAV生产的辅助基因(例如在相应质粒系统中的辅助质粒缺失一个必要辅助基因的情况下)。在一些实施例中，所述用于表达Rep蛋白的质粒包含辅助基因E2A。在一些实施例中，所述用于表达Rep蛋白的质粒包含辅助基因E4。在一些实施例中，所述用于表达Rep蛋白的质粒包含辅助基因VA RNA。
在一些实施例中，所述用于表达Rep蛋白的质粒不包含AAV反向重复序列(ITR)。
在一些实施例中，所述Rep基因表达Rep78，Rep68，Rep52，和Rep40蛋白。在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
在一些实施例中，所述Rep基因表达Rep78和Rep52蛋白。在一些实施例中，所述Rep基因不表达Rep68或Rep40蛋白。在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:1第321-2196位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
在一些实施例中，所述Rep基因表达Rep68和Rep40蛋白。在一些实施例中，所述Rep基因不表达Rep78或Rep52蛋白。在一些实施例中，所述表达Rep68和Rep40蛋白(Rep68/40)的基因包含与SEQ ID NO:69有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。在一些实施例中，所述表达Rep68和Rep40蛋白(Rep68/40)的基因包含与SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述质粒包含一个位于所述Rep基因的上游的P5启动子，所述P5启动子与所述Rep基因可操作地连接。在一些实施例中，所述质粒包含一个位于所述Rep基因的下游的P5启动子，所述P5启动子与所述Rep基因可操作地连接。在一些实施例中，所述质粒包含一个位于所述Rep基因的上游的P5启动子和一个位于所述Rep基因的下游的P5启动子，所述两个P5启动子与所述Rep基因可操作地连接。在一些实施例中，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述启动子和所述Rep基因之间没有间隔的核苷酸。在一些实施例中，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P5启动子的序列与SEQ ID NO:1第191-310位核苷酸，有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述编码Rep基因的核酸序列包含一个hGH内含子。在一些实施例中，所述内含子位于所述Rep基因的起始密码子前。在一些实施例中，所述P5启动子与所述hGH内含子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述P5启动子与所述hGH内含子之间没有间隔的核苷酸。在一些实施例中，所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述hGH内含子与所述Rep基因之间没有间隔的核苷酸。在一些实施例中，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。在一些实施例中，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT。在一些实施例中，所述donor位点的突变包含将相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
在一些实施例中，所述编码Rep基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Rep基因下游。在一些实施例中，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述Rep基因与所述polyA序列之间没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述pRep质粒包含，自5’至3’方向，P5启动子，hGH内含子序列，Rep基因，polyA序列。在一些实施例中，所述Rep基因包含与SEQ ID NO:1第321-2300位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，所述Rep基因表达Rep78/68/52/40四种蛋白。在一些实施例中，Rep基因中的内含子donor位点处的GTACCA突变为GTACGT，具体为把相对于SEQ ID NO:1第1911-1912nt CA突变为GT。在一些实施例中，hGH内含子序列包含与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，polyA序列包含与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，所述pRep质粒包含与SEQ ID NO:46(pRep6)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。在一些实施例中，该pRep质粒不能表达功能性的Cap蛋白，或不包含编码Cap基因的核酸序列。一个此类pRep质粒的非限制性实例是pRep6质粒。
pCap质粒
本文的一方面，提供了一种用于表达Cap蛋白的质粒(pCap)，其特征在于，包含编码Cap基因的核酸序列。在一些实施例中，该质粒不能表达功能性的Rep蛋白。在一些实施例中，所述质粒不包含编码Rep基因的核酸序列。
在一些实施例中，所述用于表达Cap蛋白的质粒不能表达功能性的用于AAV生产的辅助基因的表达产物。在一些实施例中，所述质粒不包含编码用于AAV生产的辅助基因的核酸序列。
在一些实施例中，所述用于表达Cap蛋白的质粒包含不超过一个用于AAV生产的辅助基因(例如在相应质粒系统中的辅助质粒缺失一个必要辅助基因的情况下)。在一些实施例中，所述用于表达Rep蛋白的质粒包含辅助基因E2A。在一些实施例中，所述用于表达Cap蛋白的质粒包含辅助基因E4。在一些实施例中，所述用于表达Cap蛋白的质粒包含辅助基因VA RNA。
在一些实施例中，所述用于表达Cap蛋白的质粒不包含AAV反向重复序列(ITR)。
pGOI质粒
本文的一方面，提供了编码包含异源核酸序列的重组腺相关病毒的质粒(pGOI质粒)。在一些实施例中，所述异源核酸序列编码一个异源目的基因(gene of interest；GOI)。在一些实施例中，所述质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。在一些实施例中，所述质粒负责生产包装于重组腺相关病毒中的编码目的基因的多核苷酸。
在一些实施例中，所述异源目的基因编码一个ND4蛋白。在一些实施例中，所述ND4蛋白包含与SEQ ID NO:112有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:104有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:105有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。
在一些实施例中，所述异源目的基因编码一个ND6蛋白。在一些实施例中，所述ND6蛋白包含与SEQ ID NO:113有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:106有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:107有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。
在一些实施例中，所述异源目的基因编码一个ND1蛋白。在一些实施例中，所述ND1蛋白包含与SEQ ID NO:114有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:108有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:109有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。
在一些实施例中，所述异源目的基因编码一个Opa1蛋白。在一些实施例中，所述Opa1蛋白包含与SEQ ID NO:115有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:110有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。在一些实施例中，所述异源目的基因包含与SEQ ID NO:111有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的核苷酸序列。
在一些实施例中，所述异源目的基因编码一个NRF2蛋白。在一些实施例中，所述NRF2蛋白包含与SEQ ID NO:116有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
pGOI-Cap质粒
本文的一方面，提供了包含Cap基因和编码包含异源核酸序列的重组腺相关病毒的质粒(pGOI-Cap)。在一些实施例中，所述异源核酸序列编码一个异源目的基因(gene of interest；GOI)。在一些实施例中，所述质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。在一些实施例中，所述质粒负责生产包装于重组腺相关病毒中的编码目的基因的多核苷酸。
在一些实施例中，所述pGOI-Cap质粒包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。在一些实施例中，所述pGOI-Cap质粒包含P5启动子，P19启动子，和P40启动子。在一些实施例中，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。在一些实施例中，所述启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述启动子之间没有间隔的核苷酸。在一些实施例中，所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述启动子和所述Cap基因之间没有间隔的核苷酸。
在一些实施例中，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。在一些实施例中，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸(除第768位核苷酸以外)100％相同。
在一些实施例中，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。在一些实施例中，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸(除第1911-1912位核苷酸以外)100％相同。
在一些实施例中，所述Cap基因编码一个功能性的Cap蛋白。在一些实施例中，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
在一些实施例中，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
在一些实施例中，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述Cap基因与所述polyA序列之间没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述pGOI-Cap质粒以本文所述的方式对Cap基因的表达进行了优化设计，包括但不限于以下优化方式的一种或多种：
(1)在所述Cap基因的下游含有一个P5启动子；
(2)修正位于所述Cap基因下游的原有P5启动子中的TATA box；
(3)删除所述Cap基因下游原有的肝脏特异性增强子-启动子序列；
(4)在所述Cap基因下游含有一个非亲本血清型启动子，与所述Cap基因可操作地连接。
在一些实施例中，所述pGOI-Cap质粒不能表达功能性的Rep蛋白。在一些实施例中，所述pGOI-Cap质粒不包含编码Rep基因的核酸序列。
在一些实施例中，所述pGOI-Cap质粒仅能表达特定的Rep蛋白，但不能表达另外的Rep蛋白。例如，在有些实施例中，所述pGOI-Cap质粒仅能表达Rep78和Rep52蛋白，但不能表达Rep68和Rep40蛋白。
三质粒AAV表达系统
在一些实施例中，本文所述的三质粒系统包含(i)辅助质粒,(ii)包装质粒，其包含Rep基因和Cap基因；和(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。(例如图1所示的一个传统三质粒系统)。在一些实施例中，本文对这个质粒系统的Rep基因和/或Cap基因以本文所述的方式进行优化设计改造，获得产量提升，成本节约，降低污染杂质等技术效果。
本发明的另一方面，通过拆分包装质粒pRC2的Rep和Cap基因，将其中一个基因置于另一个质粒上(例如辅助质粒、或用来编码目的基因的质粒)，可以产生多种质粒系统形式。同时，该设计可与本文所述的Rep基因和/或Cap基因的优化设计相结合，获得产量提升，成本节约，降低污染杂质等技术效果。
四质粒系统：Rep、Cap都成为一个单独的质粒，和辅助质粒、载体质粒共同组成四质粒系统，但增加质粒会加大投入成本，且有研究发现在转染过程中只有5％的初始质粒被转染到细胞中，而最终进入细胞核的质粒大约占总输入质粒的0.6％，那么四种质粒同时进入同一细胞核的发生概率就更低，这对于生产rAAV来说是很不利的。
新型三质粒系统：本文的一方面，提供了多种新型三质粒系统，其将Rep基因或Cap基因放在辅助质粒或载体质粒上，另外一个质粒单独表达Rep或Cap。在一些实施例中，该系统中没有任何一个质粒同时包含Rep基因和Cap基因。
本文的一方面，提供了一种三质粒系统，其特征在于，包含:(i)辅助质粒(pHelper质粒)，(ii)一个包含Rep基因或Cap基因的质粒，和(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒，其中，一个质粒包含本文所述的编码Rep基因的重组核酸，另一个质粒包含本文所述的编码Cap基因的重组核酸。在一些实施例中，(i)所述的辅助质粒包含所述Rep基因，且(ii)中所述质粒包含所述Cap基因。在一些实施例中，(ii)中所述质粒包含所述Rep基因，且(iii)中所述重组腺相关病毒的质粒包含所述Cap基因。在一些实施例中，(i)所述的辅助质粒包含所述Cap基因，且(ii)中所述质粒包含所述Rep基因。
在一些实施例中，本文所述的三质粒系统中，辅助质粒包含Rep，这样新辅助质粒可以成为公用质粒，进行其他项目时，不会增加生产成本，也不用改变目的基因所在载体质粒，更为安全。
在一些实施例中，本文所述的三质粒系统中，把Rep或Cap基因放在辅助质粒或载体质粒上，另外一个质粒单独表达Rep或Cap。在一些实施例中，本文所述的新型质粒系统将Cap拆分到辅助质粒上，Rep单独一个质粒。
在一些实施例中，该系统中没有任何一个质粒同时包含Rep基因和Cap基因。
在一些实施例中，本文所述新型三质粒系统，由pHelper-Rep质粒、pCap质粒、pGOI质粒组成。在一些实施例中，新型三质粒系统具有如图2A所示的结构。
在一些实施例中，本文所述新型三质粒系统，由pHelper质粒、pRep质粒、pGOI-Cap质粒组成。在一些实施例中，新型三质粒系统具有如图2B所示的结构。
在一些实施例中，本文所述新型三质粒系统，由pHelper-Cap质粒、pRep质粒、pGOI质粒组成。在一些实施例中，新型三质粒系统具有如图24所示的结构。
在一些实施例中，本文所述的质粒系统里，表达AAV需要的所有必须辅助基因中的一个或多个包含于表达Rep的质粒上，而剩余的必须辅助基因包含于表达Cap的质粒上。
在一些实施例中，本文所述的三质粒系统具有如下一种或多种修饰：
(1)在Rep基因的上游插入P5启动子；优选地，所述P5启动子具有如SEQ ID NO:4所示的序列。
(2)校正位于Cap基因下游的TATA box突变的P5启动子或在Cap基因下游插入P5启动子；优选地，所述突变的P5启动子具有如SEQ ID NO:8所示的序列；更优选地，校正后的P5启动子具有如SEQ ID NO:9所示的序列；更优选地，在Cap基因下游插入具有SEQ ID NO:4的序列。
(3)对内含子的donor位点进行突变；优选地，将内含子的donor位点处的GTACCA突变为GTACGT；优选地，突变前的内含子具有如SEQ ID NO:10所示的序列；更优选地，突变donor后的内含子具有如SEQ ID NO:11所示的序列。
(4)删除掉Cap基因下游包括肝脏特异性增强子-启动子的一段序列(Liver specific enhancer promotor，LSP)；优选地，删除的序列如SEQ ID NO:12所示。
(5)在Rep基因起始密码子AUG前插入hGH内含子；优选地，所述hGH内含子具有如SEQ ID NO:13所示的序列。
(6)在Rep基因起始密码子AUG前插入wtAAV2内含子；优选地，在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子；更优选地，经过改造的wtAAV2内含子具有如SEQ ID NO:14所示的序列。
(7)在Rep基因上游或在Cap基因下游插入非亲本血清型启动子；优选地，在Rep基因上游或在Cap基因下游插入wtAAV7 P5启动子；更优选地，wtAAV7 P5启动子有如SEQ ID NO:15所示的序列。
更优选地，在某些具体实施方式中，所述新型三质粒系统包括如下修饰(改造方法序号与前述具体改造方法序号保持一致)：
(1)；或
(1)和(2)；或
(3)；或
(1)(2)和(3)；或
(1)(2)(3)和(4)；或
(1)(2)(3)(4)和(5)；或
(1)和(6)；或
(1)(2)和(6)；或
(2)(3)(4)(5)和(7)；或
(1)(3)(4)(5)和(7)。
包含pHelper-Rep质粒，pCap质粒，pGOI质粒的三质粒系统
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含本文所述的：(i)一个包含Rep基因的辅助质粒(pHelper-Rep辅助质粒)；(ii)一个包含Cap基因的质粒(pCap质粒)，和(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒(pGOI质粒)。
在一些实施例中，所述三质粒系统的(i)辅助质粒不能表达功能性的Cap蛋白，和/或该质粒不包含Cap基因。
在一些实施例中，所述的三质粒系统的(ii)包含Cap基因的质粒不能表达功能性的Rep蛋白，和/或该质粒不包含Rep基因。
在一些实施例中，所述包含Rep基因的辅助质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白。在一些实施例中，三质粒系统的(ii)pCap质粒可表达Rep78和/或Rep52蛋白，但不表达Rep68和Rep40蛋白。在一些实施例中，三质粒系统的(iii)包含异源核酸序列的质粒可表达Rep78和/或Rep52蛋白,但不表达Rep68和Rep40蛋白。
在一些实施例中，所述包含Rep基因的辅助质粒表达Rep78和Rep52蛋白但不表达Rep68或Rep40蛋白。在一些实施例中，三质粒系统的(ii)pCap质粒可表达Rep68和/或Rep40蛋白，但不表达Rep78和Rep52蛋白。在一些实施例中，三质粒系统的(iii)包含异源核酸序列的质粒可表达Rep68和/或Rep40蛋白,但不表达Rep78和Rep52蛋白。
在一些实施例中，三质粒系统的(i)辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个基因。在一些实施例中，所述辅助质粒含有E2A基因。在一些实施例中，所述辅助质粒含有E4基因。在一些实施例中，所述辅助质粒含有VA RNA基因。
在一些实施例中，三质粒系统的(i)辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的至少两个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4基因，但不包含VA RNA基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，VA RNA基因，但不包含E4基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E4，VA RNA基因，但不包含E2A基因。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于质粒系统里的另一个质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于包含Cap基因的质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于包含异源核酸序列的pGOI质粒中。
在一些实施例中，所述的三质粒系统的(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
在一些实施例中，所述的三质粒系统，其特征在于，所述AAV的血清型是AAV2。
如下提供该三质粒系统的其他编号的实施方案：
实施方案A1.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Rep基因的重组核酸；
(ii)一个包含Cap基因的质粒；可选地，该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
实施方案A2.根据实施方案A1所述的质粒系统，其特征在于，所述辅助质粒不能表达功能性的Cap蛋白，和/或不包含编码Cap基因的核酸序列。
实施方案A3.根据实施方案A1-A2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78，Rep68，Rep52，和Rep40蛋白。
实施方案A4.根据实施方案A3所述的质粒系统，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案A5.根据实施方案A1-A2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案A6.根据实施方案A1-A2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep68和Rep40蛋白；可选地，所述Rep基因不表达Rep78或Rep52蛋白。
实施方案A7.根据实施方案A6所述的质粒系统，其特征在于，所述Rep基因包含与SEQ ID NO:69或SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案A8.根据实施方案A1-A7任一项所述的质粒系统，其特征在于，所述质粒系统不包含共表达Rep52蛋白和Rep40蛋白的基因。
实施方案A9.根据实施方案A1-A8任一项所述的质粒系统，其特征在于，所述Rep基因的上游含有一个hGH内含子。
实施方案A10.根据实施方案A9所述的质粒系统，其特征在于，所述hGH内含子的上游含有一个P5启动子。
实施方案A11.根据实施方案A9-A10任一项所述的质粒系统，其特征在于，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案A12.根据实施方案A10-A11任一项所述的质粒系统，其特征在于，所述P5启动子与所述hGH内含子之间，和/或所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案A13.根据实施方案A1-A12任一项所述的质粒系统，其特征在于，所述Rep基因的下游含有polyA序列；可选地，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案A14.根据实施方案A12-A13任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案A15.根据实施方案A1-A14任一项所述的质粒系统，其特征在于，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
实施方案A16.根据实施方案A15所述的质粒系统，其特征在于，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案A17.根据实施方案A1-A16任一项所述的质粒系统，其特征在于，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。
实施方案A18.根据实施方案A17所述的质粒系统，其特征在于，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT，或相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
实施方案A19.根据实施方案A1-A18任一项所述的质粒系统，其特征在于，包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。
实施方案A20.根据实施方案A19所述的质粒系统，其特征在于，包含P5启动子，P19启动子，和P40启动子；优选地，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。
实施方案A21.根据实施方案A19-A20任一项所述的质粒系统，其特征在于，所述启动子之间，和/或所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案A22.根据实施方案A19-A21任一项所述的质粒系统，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案A23.根据实施方案A19-A22任一项所述的质粒系统，其特征在于，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。
实施方案A24.根据实施方案A19-A23任一项所述的质粒系统，其特征在于，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。
实施方案A25.根据实施方案A1-A24任一项所述的质粒系统，其特征在于，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案A26.根据实施方案A1-A24任一项所述的质粒系统，其特征在于，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
实施方案A27.根据实施方案A26所述的质粒系统，其特征在于，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案A28.根据实施方案A1-A27任一项所述的质粒系统，其特征在于，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
实施方案A29.根据实施方案A28所述的质粒系统，其特征在于，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案A30.根据实施方案A28-A29任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案A31.根据实施方案A1-A30任一项所述的质粒系统，其特征在于，在所述Cap基因下游还包含一个P5启动子，与所述Cap基因可操作地连接；可选地，所述P5启动子位于Cap基因下游的polyA序列的下游；可选地，所述P5启动子与所述Cap基因之间，或所述P5启动子与所述polyA序列之间，间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案A32.根据实施方案A1-A31任一项所述的质粒系统，其特征在于，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案A33.根据实施方案A1-A32任一项所述的质粒系统，其特征在于，所述异源核酸序列编码一个异源基因。
实施方案A34.根据实施方案A1-A33任一项所述的质粒系统，其特征在于，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9；更优选地，所述AAV的血清型是AAV2。
包含pHelper质粒，pRep质粒，pGOI-Cap质粒的三质粒系统
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含本文所述的：(i)一个辅助质粒；(ii)一个包含Rep基因的质粒(pRep质粒)，和(iii)一个编码包含异源核酸序列的重组腺相关病毒并包含Cap基因的质粒(pGOI-Cap质粒)。
在一些实施例中，所述三质粒系统的(iii)pGOI-Cap质粒不能表达功能性的Rep蛋白，和/或该质粒不包含Rep。
在一些实施例中，所述的三质粒系统的(ii)包含Rep基因的质粒不能表达功能性的Cap蛋白，和/或该质粒不包含Cap基因。
在一些实施例中，所述包含Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白。在一些实施例中，三质粒系统的(i)辅助质粒可表达Rep78和/或Rep52蛋白，但不表达Rep68和Rep40蛋白。在一些实施例中，三质粒系统的(iii)包含异源核酸序列和Cap基因的质粒可表达Rep78和/或Rep52蛋白,但不表达Rep68和Rep40蛋白。
在一些实施例中，所述包含Rep基因的质粒表达Rep78和Rep52蛋白但不表达Rep68或Rep40蛋白。在一些实施例中，三质粒系统的(i)辅助质粒可表达Rep68和/或Rep40蛋白，但不表达Rep78和Rep52蛋白。在一些实施例中，三质粒系统的(iii)包含异源核酸序列和Cap基因的质粒可表达Rep68和/或Rep40蛋白,但不表达Rep78和Rep52蛋白。
在一些实施例中，三质粒系统的(i)辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个基因。在一些实施例中，所述辅助质粒含有E2A基因。在一些实施例中，所述辅助质粒含有E4基因。在一些实施例中，所述辅助质粒含有VA RNA基因。
在一些实施例中，三质粒系统的(i)辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的至少两个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4基因，但不包含VA RNA基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，VA RNA基因，但不包含E4基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E4，VA RNA基因，但不包含E2A基因。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于质粒系统里的另一个质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于包含Rep基因的质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于编码包含异源核酸序列的重组腺相关病毒并包含Cap基因的pGOI质粒中。
在一些实施例中，所述的三质粒系统的(iii)编码包含异源核酸序列的重组腺相关病毒并包含Cap基因的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
如下提供该三质粒系统的其他编号的实施方案：
实施方案B1.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)；
(ii)一个包含Rep基因的质粒；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒，且包含Cap基因的质粒。
实施方案B2.根据实施方案B1所述的质粒系统，其特征在于，所述质粒(ii)不能表达功能性的Cap蛋白，和/或不包含编码Cap基因的核酸序列。
实施方案B3.根据实施方案B1-B2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78，Rep68，Rep52，和Rep40蛋白。
实施方案B4.根据实施方案B3所述的质粒系统，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案B5.根据实施方案B1-B2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案B6.根据实施方案B1-B2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep68和Rep40蛋白；可选地，所述Rep基因不表达Rep78或Rep52蛋白。
实施方案B7.根据实施方案B6所述的质粒系统，其特征在于，所述Rep基因包含与SEQ ID NO:69或SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案B8.根据实施方案B1-B7任一项所述的质粒系统，其特征在于，所述质粒系统不包含共表达Rep52蛋白和Rep40蛋白的基因。
实施方案B9.根据实施方案B1-B8任一项所述的质粒系统，其特征在于，所述Rep基因的上游含有一个hGH内含子。
实施方案B10.根据实施方案B9所述的质粒系统，其特征在于，所述hGH内含子的上游含有一个P5启动子。
实施方案B11.根据实施方案B9-B10任一项所述的质粒系统，其特征在于，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案B12.根据实施方案B10-B11任一项所述的质粒系统，其特征在于，所述P5启动子与所述hGH内含子之间，和/或所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案B13.根据实施方案B1-B12任一项所述的质粒系统，其特征在于，所述Rep基因的下游含有polyA序列；可选地，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案B14.根据实施方案B12-B13任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案B15.根据实施方案B1-B14任一项所述的质粒系统，其特征在于，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
实施方案B16.根据实施方案B15所述的质粒系统，其特征在于，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案B17.根据实施方案B1-B16任一项所述的质粒系统，其特征在于，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。
实施方案B18.根据实施方案B17所述的质粒系统，其特征在于，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT，或相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
实施方案B19.根据实施方案B1-B18任一项所述的质粒系统，其特征在于，包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。
实施方案B20.根据实施方案B19所述的质粒系统，其特征在于，包含P5启动子，P19启动子，和P40启动子；优选地，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。
实施方案B21.根据实施方案B19-B20任一项所述的质粒系统，其特征在于，所述启动子之间，和/或所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案B22.根据实施方案B19-B21任一项所述的质粒系统，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案B23.根据实施方案B19-B22任一项所述的质粒系统，其特征在于，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。
实施方案B24.根据实施方案B19-B23任一项所述的质粒系统，其特征在于，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。
实施方案B25.根据实施方案B1-B24任一项所述的质粒系统，其特征在于，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案B26.根据实施方案B1-B24任一项所述的质粒系统，其特征在于，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
实施方案B27.根据实施方案B26所述的质粒系统，其特征在于，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案B28.根据实施方案B1-B27任一项所述的质粒系统，其特征在于，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
实施方案B29.根据实施方案B28所述的质粒系统，其特征在于，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案B30.根据实施方案B28-B29任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案B31.根据实施方案B1-B30任一项所述的质粒系统，其特征在于，在所述Cap基因下游还包含一个P5启动子，与所述Cap基因可操作地连接；可选地，所述P5启动子位于Cap基因下游的polyA序列的下游；可选地，所述P5启动子与所述Cap基因之间，或所述P5启动子与所述polyA序列之间，间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案B32.根据实施方案B1-B31任一项所述的质粒系统，其特征在于，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案B33.根据实施方案B1-B32任一项所述的质粒系统，其特征在于，所述异源核酸序列编码一个异源基因。
实施方案B34.根据实施方案B1-B33任一项所述的质粒系统，其特征在于，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9；更优选地，所述AAV的血清型是AAV2。
包含pHelper-Cap质粒，pRep质粒，pGOI质粒的三质粒系统
本文的一方面，提供了一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含本文所述的：(i)一个包含Cap基因的辅助质粒(pHelper-Cap辅助质粒)；(ii)一个包含Rep基因的质粒(pRep质粒)，和(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒(pGOI质粒)。
在一些实施例中，把Cap基因置于辅助质粒pHelper上，另外一个质粒单独表达Rep质粒，这两个质粒可以成为公用质粒，进行其他项目时，不会增加质粒生产成本，也不用在目的基因所在载体质粒上加入Rep或Cap序列，减小了包装入这两个元件的风险。还可分别对这两种质粒进行修饰，分别调控Rep和Cap基因的表达，使产量成倍增加。在一些实施例中，Cap基因和pHelper质粒三个辅助基因的大量表达对产量的提升均十分有利，放在一个质粒上易于对投料的控制。在一些实施例中，该三质粒系统利于重组腺相关病毒(rAAV)的包装。
在一些实施例中，所述三质粒系统的(i)辅助质粒不能表达功能性的Rep蛋白，和/或该辅助质粒不包含Rep基因。
在一些实施例中，所述的三质粒系统的(ii)包含Rep基因的质粒不能表达功能性的Cap蛋白，和/或该质粒不包含Cap基因。
在一些实施例中，所述包含Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白。在一些实施例中，三质粒系统的(i)辅助质粒可表达Rep78和/或Rep52蛋白，但不表达Rep68和Rep40蛋白。在一些实施例中，三质粒系统的(iii)包含异源核酸序列的质粒可表达Rep78和/或Rep52蛋白,但不表达Rep68和Rep40蛋白。
在一些实施例中，所述包含Rep基因的质粒表达Rep78和Rep52蛋白但不表达Rep68或Rep40蛋白。在一些实施例中，三质粒系统的(i)辅助质粒可表达Rep68和/或Rep40蛋白，但不表达Rep78和Rep52蛋白。在一些实施例中，三质粒系统的(iii)包含异源核酸序列的质粒可表达Rep68和/或Rep40蛋白,但不表达Rep78和Rep52蛋白。
在一些实施例中，三质粒系统的(i)辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个基因。在一些实施例中，所述辅助质粒含有E2A基因。在一些实施例中，所述辅助质粒含有E4基因。在一些实施例中，所述辅助质粒含有VA RNA基因。
在一些实施例中，三质粒系统的(i)辅助质粒含有的用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的至少两个基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，E4基因，但不包含VA RNA基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E2A，VA RNA基因，但不包含E4基因。在一些实施例中，所述辅助质粒含有的用于AAV生产的辅助基因包含E4，VA RNA基因，但不包含E2A基因。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于质粒系统里的另一个质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于包含Rep基因的质粒中。在一些实施例中，该辅助质粒中不包含的那个辅助基因存在于编码包含异源核酸序列的重组腺相关病毒的pGOI质粒中。
在一些实施例中，表达Rep的质粒上也可包含一个或多个辅助基因。在一些实施例中，所述的质粒系统里，表达AAV需要的所有必须辅助基因中的一个或多个包含于表达Rep的质粒上，而剩余的必须辅助基因包含于表达Cap的质粒上。
在一些实施例中，所述的三质粒系统的(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
在一些实施例中，所述的三质粒系统包含(i)一个包含Cap基因的辅助质粒(pHelper-Cap辅助质粒)；(ii)一个包含Rep基因的质粒(pRep质粒)，和(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒(pGOI质粒)。其中：
-所述包含Cap基因的辅助质粒包含，从5’到3’方向，P5启动子(例如wtAAV2第191-320nt)，P19启动子(例如wtAAV2第741-890nt，其中第768G突变为T)，P40启动子(例如wtAAV2第1701-2202nt，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，Cap基因，polyA序列(例如wtAAV2第4411-4460nt)；
-所述包含Rep基因的质粒(ii)包含P5启动子和Rep基因(相对于wtAAV2第191-2300nt的序列，表达Rep78/68/52/40四种蛋白，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，再在Rep基因起始密码子AUG前(相对于wtAAV2第321nt处)插入hGH内含子序列(例如SEQ ID NO:13)，在相对于wtAAV2第2300nt后加上polyA序列(wtAAV2第4411-4460nt)。
如下提供该三质粒系统的其他编号的实施方案：
实施方案C1.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Cap基因的重组核酸；
(ii)一个包含Rep基因的质粒；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
实施方案C2.根据实施方案C1所述的质粒系统，其特征在于，所述(i)辅助质粒不能表达功能性的Rep蛋白，和/或所述(ii)编码Rep基因的质粒不能表达功能性的Cap蛋白。
实施方案C3.根据实施方案C1-C2任一项所述的质粒系统，其特征在于，所述Cap基因的上游含有三个启动子，从5’至3’方向分别为：P5启动子，P19启动子，和P40启动子，均与所述Cap基因可操作地连接，所述Cap基因的下游含有polyA序列。
实施方案C4.根据实施方案C1-C3任一项所述的质粒系统，其特征在于，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9；更优选地，所述AAV的血清型是AAV2。
实施方案C5.根据实施方案C1-C4任一项所述的质粒系统，其特征在于，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。
实施方案C6.根据实施方案C5所述的质粒系统，其特征在于，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT，或相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
实施方案C7.根据实施方案C1-C6任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78、Rep68、Rep52和Rep40蛋白。
实施方案C8.根据实施方案C7所述的质粒系统，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案C9.根据实施方案C1-C8任一项所述的质粒系统，其特征在于，其特征在于，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案C10.根据实施方案C1-C9所述的质粒系统，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2196位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案C11.根据实施方案C1-C10任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep68和Rep40蛋白；可选地，所述Rep基因不表达Rep78或Rep52蛋白。
实施方案C12.根据实施方案C11所述的质粒系统，其特征在于，所述Rep基因包含与SEQ ID NO:69或SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案C13.根据实施方案C1-C12任一项所述的质粒系统，其特征在于，所述质粒系统不包含共表达Rep52蛋白和Rep40蛋白的基因。
实施方案C14.根据实施方案C1-C13任一项所述的质粒系统，其特征在于，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(i)辅助质粒包含一个表达Rep78和/或Rep52蛋白的基因；可选地，该基因不表达Rep68或Rep40。
实施方案C15.根据实施方案C1-C14任一项所述的质粒系统，其特征在于，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒包含一个表达Rep78和/或Rep52蛋白的基因；可选地，该基因不表达Rep68或Rep40。
实施方案C16.根据实施方案C1-C15任一项所述的质粒系统，其特征在于，所述(i)辅助质粒包含E4基因和VA RNA基因；所述(ii)编码Rep基因的质粒包含E2A基因。
实施方案C17.根据实施方案C1-C16任一项所述的质粒系统，其特征在于，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
实施方案C18.根据实施方案C17所述的质粒系统，其特征在于，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案C19.根据实施方案C17-C18任一项所述的质粒系统，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸，或与SEQ ID NO:1第191-310位核苷酸，有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案C20.根据实施方案C1-C19任一项所述的质粒系统，其特征在于，所述Rep基因的上游含有一个hGH内含子。
实施方案C21.根据实施方案C20所述的质粒系统，其特征在于，所述hGH内含子的上游含有一个P5启动子。
实施方案C22.根据实施方案C20-C21任一项所述的质粒系统，其特征在于，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案C23.根据实施方案C21-C22任一项所述的质粒系统，其特征在于，所述P5启动子与所述hGH内含子之间，和/或所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案C24.根据实施方案C1-C23任一项所述的质粒系统，其特征在于，所述Rep基因的下游含有polyA序列。
实施方案C25.根据实施方案C24所述的质粒系统，其特征在于，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案C26.根据实施方案C24-C25任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案C27.根据实施方案C1-C26任一项所述的质粒系统，其特征在于，所述异源核酸序列编码一个异源基因。
实施方案C28.根据实施方案C1-C27任一项所述的质粒系统，其特征在于，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案C29.根据实施方案C1-C28任一项所述的质粒系统，其特征在于，包含P5启动子，P19启动子，和P40启动子；优选地，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。
实施方案C30.根据实施方案C29所述的质粒系统，其特征在于，所述启动子之间，和/或所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案C31.根据实施方案C29-C30任一项所述的质粒系统，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案C32.根据实施方案C29-C31任一项所述的质粒系统，其特征在于，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。
实施方案C33.根据实施方案C29-C32任一项所述的质粒系统，其特征在于，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。
实施方案C34.根据实施方案C1-C33任一项所述的质粒系统，其特征在于，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案C35.根据实施方案C1-C34任一项所述的质粒系统，其特征在于，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
实施方案C36.根据实施方案C35所述的质粒系统，其特征在于，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案C37.根据实施方案C1-C36任一项所述的质粒系统，其特征在于，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
实施方案C38.根据实施方案C37所述的质粒系统，其特征在于，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案C39.根据实施方案C37-C38任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案C40.根据实施方案C1-C39任一项所述的质粒系统，其特征在于，在所述Cap基因下游还包含一个P5启动子，与所述Cap基因可操作地连接；可选地，所述P5启动子位于Cap基因下游的polyA序列的下游；可选地，所述P5启动子与所述Cap基因之间，或所述P5启动子与所述polyA序列之间，间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
二质粒AAV表达系统
本文的一方面，提供了一种二质粒系统，其特征在于，包含本文的含所述Rep基因的第一质粒和所述Cap基因的第二质粒，且所述Rep和所述Cap基因分别位于不同质粒上。在一些实施例中，本文所述的二质粒系统分别拆分Rep和Cap基因到辅助质粒和载体质粒上。在一些实施例中，该系统将Rep放在辅助质粒(即形成pHelper-Rep质粒)上，将Cap放在载体质粒上。在一些实施例中，这种质粒系统形式更为简单和灵活，pHelper-Rep可以作为一个共用质粒，不仅改进了生产制造的经济性、也不用改变目的基因所在载体质粒，增强了载体制剂的安全性和质量属性。
本文的一方面，提供了一种二质粒系统，其特征在于，包含一个含有AAV的Rep基因的第一质粒和一个含有AAV的Cap基因的第二质粒，所述Rep基因和所述Cap基因分别位于不同质粒上。在一些实施例中，所述的二质粒系统特征在于，含有所述Rep基因的第一质粒为辅助质粒(pHelper质粒)，且含有所述Cap基因的第二质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，含有所述Cap基因的第二质粒为辅助质粒(pHelper质粒)，且含有所述Rep基因的第一质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒。
在一些实施例中，所述二质粒系统，Rep和Cap基因分别位于两个不同质粒上。在一些实施例中，所述任一质粒仅包含Rep基因和Cap基因中的一个，而不同时包含Rep基因和Cap基因。
在一些实施例中，所述二质粒系统由pHelper-Rep质粒或pGOI-Cap质粒组成。其中，pHelper-Rep质粒为含有Rep基因的辅助质粒，pGOI-Cap质粒为含有Cap基因和编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。
在一些实施例中，所述二质粒系统由pGOI-Rep和pHelper-Cap组成。其中，pHelper-Cap质粒为含有Cap基因的辅助质粒，pGOI-Rep质粒为含有Rep基因和编码包含异源核酸序列的重组腺相关病毒的质粒。在一些实施例中，所述异源核酸序列编码一个异源基因。
在一些实施例中，所述二质粒系统具有如下一种或多种修饰：
(1)在Rep基因的上游插入P5启动子；优选地，所述P5启动子具有如SEQ ID NO:4所示的序列。
(2)校正位于Cap基因下游的TATA box突变的P5启动子或在Cap基因下游插入P5启动子；优选地，所述突变的P5启动子具有如SEQ ID NO:8所示的序列；更优选地，校正后的P5启动子具有如SEQ ID NO:9所示的序列；在Cap基因下游插入具有SEQ ID NO:4的序列。
(3)对内含子的donor位点进行突变；优选地，将内含子的donor位点处的GTACCA突变为GTACGT；优选地，突变前的内含子具有如SEQ ID NO:10所示的序列；更优选地，突变donor后的内含子具有如SEQ ID NO:11所示的序列。
(4)删除掉Cap基因下游包括肝脏特异性增强子-启动子的一段序列(Liver specific enhancer promotor，LSP)；优选地，删除的序列如SEQ ID NO:12所示。
(5)在Rep基因起始密码子AUG前插入hGH内含子；优选地，所述hGH内含子具有如SEQ ID NO:13所示的序列。
(6)在Rep基因起始密码子AUG前插入wtAAV2内含子；优选地，在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子；更优选地，经过改造的wtAAV2内含子具有如SEQ ID NO:14所示的序列。
(7)在Rep基因上游或在Cap基因下游插入非亲本血清型启动子；优选地，在Rep基因上游或在Cap基因下游插入wtAAV7 P5启动子；更优选地，wtAAV7 P5启动子有如SEQ ID NO:15所示的序列。
更优选地，在某些具体实施方式中，所述二质粒系统包括如下修饰(改造方法序号与前述具体改造方法序号保持一致)：
(1)；或
(1)和(2)；或
(3)；或
(1)(2)和(3)；或
(1)(2)(3)和(4)；或
(1)(2)(3)(4)和(5)；或
(1)和(6)；或
(1)(2)和(6)；或
(2)(3)(4)(5)和(7)；或
(1)(3)(4)(5)和(7)。
如下提供该二质粒系统的其他编号的实施方案：
实施方案D1.一种二质粒系统，其特征在于，包含一个含有AAV的Rep基因的第一质粒和一个含有AAV的Cap基因的第二质粒，所述Rep基因和所述Cap基因分别位于不同质粒上，含有所述Rep基因的第一质粒为辅助质粒(pHelper质粒)，且含有所述Cap基因的第二质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒。
实施方案D2，根据实施方案D1所述的质粒系统，其特征在于，所述辅助质粒包含选自E2A，E4，和VA RNA的一个或多个辅助基因。
实施方案D3.根据实施方案D1-D2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78，Rep68，Rep52，和Rep40蛋白。
实施方案D4.根据实施方案D3所述的质粒系统，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案D5.根据实施方案D1-D2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案D6.根据实施方案D1-D2任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep68和Rep40蛋白；可选地，所述Rep基因不表达Rep78或Rep52蛋白。
实施方案D7.根据实施方案D6所述的质粒系统，其特征在于，所述Rep基因包含与SEQ ID NO:69或SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案D8.根据实施方案D1-D7任一项所述的质粒系统，其特征在于，所述质粒系统不包含共表达Rep52蛋白和Rep40蛋白的基因。
实施方案D9.根据实施方案D1-D8任一项所述的质粒系统，其特征在于，所述Rep基因的上游含有一个hGH内含子。
实施方案D10.根据实施方案D9所述的质粒系统，其特征在于，所述hGH内含子的上游含有一个P5启动子。
实施方案D11.根据实施方案D9-D10任一项所述的质粒系统，其特征在于，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案D12.根据实施方案D10-D11任一项所述的质粒系统，其特征在于，所述P5启动子与所述hGH内含子之间，和/或所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案D13.根据实施方案D1-D12任一项所述的质粒系统，其特征在于，所述Rep基因的下游含有polyA序列；可选地，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案D14.根据实施方案D12-D13任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案D15.根据实施方案D1-D14任一项所述的质粒系统，其特征在于，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
实施方案D16.根据实施方案D15所述的质粒系统，其特征在于，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案D17.根据实施方案D1-D16任一项所述的质粒系统，其特征在于，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。
实施方案D18.根据实施方案D17所述的质粒系统，其特征在于，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT，或相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
实施方案D19.根据实施方案D1-D18任一项所述的质粒系统，其特征在于，包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。
实施方案D20.根据实施方案D19所述的质粒系统，其特征在于，包含P5启动子，P19启动子，和P40启动子；优选地，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。
实施方案D21.根据实施方案D19-D20任一项所述的质粒系统，其特征在于，所述启动子之间，和/或所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案D22.根据实施方案D19-D21任一项所述的质粒系统，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案D23.根据实施方案D19-D22任一项所述的质粒系统，其特征在于，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。
实施方案D24.根据实施方案D19-D23任一项所述的质粒系统，其特征在于，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。
实施方案D25.根据实施方案D1-D24任一项所述的质粒系统，其特征在于，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案D26.根据实施方案D1-D24任一项所述的质粒系统，其特征在于，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
实施方案D27.根据实施方案D26所述的质粒系统，其特征在于，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案D28.根据实施方案D1-D27任一项所述的质粒系统，其特征在于，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
实施方案D29.根据实施方案D28所述的质粒系统，其特征在于，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案D30.根据实施方案D28-D29任一项所述的质粒系统，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案D31.根据实施方案D1-D30任一项所述的质粒系统，其特征在于，在所述Cap基因下游还包含一个P5启动子，与所述Cap基因可操作地连接；可选地，所述P5启动子位于Cap基因下游的polyA序列的下游；可选地，所述P5启动子与所述Cap基因之间，或所述P5启动子与所述polyA序列之间，间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案D32.根据实施方案D1-D31任一项所述的质粒系统，其特征在于，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案D33.根据实施方案D1-D32任一项所述的质粒系统，其特征在于，所述异源核酸序列编码一个异源基因。
实施方案D34.根据实施方案D1-D33任一项所述的质粒系统，其特征在于，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9；更优选地，所述AAV的血清型是AAV2。
包含Rep基因和/或Cap基因的重组核酸或质粒的优化设计
本文的一方面，提供了一种重组核酸或包含该重组核酸的质粒，其包含腺相关病毒(AAV)的Rep基因和/或Cap基因，且有如下的一种或多种特征：
(1)在所述Rep基因的上游和/或所述Cap基因的下游含有一个P5启动子；
(2)位于所述Cap基因下游的原有P5启动子中的TATA box突变被修正，或在所述Cap基因下游插入一个P5启动子，与所述Cap基因可操作地连接；
(3)所述腺相关病毒的病毒基因组原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:7的第1717-1722核苷酸位点；
(4)所述Cap基因下游原有的肝脏特异性增强子-启动子序列被删除；
(5)在所述Rep基因起始密码子前含有一个hGH内含子；
(6)在所述Rep基因起始密码子前含有一个腺相关病毒基因组的内含子；和/或
(7)在所述Rep基因上游或在所述Cap基因下游含有一个非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
在一些实施例中，所述重组核酸或质粒包括所述特征：
(1)；或
(1)和(2)；或
(3)；或
(1)(2)和(3)；或
(1)(2)(3)和(4)；或
(1)(2)(3)(4)和(5)；或
(1)和(6)；或
(1)(2)和(6)；或
(2)(3)(4)(5)和(7)；或
(1)(3)(4)(5)和(7)。
在一些实施例中，所述重组核酸或质粒同时包含Rep基因和Cap基因。
在一些实施例中，所述重组核酸或质粒仅包含Rep基因和Cap基因中的一个(例如在一些实施例中的pHelper-Cap辅助质粒以及pRep质粒)。
在一些实施例中，所述重组核酸或质粒包含特征(1)，且位于所述Rep基因上游的所述P5启动子的插入位置紧接在相当于SEQ ID NO:7的腺相关病毒基因组的120bp的核苷酸位点后，与所述Rep基因可操作地连接。在一些实施例中，所述P5启动子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
在一些实施例中，所述P5启动子的序列与SEQ ID NO:4有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述重组核酸或质粒包含特征(2)，且所述原有P5启动子的TATA box突变的修正包含将相当于SEQ ID NO:7第4469位核苷酸的T突变为A，和/或将相当于SEQ ID NO:7第4477-4483位核苷酸的GGGGGGG突变为TATTTAA。在一些实施例中，所述原有P5启动子修正后的序列与SEQ ID NO:9有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述重组核酸或质粒在所述Cap基因下游插入一个P5启动子，与所述Cap基因可操作地连接，且所述P5启动子的序列与SEQ ID NO:4有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的重组核酸或质粒包含特征(3)，且所述donor位点的突变包含将相当于SEQ ID NO:7第1721-1722位核苷酸的CA突变为GT，或相当于SEQ ID NO:7第1717-1722位核苷酸的donor位点序列突变为GTACGT。在一些实施例中，突变后的所述内含子序列与SEQ ID NO:11有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述的重组核酸或质粒包含特征(4)，且所述肝脏特异性增强子-启动子序列被删除。
在一些实施例中，肝脏特异性增强子-启动子序列与SEQ ID NO:12或SEQ ID NO:35有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述重组核酸或质粒包含特征(5)，且所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述hGH内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。在一些实施例中，所述hGH内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
在一些实施例中，所述重组核酸或质粒的所述hGH内含子与所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述hGH内含子与所述Rep基因起始密码子之间没有间隔的核苷酸。
在一些实施例中，所述重组核酸或质粒包含特征(6)，且所述腺相关病毒基因组的内含子的序列与SEQ ID NO:14有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述腺相关病毒基因组的内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。在一些实施例中，所述腺相关病毒基因组的内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
在一些实施例中，所述腺相关病毒基因组的内含子和所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
在一些实施例中，所述的重组核酸或质粒包含特征(7)，且所述非亲本血清型启动子的序列与SEQ ID NO:15有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述非亲本血清型启动子位于所述Cap基因的下游。在一些实施例中，所述非亲本血清型启动子位于相当于SEQ ID NO:7第4270-4271位核苷酸之间的位置。
在一些实施例中，所述重组核酸或质粒不含有相当于SEQ ID NO:7第4368-4532位核苷酸的序列，或不含有相当于SEQ ID NO:7第4373-4532位核苷酸的序列。
在一些实施例中，所述非亲本血清型启动子位于所述Rep基因上游，且所述所述非亲本血清型启动子位于所述hGH内含子的上游。
在一些实施例中，所述重组核酸或质粒不含有相当于SEQ ID NO:7第121-130位核苷酸的序列。
本文的一方面，提供了一种质粒系统的改造方法，其特征在于，具有根据下一种或多种修饰方法：
(1)在Rep基因的上游插入P5启动子，与所述Rep基因可操作地连接；
(2)校正位于Cap基因下游的TATA box突变的P5启动子或在Cap基因下游插入P5启动子，与所述Cap基因可操作地连接；
(3)对内含子的donor位点进行突变；
(4)删除掉Cap基因下游包括肝脏特异性增强子-启动子的一段序列；
(5)在Rep基因起始密码子AUG前插入hGH内含子；
(6)在Rep基因起始密码子AUG前插入wtAAV2内含子；
(7)在Rep基因上游或在Cap基因下游插入非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
在一些实施例中，所述质粒系统的改造方法的特征在于：
(1)中所述在Rep基因的上游插入P5启动子具有如SEQ ID NO:4所示的序列；
(2)中所述突变的P5启动子具有如SEQ ID NO:8所示的序列，在Cap基因下游插入的所述P5启动子具有SEQ ID NO:4的序列；
(3)对内含子的donor位点进行突变是将内含子的donor位点处的GTACCA突变为GTACGT，优选地，突变前的内含子具有如SEQ ID NO:10所示的序列；突变donor后的内含子具有如SEQ ID NO:11所示的序列；
(4)中删除的序列如SEQ ID NO:12所示；
(5)中插入的hGH内含子具有如SEQ ID NO:13所示的序列；
(6)中在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子，优选地，经过改造的wtAAV2内含子具有如SEQ ID NO:14所示的序列；
(7)中非亲本血清型启动子为wtAAV7 P5启动子，其有如SEQ ID NO:15所示的序列。
重组腺相关病毒的生产方法和宿主细胞
本文的一方面，提供了一种重组腺相关病毒，其特征在于，采用根据本文所述的重组腺相关病毒的生产方法获得。
本文的一方面，提供了一种重组腺相关病毒的生产方法，其特征在于，将本文所述的质粒或本文所述的质粒系统中的所有质粒转染至宿主细胞，培养，分离得到所述重组腺相关病毒。在一些实施例中，所述(i)辅助质粒与所述(ii)编码Rep基因的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1；优选地，至少3:1。在一些实施例中，所述(i)辅助质粒与所述(ii)编码Rep基因的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1；优选地，约3:1。在一些实施例中，所述(ii)编码Rep基因的质粒与所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1；优选地，至少2.5:1。在一些实施例中，所述(ii)编码Rep基因的质粒与所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1；优选地，约2.5:1。
在一些实施例中，例如利用本文所述的新型二质粒系统时，所述(i)含目的基因和Cap基因的质粒与所述(ii)含Rep基因的辅助质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1；优选地，至少3:1。在一些实施例中，所述所述(i)含目的基因和Cap基因的质粒与所述(ii)含Rep基因的辅助质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1；优选地，约3:1。
在一些实施例中，所述宿主细胞的培养方式包括贴壁培养或悬浮培养。
本文的一方面，提供了一种宿主细胞，其特征在于，所述宿主细胞含有采用根据本文所述的一个质粒系统中的所有质粒。本文的一方面，提供了一种宿主细胞，其特征在于，所述宿主细胞含有采用本文所述的质粒。
在一些实施例中，所述宿主细胞选自HEK293细胞、HEK293T细胞、HEK293F细胞、VPC2.0细胞、Hela细胞和A549细胞。
在一些实施例中，本文所述的生产方法相比基于传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统的生产方法产生的重组腺相关病毒的实心率更高。在一些实施例中，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少0.70,至少0.75,或至少0.80。
在一些实施例中，本文所述的生产方法相比基于传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统的生产方法产生的重组腺相关病毒的基因组滴度更高。在一些实施例中，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍。
在一些实施例中，本文所述的生产方法相比基于传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统的生产方法产生的重组腺相关病毒的Cap编码序列的相关杂质残留更低。在一些实施例中，Cap编码序列的相关杂质残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
在一些实施例中，本文所述的生产方法相比基于传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统的生产方法产生的重组腺相关病毒的具有复制能力的AAV(rcAAV)残留更低。在一些实施例中，rcAAV残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
腺相关病毒
腺相关病毒AAV基因组长约4.7kb，是线性单链DNA。野生型AAV基因组包括两个开放阅读框(ORF)，即Rep和Cap，其两端是反向重复序列(ITR)，长度约100-200bp(例如145bp)。ITR由一个约125bp回文序列组成，通过互补碱基配对形成T型发夹结构。剩下的约20个碱基没有配对，称为D序列。ITR一般还具有一个Rep蛋白结合位点(RBS)和一个末端拆分位点(TRS)，两者都是复制的起始点。野生型AAV2(wtAAV2,SEQ ID NO:1)是一种复制缺陷型细小病毒，需辅助病毒帮助其在体内复制扩增。
本文的一方面，提供了一种重组腺相关病毒。在一些实施例中，所述重组腺相关病毒是采用根据本文所述的生产方法获得。在一些实施例中，所述重组腺相关病毒是基于本文所述的质粒系统生产的。在一些实施例中，所述重组腺相关病毒的生产中使用到了本文所述的质粒。在一些实施例中，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ。在一些实施例中，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12或AAV-DJ。在一些实施例中，所述AAV血清型是AAV2、AAV5、AAV8、或AAV9。在一些实施例中，所述AAV的血清型是AAV2。
在一些实施例中，本文提供的重组腺相关病毒相比由传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统生产的重组腺相关病毒的实心率更高。在一些实施例中，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少0.70,至少0.75,或至少0.80。
在一些实施例中，本文提供的重组腺相关病毒相比由传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统生产的重组腺相关病毒的基因组滴度更高。在一些实施例中，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍。
在一些实施例中，本文提供的重组腺相关病毒相比由传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统生产的重组腺相关病毒的Cap编码序列的相关杂质残留更低。在一些实施例中，Cap编码序列的相关杂质残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
在一些实施例中，本文提供的重组腺相关病毒相比由传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统生产的重组腺相关病毒的具有复制能力的AAV(rcAAV)残留更低。在一些实施例中，rcAAV残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
Rep基因
Rep基因编码AAV病毒DNA复制和包装所需的四种蛋白质，按照分子量大小命名，分别是Rep78、Rep68、Rep52和Rep40。Rep78/68表达受P5启动子控制，P19启动子则控制较小的Rep蛋白Rep52/40的表达，Rep68和Rep40分别作为剪接异构体产生，且Rep52和Rep40的C末端分别与Rep78和Rep68相同。Rep78和Rep68促进AAV DNA拯救和随后的复制，参与AAV基因表达的正负调控。Rep52和Rep40参与单链DNA和预形成的衣壳相互作用，促进病毒DNA被封装进衣壳。在一些实施例中，Rep基因包含与SEQ ID NO:2有至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，本文所述的Rep基因表达Rep78、Rep68、Rep52和Rep40蛋白。
在一些实施例中，Rep68蛋白包含与SEQ ID NO:89有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
在一些实施例中，Rep78蛋白包含与SEQ ID NO:90有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
在一些实施例中，Rep40蛋白包含与SEQ ID NO:91有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
在一些实施例中，Rep52蛋白包含与SEQ ID NO:92有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。在一些实施例中，所述Rep基因的序列包含与SEQ ID NO:2有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
在一些实施例中，所述Rep基因表达Rep78和Rep52蛋白，而不表达Rep68和Rep40蛋白。在一些实施例中，所述表达Rep78和Rep52蛋白(Rep78/52)的基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述Rep基因表达Rep68和Rep40蛋白，而不表达Rep78和Rep52蛋白。在一些实施例中，所述表达Rep68和Rep40蛋白(Rep68/40)的基因包含与SEQ ID NO:69有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。在一些实施例中，所述表达Rep68和Rep40蛋白(Rep68/40)的基因包含与SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，所述Rep基因表达Rep52和Rep40蛋白，而不表达Rep78和Rep68蛋白。在一些实施例中，所述表达Rep52和Rep40蛋白(Rep52/40)的基因包含与SEQ ID NO:70有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。。
在一些实施例中，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
在一些实施例中，所述Rep基因的上游含有一个P5启动子。在一些实施例中，该P5启动子的插入位置紧接在相当于SEQ ID NO:7的腺相关病毒基因组的120bp的核苷酸位点后，与所述Rep基因可操作地连接。在一些实施例中，该P5启动子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述P5启动子与所述Rep基因之间没有间隔的核苷酸。在一些实施例中，所述P5启动子的序列与SEQ ID NO:4有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。在一些实施例中，所述donor位点的突变包含将相当于SEQ ID NO:7第1721-1722位核苷酸的CA突变为GT。在一些实施例中，所述donor位点的突变包含将相当于SEQ ID NO:7第1717-1722位核苷酸的donor位点序列突变为GTACGT。在一些实施例中，突变后的内含子序列与SEQ ID NO:11有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Rep基因的上游含有一个hGH内含子。在一些实施例中，所述内含子位于所述Rep基因的起始密码子前。在一些实施例中，所述hGH内含子的上游含有一个P5启动子。在一些实施例中，所述P5启动子与所述hGH内含子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述P5启动子与所述hGH内含子之间没有间隔的核苷酸。在一些实施例中，所述hGH内含子与所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述hGH内含子与所述Rep基因起始密码子之间没有间隔的核苷酸。在一些实施例中，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，在所述Rep基因起始密码子前含有一个腺相关病毒基因组的内含子。在一些实施例中，所述腺相关病毒基因组的内含子的序列与SEQ ID NO:14有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述腺相关病毒基因组的内含子位于P5启动子的下游，并位于所述Rep基因的上游。在一些实施例中，所述腺相关病毒基因组的内含子和P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述腺相关病毒基因组的内含子和P5启动子之间没有间隔的核苷酸。在一些实施例中，所述腺相关病毒基因组的内含子和所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述腺相关病毒基因组的内含子和所述Rep基因起始密码子之间没有间隔的核苷酸。
在一些实施例中，所述Rep基因上游含有一个非亲本血清型启动子，与所述Rep基因可操作地连接。在一些实施例中，所述非亲本血清型启动子的序列与SEQ ID NO:15有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述Rep基因的上游含有一个hGH内含子，且所述非亲本血清型启动子位于所述hGH内含子的上游。
在一些实施例中，所述Rep基因的下游含有polyA序列。在一些实施例中，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述Rep基因与所述polyA序列之间没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
Cap基因
在一些实施例中，Cap基因与选自P5启动子，P19启动子，和P40启动子的至少一个启动子可操作地连接。在一些实施例中，Cap基因与P5启动子，P19启动子，和P40启动子可操作地连接。在一些实施例中，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。在一些实施例中，所述启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述启动子之间没有间隔的核苷酸。在一些实施例中，所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述启动子和所述Cap基因之间没有间隔的核苷酸。
在一些实施例中，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。在一些实施例中，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸(除第768位核苷酸以外)100％相同。
在一些实施例中，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。在一些实施例中，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸(除第1911-1912位核苷酸以外)100％相同。
在一些实施例中，本文所述的Cap基因的下游含有一个P5启动子。在一些实施例中，该P5启动子与所述Cap基因可操作地连接。在一些实施例中，所述P5启动子与所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述P5启动子与所述Cap基因之间没有间隔的核苷酸。在一些实施例中，所述P5启动子与所述Cap基因下游的polyA之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。在一些实施例中，所述P5启动子与所述Cap基因下游的polyA之间没有间隔的核苷酸。在一些实施例中，所述P5启动子的序列与SEQ ID NO:4有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，位于所述Cap基因下游的原有P5启动子中的TATA box突变被修正。在一些实施例中，所述原有P5启动子的TATA box突变的修正包含将相当于SEQ ID NO:7第4469位核苷酸的T突变为A。在一些实施例中，所述原有P5启动子的TATA box突变的修正包含将相当于SEQ ID NO:7第4477-4483位核苷酸的GGGGGGG突变为TATTTAA。在一些实施例中，所述原有P5启动子修正后的序列与SEQ ID NO:9有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Cap基因下游原有的肝脏特异性增强子-启动子序列被删除。在一些实施例中，所述肝脏特异性增强子-启动子序列与SEQ ID NO:12有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述肝脏特异性增强子-启动子序列与SEQ ID NO:35有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Cap基因下游含有一个非亲本血清型启动子，与所述Cap基因可操作地连接。在一些实施例中，所述非亲本血清型启动子的序列与SEQ ID NO:15有至少90％，至少95％，至少98％，至少99％，或100％的相同性。在一些实施例中，所述非亲本血清型启动子位于相当于SEQ ID NO:7第4270-4271位核苷酸之间的位置。
在一些实施例中，所述Cap基因下游包含一个polyA序列。在一些实施例中，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸。在一些实施例中，所述Cap基因与所述polyA序列之间没有间隔的核苷酸。在一些实施例中，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
在一些实施例中，所述Cap基因编码一个功能性的Cap蛋白。
Cap基因通过来自不同起始密码子的可变剪接和翻译编码三种衣壳蛋白VP1、VP2、VP3和组装激活蛋白AAP，受P40启动子调控。这三种结构蛋白在AAP的协助下组装，大约60个VP1,VP2和VP3的拷贝被组装成一个直径约26nm的二十面体粒子，摩尔比为1:1:10。不同的衣壳蛋白决定了AAV的血清型，这些血清型组成了不同的组织倾向性。在一些实施例中，Cap基因编码的衣壳蛋白的血清型为AAV1(SEQ ID NO:48)，AAV2(SEQ ID NO:49)，AAV3(SEQ ID NO:50)，AAV4(SEQ ID NO:51)，AAV5(SEQ ID NO:52)，AAV6(SEQ ID NO:53)，AAV7(SEQ ID NO:54)，AAV8(SEQ ID NO:55)，AAV9(SEQ ID NO:56)，AAV10(SEQ ID NO:57)，AAVrh.10，或基于其中一个血清型的衍生衣壳蛋白。在一些实施例中，Cap基因编码的衣壳蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:48(AAV1)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:58有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:49(AAV2)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:59有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:50(AAV3)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:60有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:51(AAV4)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:61有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:52(AAV5)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:62有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:53(AAV6)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:63有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:54(AAV7)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:64有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:55(AAV8)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:65有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:56(AAV9)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:66有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，Cap基因编码的衣壳蛋白包含与SEQ ID NO:57(AAV10)有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。在一些实施例中，该Cap基因包含与SEQ ID NO:67有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
衣壳蛋白包括通过天然存在的AAV血清型的两个或更多个衣壳编码序列之间的重组产生的那些。这可例如通过标志补救方法来进行，其中一种血清型的非感染衣壳序列与不同血清型的衣壳序列共转染，并采用定向选择来选择具有所需特性的衣壳序列。可通过细胞内的同源重组来改变不同血清型的衣壳序列，以产生新型衣壳蛋白。
衣壳蛋白还包括通过对衣壳蛋白序列进行工程改造以在两种或更多种衣壳蛋白之间(例如在不同血清型的两种或更多种衣壳蛋白之间)转移特定的衣壳蛋白结构域、表面环或特定的氨基酸残基而产生的那些。
也可通过DNA改组或通过易错PCR产生改组或嵌合衣壳蛋白。可通过如下方式来产生杂合AAV衣壳基因，即随机片段化相关AAV基因的序列(例如编码多种不同血清型的衣壳蛋白的序列)，然后在自引发聚合酶反应中后续重新装配片段，这也可能在序列同源性的区域中导致交叉。可筛选以这种方式通过改组若干血清型的衣壳基因而产生的杂合AAV基因的文库，以鉴定具有所需功能的病毒克隆。类似地，易错PCR可用于使AAV衣壳基因随机突变，以产生变体的多样文库，然后可针对所需特性对其进行选择。
也可以对衣壳基因的序列进行遗传修饰，以相对于天然野生型序列引入特定的缺失、取代或插入。特别地，可通过在衣壳编码序列的开放阅读框内或者在衣壳编码序列的N和/或C末端插入无关蛋白或肽的序列来修饰衣壳基因。
无关蛋白或肽可有利地为充当特定细胞类型的配体的蛋白或肽，从而改善与靶细胞的结合，或提高载体靶向特定细胞群的特异性。一个实例可包括使用RGD肽来阻止视网膜色素上皮细胞中的摄取，从而增强周围视网膜组织的转导。无关蛋白也可以是有助于作为生产过程的一部分的病毒颗粒的纯化的蛋白质，即表位或亲和标签。通常选择插入的位点，以便不干扰病毒颗粒的其它功能，例如病毒颗粒的内化、转运。技术人员可基于他们的公知常识来鉴定合适的插入位点。本文另外涵盖以与天然AAV基因组不同的顺序和构型提供AAV基因组的序列。本文还涵盖用来自另一种病毒的序列或用由来自一种以上病毒的序列组成的嵌合基因来替换一个或多个AAV序列或基因。这类嵌合基因可由来自不同病毒种类的两种或更多种相关病毒蛋白的序列组成。
辅助基因
辅助病毒(例如，腺病毒或疱疹病毒)可提供允许AAV病毒在被感染的细胞中生产的辅助基因。在腺病毒的情况下，基因E1A、E1B、E2A、E4和VA可提供辅助功能。
在一些实施例中，本文所述的辅助基因包含选自E1A、E1B、E2A、E4和VA RNA基因的一个或多个基因。在一些实施例中，本文所述的辅助基因包含选自E2A、E4和VA RNA基因的一个或多个基因。
在一些实施例中，本文所述的质粒或质粒系统包含E2A、E4和VA RNA基因。
在一些实施例中，本文所述的质粒系统的所有辅助基因都位于辅助质粒上。在一些实施例中，本文所述的质粒系统的至少一个辅助基因不在辅助质粒上。
在一些实施例中，本文所述的质粒或质粒系统的辅助基因来源于腺病毒。
在一些实施例中，本文所述的质粒或质粒系统的辅助基因来源于腺病毒2型(Ad2)。在一些实施例中，本文所述的质粒或质粒系统包含的E2A基因包含与SEQ ID NO:93有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。在一些实施例中，本文所述的质粒或质粒系统包含的E4基因包含与SEQ ID NO:94有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。在一些实施例中，本文所述的质粒或质粒系统包含的VA RNA基因包含与SEQ ID NO:95有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
在一些实施例中，本文所述的质粒系统的辅助基因来源于腺病毒2型(Ad2)。
在一些实施例中，本文所述的质粒系统的辅助基因来源于疱疹病毒。
在一些实施例中，所述辅助基因与相应病毒中的基因相同。在一些实施例中，所述辅助基因是相应病毒中的基因的衍生序列。
与现有技术相比，本文所述质粒和质粒系统的有益效果包括但不限于：
本文提供了几种改造传统三质粒系统中pRC2质粒的方法，经过改造的质粒组成的新型质粒系统不仅可以提高病毒基因组滴度和实心率，还降低了rcAAV的产生。既可以用于转染贴壁培养HEK293细胞，也可以用于可放大的悬浮系统生产rAAV；不只适用于rAAV2载体的生产，还可以用于其他血清型rAAV的生产；满足目前临床试验和市场供应对rAAV载体材料的需求，快速简便应用于生产系统中，极大地降低了生产成本，可以减少很多的人力和物力的投入，提高生产效率。
制剂、施用途径和有效剂量
本文的又一方面涉及药物制剂、施用途径和有效剂量。这类药物制剂可用于治疗疾病(如眼部疾病)。
本文的一方面，提供了一种药物制剂，其特征在于，所述药物制剂包括根据本文所述的重组腺相关病毒，以及药学上可接受的载体和赋形剂。
本文的药物制剂的施用，包括适于口服(包括经颊和舌下)、经直肠、经鼻、局部、透皮贴剂、经肺、经阴道、栓剂或肠胃外(包括眼内、玻璃体内、肌内、动脉内、鞘内、皮内、腹膜内、皮下和静脉内)施用或呈适于通过雾化、吸入或吹入施用的形式的药物制剂。关于药物递送系统的一般信息可见于Ansel等人，Pharmaceutical Dosage Forms and Drug Delivery Systems(Lippencott Williams&Wilkins,Baltimore Md.(1999))。
在各种实施方案中，药物制剂包括载剂和赋形剂(包括但不限于缓冲剂、碳水化合物、甘露糖醇、多肽、氨基酸、抗氧化剂、抑菌剂、螯合剂、悬浮剂、增稠剂和/或防腐剂)、水、油(包括石油、动物、植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等)、盐水溶液、葡萄糖水溶液和甘油溶液、调味剂、着色剂、防粘剂及其它可接受的添加剂、佐剂或粘结剂、用以接近生理条件的其它药学上可接受的辅助物质如pH缓冲剂、张力调节剂、乳化剂、润湿剂等。赋形剂的实例包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、甘油、丙烯、二醇、水、乙醇等。在一些实施方案中，药物制剂基本上不含防腐剂。在其它实施方案中，药物制剂可含有至少一种防腐剂。关于药物剂型的一般方法见于Ansel等人，Pharmaceutical Dosage Forms and Drug Delivery Systems(Lippencott,Williams,&Wilkins,Baltimore Md.(1999))。可以认识到，虽然可以使用本领域普通技术人员已知的任意合适的载体来施用本公开的组合物或制剂，但载体的类型可根据施用方式而变化。
也可采用熟知的技术将药物包封在脂质体内。也可以使用生物可降解的微球作为本公开的药物制剂的载剂。合适的生物可降解微球公开在例如美国专利第4,897,268、5,075,109、5,928,647、5,811,128、5,820,883、5,853,763、5,814,344和5,942,252号中。
可以脂质体或微球(或微颗粒)形式施用药物。用于向受试者施用的脂质体和微球的制备方法是本领域技术人员熟知的。美国专利第4,789,734号(其内容特此以引用的方式并入)描述了将生物物质包封在脂质体中的方法。基本上，将物质溶解在水溶液中，添加适当的磷脂和脂质，如果需要的话连同表面活性剂一起添加，并且必要时对物质进行渗析或声处理。G.Gregoriadis，第14章，“Liposomes,”Drug Carriers in Biology and Medicine，第2页、增补87-341页(Academic Press,1979)提供了已知方法的综述。
由聚合物或多肽形成的微球是本领域技术人员熟知的，并且可以被设计成通过胃肠道直接进入血流。或者，可以掺入化合物，并植入微球或微球复合物，以在数天至数月的时间段内缓慢释放。参见例如美国专利第4,906,474、4,925,673和3,625,214号以及Jein,TIPS 19:155-157(1998)，上述内容特此以引用的方式并入。
可调节药物的浓度，缓冲溶液的pH，并调节等渗性以与眼内注射或玻璃体内注射相容。
可将本公开的药物配制为在合适媒介物中的无菌溶液或悬浮液。药物制剂可通过常规的熟知灭菌技术进行灭菌，或者可进行无菌过滤。所得到的水溶液可原样包装使用或冻干，冻干制剂在施用之前与无菌溶液合并。Remington“The Science and Practice of Pharmacy”(第20版，Lippincott Williams&Wilkins,Baltimore MD)中描述了合适的制剂和另外的载剂。
剂或其药学上可接受的盐可单独或与一种或多种其它剂组合提供，或者以一种或多种其它形式提供。例如，制剂可包含特定比例的一种或多种剂，这取决于每种剂的相对效力和预期的适应症。例如，在用于靶向两种不同宿主靶标的组合物或制剂中，并且在效力相似的情况下，可以使用约1:1比率的剂。可将两种形式一起配制在同一剂量单位中，例如在一种霜剂、栓剂、片剂、胶囊、气溶胶喷雾剂或要在饮料中溶解的粉末包中；或者可将每种形式配制在单独的单位中，例如两种霜剂、两种栓剂、两种片剂、两种胶囊、片剂和用于溶解片剂的液体、两种气溶胶喷雾剂或粉末包和用于溶解粉末的液体等。
术语“药学上可接受的盐”意指那些保留了在本公开中使用的剂的生物有效性和特性并且不是生物学上或其它方面上不可取的盐。
典型的盐为无机离子比如钠、钾、钙、镁离子等的盐。这类盐包括与无机或有机酸如盐酸、氢溴酸、磷酸、硝酸、硫酸、甲磺酸、对甲苯磺酸、乙酸、富马酸、琥珀酸、乳酸、扁桃酸、苹果酸、柠檬酸、酒石酸或马来酸形成的盐。此外，如果剂含有羧基或其它酸性基团，则可以用无机或有机碱将其转化成药学上可接受的加成盐。合适的碱的实例包括氢氧化钠、氢氧化钾、氨、环己胺、二环己胺、乙醇胺、二乙醇胺、三乙醇胺等。
药学上可接受的酯或酰胺是指保留了在本公开中使用的剂的生物有效性和特性并且不是生物学上或其它方面上不可取的那些酯和酰胺。典型的酯包括乙酯、甲酯、异丁酯、乙二醇酯等。典型的酰胺包括未取代的酰胺、烷基酰胺、二烷基酰胺等。
在一些实施方案中，药物可以与一种或多种例如如上所述的其它药物、形式和/或剂组合施用。可将含有一种或多种其它活性剂的药物制剂配制成包含一定的摩尔比。例如，第一活性剂与其它活性剂的摩尔比可以是约99:1至约1:99。在实施方案的一些子集中，第一活性剂:其它活性剂的摩尔比范围选自约80:20至约20:80；约75:25至约25:75、约70:30至约30:70、约66:33至约33:66、约60:40至约40:60；约50:50；和约90:10至约10:90。第一活性剂:其它活性剂的摩尔比可以为约1:9，并且在一些实施方案中可以为约1:1。可将两种剂、形式和/或药物一起配制在同一剂量单位中，例如在一种霜剂、栓剂、片剂、胶囊或要在饮料中溶解的粉末包中；或者可将每种剂、形式和/或药物配制在单独的单位中，例如两种霜剂、栓剂、片剂、两种胶囊、片剂和用于溶解片剂的液体、气溶胶喷雾剂、粉末包和用于溶解粉末的液体等。
如果必要或需要的话，剂和/或剂组合可再与其它剂一起施用。可与本公开的剂和/或剂组合共同施用的剂的选择可至少部分地取决于所治疗的疾患。
剂(或其药学上可接受的盐、酯或酰胺)可单独或以药物制剂的形式施用，其中活性剂与一种或多种药学上可接受的载剂相掺合或混合。如本文所用的药物制剂可以是制备用于向受试者施用的任何组合物或制剂。可以使用一种或多种生理学上可接受的载剂以常规方式配制根据本公开使用的药物制剂，所述载剂包括赋形剂、稀释剂和/或助剂，其例如有助于将活性剂加工成可施用的制剂。适当的制剂可至少部分地取决于所选择的施用途径。可采用多种施用途径或模式向受试者递送用于本公开的剂或其药学上可接受的盐、酯或酰胺，包括口服、经颊、局部、经直肠、经皮、经粘膜、皮下、静脉内、眼内、玻璃体内和肌内施加以及通过吸入。
在一些实施方案中，由于例如存在大的亲脂性部分，可使用油或非水溶剂将剂带入溶液中。或者，可以使用乳液、悬浮液或其它制剂，例如脂质体制剂。关于脂质体制剂，可以采用任何已知的方法制备用于治疗疾患的脂质体。参见例如以引用的方式并入本文的Bangham等人，J.Mol.Biol.23:238-252(1965)和Szoka等人，Proc.Natl Acad.Sci.USA 75:4194-4198(1978)。也可以使配体与脂质体附接，以将这些组合物导向特定的作用位点。也可将本公开的剂并入到食品例如奶油干酪、黄油、色拉酱或冰淇淋中，以在某些受试者群体中促进增溶、施用和/或顺应性。
本公开的化合物可以被配制成用于肠胃外施用(例如，通过注射，例如眼内注射或玻璃体内注射)，并且可以单位剂型存在于安瓿、预填充注射器、小体积输注液中，或者存在于添加有防腐剂的多剂量容器中。组合物或制剂可采取诸如在油性或水性媒介物中的悬浮液、溶液或乳液的形式，例如在含水聚乙二醇中的溶液。
对于可注射制剂，媒介物可选自本领域中已知合适的那些，包括水溶液或油悬浮液或乳液，以及芝麻油、玉米油、棉籽油或花生油，以及酏剂、甘露糖醇、葡萄糖，或无菌水溶液及类似的药物媒介物。制剂还可以包含生物相容性可生物降解的聚合物组合物，如聚(乳酸-共-乙醇酸)。可将这些物质制成微球或纳米球，装载药物并进一步包衣或衍生化，以提供优异的持续释放性能。适用于眼周或眼内注射的媒介物包括例如治疗剂在注射级水、脂质体和适用于亲脂性物质的媒介物中的悬浮液。用于眼周或眼内注射的其它媒介物是本领域中熟知的。
在一些实施方案中，根据常规程序将组合物配制为适于向人静脉内施用的药物制剂。通常，用于静脉内施用的组合物是在无菌等渗水性缓冲剂中的溶液。在必要的情况下，组合物或制剂还可包括增溶剂和局部麻醉剂如利多卡因，以减轻注射部位的疼痛。通常，各成分以单位剂型单独或混合在一起提供，例如作为在密闭密封容器中的干燥的冻干粉末或无水浓缩物，所述容器如指示活性剂的量的安瓿或小药囊。在组合物或制剂要通过输注施用的情况下，可将其用含有无菌药物级水或盐水的输注瓶进行分配。在通过注射施用组合物或制剂的情况下，可提供注射用无菌水或盐水的安瓿，以便可以在施用之前将各成分混合。
当通过注射施用时，可将活性化合物配制在水溶液中，特别是在生理相容的缓冲液中，如汉克斯溶液、林格氏溶液或生理盐水缓冲液。溶液可含有配制剂，如悬浮剂、稳定剂和/或分散剂。或者，活性化合物可呈粉末形式，在使用前用合适的媒介物例如无菌无热原水进行复原。在一些实施方案中，药物制剂不包含添加以增强由肽刺激的免疫反应的佐剂或任何其它物质。在一些实施方案中，药物制剂包含抑制对肽的免疫反应的物质。配制方法是本领域中已知的，例如公开于Remington’s Pharmaceutical Sciences，最新版，Mack Publishing Co.,Easton P中。
在一些实施方案中，可用包含本公开的剂或的剂组合的眼用溶液、悬浮液、油膏或插入物来有效地治疗眼部病症。可通过将活性成分溶解在无菌水溶液如生理盐水、缓冲溶液等中或者通过在使用前将要溶解的粉末组合物合并来制备眼药水。可选择本领域中已知的其它媒介物，包括但不限于：平衡盐溶液、盐水溶液、水溶性聚醚如聚乙二醇、聚乙烯类化合物如聚乙烯醇和聚维酮、纤维素衍生物如甲基纤维素和羟丙基甲基纤维素、石油衍生物如矿物油和白凡士林、动物脂肪如羊毛脂、丙烯酸的聚合物如羧基聚亚甲基凝胶、植物脂肪如花生油和多糖如葡聚糖以及糖胺聚糖如透明质酸钠。如果需要的话，可以添加眼药水中常用的添加剂。这类添加剂包括等渗剂(例如，氯化钠等)、缓冲剂(例如，硼酸、磷酸一氢钠、磷酸二氢钠等)、防腐剂(例如，苯扎氯铵、苄索氯铵、氯丁醇等)、增稠剂(例如，糖类，如乳糖、甘露糖醇、麦芽糖等；例如，透明质酸或其盐，如透明质酸钠、透明质酸钾等；例如，粘多糖，如硫酸软骨素等；例如，聚丙烯酸钠、羧乙烯基聚合物、交联聚丙烯酸酯、聚乙烯醇、聚乙烯吡咯烷酮、甲基纤维素、羟丙基甲基纤维素、羟乙基纤维素、羧甲基纤维素、羟丙基纤维素或本领域技术人员已知的其它剂)。
可通过组合物或制剂中的表面活性剂或其它适当的共溶剂来提高本文组合物或制剂的组分的溶解度。这类共溶剂包括聚山梨醇酯20、60和80；Pluronic F68、F-84和P-103；环糊精或本领域技术人员已知的其它试剂。这类共溶剂的使用水平可以是约0.01重量％至2重量％。
可按多剂量形式包装本公开的组合物或制剂。防腐剂可优选防止使用期间的微生物污染。合适的防腐剂包括：苯扎氯铵、硫柳汞、氯丁醇、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯乙醇、依地酸二钠、山梨酸、Onamer M或本领域技术人员已知的其它剂。在一些实施例中，这类防腐剂的使用水平可以是0.004％至0.02％。在本申请的组合物或制剂中，可以按0.001重量％至少于0.01重量％(例如0.001重量％至0.008重量％，优选约0.005重量％)的水平使用防腐剂，优选苯扎氯铵。已发现0.005％浓度的苯扎氯铵可足以保护本公开的组合物或制剂免受微生物攻击。
在一些实施方案中，以可溶性而不是悬浮液形式递送本公开的剂，这样允许更快速和定量地吸收至作用位点。一般来说，诸如凝胶剂、霜剂、洗剂、栓剂和油膏的制剂可提供更长时间暴露于本公开的剂的区域，而溶液形式的制剂例如喷雾剂则提供更直接的短期暴露。
另外可以预见，本公开的化合物可以可释放的方式附着于生物相容性聚合物上，用于在持续释放的制剂中使用，所述持续释放的制剂在用于局部、眼内、眼周或全身施用的插入物之上、之中或附着于其上。从生物相容性聚合物中的受控释放也可以水溶性聚合物利用以形成可滴注的制剂。从生物相容性聚合物比如PLGA微球或纳米球中的受控释放可用于适于眼内植入或注射以进行持续释放施用的制剂中，也可以使用任意合适的可生物降解且是生物相容性的聚合物。
疾病治疗、试剂盒
本文的一方面，提供了根据本文所述的药物制剂在治疗疾病中的应用。本文的一方面，提供了根据本文所述的质粒在制备药物中的应用。本文的一方面，提供了根据本文所述的质粒系统在制备药物中的应用。本文的一方面，提供了根据本文所述的生产方法在制备药物中的应用。本文的一方面，提供了根据本文所述的重组腺相关病毒在制备药物中的应用。本文的一方面，提供了根据本文所述的宿主细胞在制备药物中的应用。
本文的一方面，提供了一种治疗疾病的方法，其包括向有需要的患者施用根据本文所述的重组腺相关病毒或药物制剂。
在一些实施例中，所述疾病是由于生物体内的靶基因发生基因突变和/或基因缺失导致的。在一些实施例中，患有疾病的个体是人。
本文的一方面，提供了一种试剂盒，其包含本文所述的质粒。本文的一方面，提供了一种试剂盒，其包含本文所述的质粒系统。本文的一方面，提供了一种试剂盒，其包含本文所述的重组腺相关病毒。本文的一方面，提供了一种试剂盒，其包含本文所述的宿主细胞。
在一些实施例中，所述宿主细胞选自HEK293细胞、HEK293T细胞、HEK293F细胞、VPC2.0细胞、Hela细胞和A549细胞。
定义
除非另有定义，这里使用的所有技术和科学术语与本申请所属领域的普通技术人员通常理解的含义相同。尽管与本文描述的那些方法或材料相似或等同的任何方法和材料都可以用于本文的制剂或单位剂量的实践或测试中，但是现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料，方法和实施例仅是说明性的而不是限制性的。
如本文和所附权利要求书中所使用的，单数形式“一个”、“一种”和“该”包括复数指示物，除非上下文另外明确指出。
如本文所用，除非另外指出，否则术语“或”可以是连接词或反意连接词(disjunctive)。如本文所用，除非另外指出，否则任何实施例都可以与任何其他实施例结合。
如本文所用，除非另外指出，否则本文中的某些申请实施例预期数值范围。存在范围时，范围包括范围端点。此外，该范围内的每个子范围和值都存在，就好像已明确写出一样。
术语“约”及其与参考数值相关的语法对等词以及其如本文所用的语法对等词可包括值的范围加上或减去该值的10％，如值的范围加上或减去该值的10％、9％、8％、7％、6％、5％、4％、3％、2％或1％。例如，量“约10”包括9至11的量。
术语“包含”(以及相关术语，例如“包含”或“具有”或“包括”)并不旨在排除其他某些实施例中的内容，例如，本文描述的物质、组成、方法或过程等的任何组成的实施例可以“由所描述的特征组成”或“基本上由所描述的特征组成”。
术语“受试者”是指已经或将成为治疗，观察或实验对象的哺乳动物。术语“哺乳动物”旨在具有其标准含义，并且包括例如人类、狗、猫、绵羊和牛。本文所述的方法可用于人类治疗和兽医应用。在一些实施例中，主体是人。
术语“治疗”或“治疗”包括将至少一种本申请公开的化合物或其药学上可接受的盐给予哺乳动物受试者，特别是人类受试者，并且包括(i)阻止疾病临床症状的发展，(ii)使该疾病的临床症状消退和/或(iii)预防该疾病发作的预防性治疗。
术语“治疗有效量”是指有效量，当施用于人类或非人类受试者时，提供治疗益处，例如症状改善、疾病进展减慢或疾病预防。
如本文所用，除非另有说明，否则术语“核酸”和“多核苷酸”可以互换使用。
如本文所用，除非另有说明，否则在描述基因或核酸位点时，术语“上游”是指该基因或核酸位点的5’端，术语“下游”是指该基因或核酸位点的3’端。
术语“启动子”是一段位于结构基因5’-端上游区的DNA序列，能活化RNA聚合酶，使之与模板DNA准确地结合，并具有转录起始的特异性。
术语“肝脏特异性增强子-启动子”(Liver specific enhancer promotor，LSP)是指能增强外源基因在肝脏的表达的启动子，位于cap基因的终止密码子与右侧末端反向重复序列之间(见Logan et al.,Identification of liver-specific enhancer–promoter activity in the 3’untranslated region of the wild-type AAV2 genome)。在一些情况下，由于AAV对肝脏天然的亲嗜性，含有肝脏特异性增强子-启动子的AAV表达系统容易造成AAV在肝脏累积，有可能会对AAV载体安全性造成影响。
术语“可操作地连接”是指第一多核苷酸组件，如启动子，与第二可转录的多核苷酸组件，如一个基因的读码框，被布置成使得第一多核苷酸组件影响第二多核苷酸组件的功能。两个多核苷酸组件可以是单个连续多核苷酸分子的一部分并且可以是相邻的。然而，不连续或不相邻的第一和第二多核苷酸组件也可以被布置成可操作地连接。在一些实施例中，术语“可操作地连接”还指在重组之后(例如，由重组酶介导，或在内含子被去除后)，而非在初始布置中，可操作地连接的两个多核苷酸组件。除非另有说明，当本文描述一个启动子和相对应的基因时，这两者可操作地连接。
术语“宿主细胞”是指目的基因(GOI)进行克隆的细胞，质粒能在宿主细胞提供的条件下产生与自身序列基本相同的复制品，质粒在宿主细胞内进行复制时，由宿主细胞提供能量、材料、酶等条件。例如，宿主细胞可以选自HEK293细胞、HEK293T细胞、HEK293F细胞、VPC2.0细胞、Hela细胞和A549细胞等。
术语“序列相同性”是指两个多核苷酸或多肽序列之间相同且处于相同相对位置的碱基或氨基酸的百分比。因此，一个多核苷酸或多肽序列与另一个多核苷酸或多肽序列相比具有一定百分比的序列同一性。对于序列比较，通常一个序列充当参考序列，测试序列与所述参考序列进行比较。术语“参考序列”是指与测试序列进行比较的分子。除非另有说明，否则权利要求中的术语“序列相同性”是指如通过Clustal版本1.2.4使用默认参数计算的序列同一性。
 附图说明
图1是传统三质粒系统结构示意图。
图2A是新型三质粒系统I结构示意图。
图2B是新型三质粒系统II结构示意图。
图3是二质粒系统结构示意图。
图4是wtAAV2基因结构示意图。
图5是pHelper-R2质粒结构示意图。
图6是pHelper-R10质粒结构示意图。
图7是pRC-2A质粒结构示意图。
图8是pRC-9A质粒结构示意图。
图9是pRep1质粒结构示意图。
图10是pRep3质粒结构示意图。
图11是pAAV-GOI1-C1结构示意图。
图12是pRC2、pRC2-1、pRC2-4、pRC2-5包装的rAAV2-ND4载体基因组产量倍数变化图。
图13是pRC2、pRC2-4、pRC2-8包装的rAAV2-ND4载体基因组产量倍数变化图。
图14是pRC2、pRC2-4、pRC2-8包装的rAAV2-ND4载体的实心率。
图15是pRC2、pRC2-4、pRC2-8包装的rAAV2-OPA1载体基因组产量倍数变化图。
图16是pRC2、pRC2-4、pRC2-8包装的rAAV2-OPA1载体的实心率。
图17是pRC2、pRC2-4、pRC2-8包装的rAAV2-GOI1载体基因组产量倍数变化图。
图18是pRC2、pRC2-53、pRC2-58包装的rAAV2-GOI1载体基因组产量倍数变化图。
图19是pRC2、pJSD33、pJSD34包装的rAAV2-GOI1载体基因组产量倍数变化图。
图20是pRC2、pRC2-64、pRC2-69包装的rAAV2-GOI1载体基因组产量倍数变化图。
图21是pHelper:pRC2:pAAV-EGFP、pHelper-R10:pRC-2A:pAAV-EGFP包装的rAAV2-EGFP载体基因组产量倍数变化图。
图22是pHelper-R10:pRC-2A:pAAV-hNRF2三质粒比例优化包装的rAAV2-hNRF2载体的病毒基因组滴度。
图23是pHelper:pRC2:pAAV-GOI1、pHelper:pRep1:pAAV-GOI1-C1、pHelper:pRep3:pAAV-GOI1-C1包装的rAAV2-GOI1载体基因组产量倍数变化图。
图24是pHelper-Cap、pRep和pAAV-GOI三质粒系统结构示意图。
图25是wtAAV2基因组结构示意图。
图26是pHelper-Cap2质粒结构示意图。
图27是pRep5质粒结构示意图。
图28是pRep6质粒结构示意图。
图29是pHelper-Cap2质粒分别和pRep1、pRep3、pRep5、pRep6质粒包装的rAAV2-GOI1载体基因组滴度图。
图30是pHelper-Cap2质粒和pRep5包装rAAV2-GOI1工艺优化的病毒基因组滴度图。
图31是新型三质粒系统III和传统三质粒系统包装rAAV2-GOI1载体实心率图。
图32A是多个二质粒系统的载体产量相对比值图。图32B是多个二质粒系统的Cap残留量相对比值图。图32C是多个二质粒系统的质粒残留量相对比值图。
图33是两个二质粒系统的相应质粒在不同比例下生产的病毒基因组滴度图。
图34A是多个三质粒系统I的载体产量相对比值图。图34B是多个三质粒系统I生产的病毒实心率图。图34C是传统三质粒系统与三质粒系统I生产的不同血清型AAV病毒的基因组滴度比较图。
图35A是两个三质粒系统II的载体产量相对比值图。图35B是多个三质粒系统II的相应质粒在不同比例下生产的病毒基因组滴度图。图35C是传统三质粒系统与三质粒系统II生产的不同血清型AAV病毒的基因组滴度比较图。
图36A是传统三质粒系统与三质粒系统III生产的不同血清型AAV病毒的基因组滴度比较图。图36B是传统三质粒系统与三质粒系统III生产的不同血清型AAV病毒的实心率比较图。
图37是三质粒系统III的相应质粒在不同比例下生产的病毒基因组滴度图。
图38A显示了对三质粒系统III的Rep基因改造，将Rep78/52编码基因置于pHelper-Cap质粒或者编码异源基因序列的AAV质粒后的新型质粒系统的病毒基因组滴度图。图38B是该改造后的新型三质粒系统III扩展系统包装rAAV2-EGFP病毒的实心率结果。
图39A和图39B显示了将三质粒系统III的pHelper-Cap质粒上的三个辅助基因的一个或两个移至pRep6质粒后形成的新型质粒系统的病毒基因组滴度图(图39A)和实心率(图39B)。
图40A是传统三质粒系统与三质粒系统III生产的不同血清型rAAV-scEGFP病毒的基因组滴度比较图。图40B是新型三质粒系统III和传统三质粒系统包装不同血清型rAAV-scEGFP病毒相对实心率对比图(根据传统三质粒系统生产的各种血清型的rAAV-scEGFP的相对实心率值定为1)。
图41是pHelper-R5质粒结构示意图。
图42是pRC-5A质粒结构示意图。
图43是pRC-8A质粒结构示意图。
图44是pHelper-Cap5质粒结构示意图。
图45是pHelper-Cap8质粒结构示意图。
图46是pHelper-Cap9质粒结构示意图。
图47是pHelper-C2R4质粒结构示意图。
图48是VA-Cap2质粒结构示意图。
图49是VAE4-Cap2质粒结构示意图。
图50是pRep6-E2A质粒结构示意图。
图51是pRep6-E4E2A质粒结构示意图。
图52是pRep4质粒结构示意图。
图53是pEGFP-Cap2质粒结构示意图。
图54是pEGFP-Cap5质粒结构示意图。
图55是pEGFP-Cap8质粒结构示意图。
图56是pEGFP-Cap9质粒结构示意图。
其他编号的实施方案
如下提供本发明的其他编号的实施方案：
实施方案1.一种重组核酸，包含腺相关病毒(AAV)的Rep基因和/或Cap基因，且有如下的一种或多种特征：
(1)在所述Rep基因的上游和/或所述Cap基因的下游含有一个P5启动子；
(2)位于所述Cap基因下游的原有P5启动子中的TATA box突变被修正，或在所述Cap基因下游插入一个所述P5启动子，与所述Cap基因可操作地连接；
(3)所述腺相关病毒的病毒基因组原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:7的第1717-1722核苷酸位点；
(4)所述Cap基因下游原有的肝脏特异性增强子-启动子序列被删除；
(5)在所述Rep基因起始密码子前含有一个hGH内含子；
(6)在所述Rep基因起始密码子前含有一个腺相关病毒基因组的内含子；
和/或
(7)在所述Rep基因上游或在所述Cap基因下游含有一个非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
实施方案2.根据实施方案1所述的重组核酸，其特征在于，包含特征(1)，且位于所述Rep基因上游的所述P5启动子的插入位置紧接在相当于SEQ ID NO:7的腺相关病毒基因组的120bp的核苷酸位点后，与所述Rep基因可操作地连接。
实施方案3.根据实施方案1-2任一项所述的重组核酸，其特征在于，在所述Cap基因下游插入一个P5启动子，与所述Cap基因可操作地连接。
实施方案4.根据实施方案2-3任一项所述的重组核酸，其特征在于，所述P5启动子与所述Rep基因或所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案5.根据实施方案1-4任一项所述的重组核酸，其特征在于，所述P5启动子的序列与SEQ ID NO:4有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案6.根据实施方案1-5任一项所述的重组核酸，其特征在于，包含特征(2)，且所述原有P5启动子的TATA box突变的修正包含将相当于SEQ ID NO:7第4469位核苷酸的T突变为A，和/或将相当于SEQ ID NO:7第4477-4483位核苷酸的GGGGGGG突变为TATTTAA。
实施方案7.根据实施方案6所述的重组核酸，其特征在于，所述原有P5启动子修正后的序列与SEQ ID NO:9有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案8.根据实施方案1-7任一项所述的重组核酸，其特征在于，包含特征(3)，且所述donor位点的突变包含将相当于SEQ ID NO:7第1721-1722位核苷酸的CA突变为GT，或相当于SEQ ID NO:7第1717-1722位核苷酸的donor位点序列突变为GTACGT。
实施方案9.根据实施方案8所述的重组核酸，其特征在于，突变后的内含子序列与SEQ ID NO:11有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案10.根据实施方案1-9任一项所述的重组核酸，其特征在于，包含特征(4)，且所述肝脏特异性增强子-启动子序列被删除。
实施方案11.根据实施方案1-10任一项所述的重组核酸，其特征在于，所述肝脏特异性增强子-启动子序列与SEQ ID NO:12或SEQ ID NO:35有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案12.根据实施方案1-11任一项所述的重组核酸，其特征在于，包含特征(5)，且所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案13.根据实施方案12任一项所述的重组核酸，其特征在于，所述hGH内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。
实施方案14.根据实施方案13所述的重组核酸，其特征在于，所述hGH内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案15.根据实施方案1-14任一项所述的重组核酸，其特征在于，所述hGH内含子与所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案16.根据实施方案15所述的重组核酸，其特征在于，所述hGH内含子与所述Rep基因起始密码子之间没有间隔的核苷酸。
实施方案17.根据实施方案1-16任一项所述的重组核酸，其特征在于，包含特征(6)，且所述腺相关病毒基因组的内含子的序列与SEQ ID NO:14有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案18.根据实施方案1-17任一项所述的重组核酸，其特征在于，所述腺相关病毒基因组的内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。
实施方案19.根据实施方案18所述的重组核酸，其特征在于，所述腺相关病毒基因组的内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案20.根据实施方案1-19任一项所述的重组核酸，其特征在于，所述腺相关病毒基因组的内含子和所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案21.根据实施方案1-20任一项所述的重组核酸，其特征在于，包含特征(7)，且所述非亲本血清型启动子的序列与SEQ ID NO:15有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案22.根据实施方案1-21任一项所述的重组核酸，其特征在于，(i)所述非亲本血清型启动子位于所述Cap基因的下游，或(ii)所述非亲本血清型启动子位于所述Rep基因上游，且所述所述非亲本血清型启动子位于所述hGH内含子的上游。
实施方案23.根据实施方案22所述的重组核酸，其特征在于，所述非亲本血清型启动子位于相当于SEQ ID NO:7第4270-4271位核苷酸之间的位置。
实施方案24.根据实施方案1-23任一项所述的重组核酸，其特征在于，所述重组核酸不含有相当于SEQ ID NO:7第4368-4532位核苷酸的序列，或不含有相当于SEQ ID NO:7第4373-4532位核苷酸的序列。
实施方案25.根据实施方案1-24任一项所述的重组核酸，其特征在于，所述重组核酸不含有相当于SEQ ID NO:7第121-130位核苷酸的序列。
实施方案26.根据实施方案1-25任一项所述的重组核酸，其特征在于：
(i)所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、AAV9；更优选地，所述AAV的血清型是AAV2；
(ii)所述Rep基因的序列与SEQ ID NO:2有至少90％，至少95％，至少98％，至少99％，或100％的相同性；和/或
(iii)所述Cap基因的序列与SEQ ID NO:3有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案27.根据实施方案1-26任一项所述的重组核酸，其特征在于：所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
实施方案28.根据实施方案1-27任一项所述的重组核酸，其特征在于,所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案29.根据实施方案1-28任一项所述的重组核酸，其特征在于，包括所述特征：
(1)；或
(1)和(2)；或
(3)；或
(1)(2)和(3)；或
(1)(2)(3)和(4)；或
(1)(2)(3)(4)和(5)；或
(1)和(6)；或
(1)(2)和(6)；或
(2)(3)(4)(5)和(7)；或
(1)(3)(4)(5)和(7)。
实施方案30.根据实施方案1-29任一项所述的重组核酸，其特征在于，包含所述Rep基因和所述Cap基因。
实施方案31.根据实施方案1,3-7,10-11,21-29任一项所述的重组核酸，其特征在于，包含所述Cap基因，但不包含编码Rep基因的核酸序列。
实施方案32.根据实施方案1-2,4-5,8-9,12-29任一项所述的重组核酸，其特征在于，包含所述Rep基因，但不包含编码Cap基因的核酸序列。
实施方案33.一种质粒，包含根据实施方案1-32任一项所述的重组核酸。
实施方案34.根据实施方案33所述的质粒，其特征在于，所述质粒为重组腺相关病毒无辅助病毒系统中的包装质粒(RC质粒)，或辅助质粒(pHelper质粒)，或编码包含异源核酸序列的重组腺相关病毒的质粒。
实施方案35.根据实施方案33-34所述的质粒，其特征在于，所述质粒包含所述Cap基因，但不包含编码Rep基因的核酸序列，也不包含编码用于AAV生产的辅助基因的核酸序列，也不包含AAV反向重复序列(ITR)。
实施方案36.一种质粒，其特征在于，包含Cap基因，但不包含编码Rep基因的核酸序列，也不包含编码用于AAV生产的辅助基因的核酸序列，也不包含AAV反向重复序列(ITR)。
实施方案37.根据实施方案33-34任一项所述的质粒，其特征在于，所述质粒为编码包含异源核酸序列的重组腺相关病毒的质粒，且包含所述Cap基因。
实施方案38.根据实施方案37所述的质粒，其特征在于，所述目的基因质粒包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案39.一种辅助质粒，其特征在于，包含根据实施方案1-31任一项所述的包含所述Cap基因的重组核酸，且所述辅助质粒包含编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。
实施方案40.一种辅助质粒，其特征在于，包含编码Cap基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列，所述Cap基因编码一个功能性的Cap蛋白。
实施方案41.根据实施方案39-40任一项所述的辅助质粒，其特征在于，所述辅助质粒包含Rep基因；可选地，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案42.根据实施方案39-41任一项所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。
实施方案43.根据实施方案42所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的两个基因；可选地，所述辅助质粒包含E4基因和VA RNA基因。
实施方案44.根据实施方案42所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含E2A，E4，和VA RNA。
实施方案45.根据实施方案42-44任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)；可选地，所述E2A基因包含与SEQ ID NO:93有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列；可选地，所述E4基因包含与SEQ ID NO:94有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列；可选地，所述VA RNA基因包含与SEQ ID NO:95有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案46.根据实施方案42-44任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)。
实施方案47.根据实施方案1-31任一项所述的重组核酸，或根据实施方案31-41任一项所述的质粒，其特征在于，包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。
实施方案48.根据实施方案47所述的重组核酸或质粒，其特征在于，包含P5启动子，P19启动子，和P40启动子；优选地，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。
实施方案49.根据实施方案47-48任一项所述的重组核酸或质粒，其特征在于，所述启动子之间，和/或所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案50.根据实施方案47-49任一项所述的重组核酸或质粒，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案51.根据实施方案47-50任一项所述的重组核酸或质粒，其特征在于，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。
实施方案52.根据实施方案47-51任一项所述的重组核酸或质粒，其特征在于，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核苷酸的位置是GT。
实施方案53.根据实施方案1-31任一项所述的重组核酸，根据实施方案33-46任一项所述的质粒，或根据实施方案47-52任一项所述的重组核酸或质粒，其特征在于，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案54.根据实施方案1-31任一项所述的重组核酸，根据实施方案33-46任一项所述的质粒，或根据实施方案47-52任一项所述的重组核酸或质粒，其特征在于，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
实施方案55.根据实施方案54所述的重组核酸或质粒，其特征在于，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案56.根据实施方案1-31任一项所述的重组核酸，根据实施方案33-46任一项所述的质粒，或根据实施方案47-55任一项所述的重组核酸或质粒，其特征在于，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
实施方案57.根据实施方案56所述的重组核酸或质粒，其特征在于，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案58.根据实施方案56-57任一项所述的重组核酸或质粒，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案59.根据实施方案1-31任一项所述的重组核酸，根据实施方案33-46任一项所述的质粒，或根据实施方案47-58任一项所述的重组核酸或质粒，其特征在于，在所述Cap基因下游还包含一个P5启动子，与所述Cap基因可操作地连接；可选地，所述P5启动子位于Cap基因下游的polyA序列的下游；可选地，所述P5启动子与所述Cap基因之间，或所述P5启动子与所述polyA序列之间，间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
实施方案60.根据实施方案1-31任一项所述的重组核酸，根据实施方案33-46任一项所述的质粒，或根据实施方案47-59任一项所述的重组核酸或质粒，其特征在于，所述Cap基因编码一个功能性的Cap蛋白。
实施方案61.根据实施方案33-60任一项所述的质粒，其特征在于，所述质粒不能表达功能性的Rep蛋白。
实施方案62.根据实施方案33-61任一项所述的质粒，其特征在于，所述质粒不包含编码Rep基因的核酸序列。
实施方案63.一种辅助质粒，其特征在于，包含根据实施方案1-30,32任一项所述的包含所述Rep基因的重组核酸，且所述辅助质粒包含编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。
实施方案64.一种辅助质粒，其特征在于，包含编码Rep基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列，
实施方案65.根据实施方案63-64任一项所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。
实施方案66.根据实施方案65所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的两个基因；可选地，所述辅助质粒包含E4基因和VA RNA基因。
实施方案67.根据实施方案65所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含E2A，E4，和VA RNA。
实施方案68.根据实施方案65-67任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)；可选地，所述E2A基因包含与SEQ ID NO:93有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列；可选地，所述E4基因包含与SEQ ID NO:94有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列；可选地，所述VA RNA基因包含与SEQ ID NO:95有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案69.根据实施方案65-67任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)。
实施方案70.根据实施方案63-69任一项所述的辅助质粒，其特征在于，包含编码所述Rep基因的核酸序列，并且所述质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列。
实施方案71.根据实施方案33-34任一项所述的质粒，其特征在于，包含编码所述Rep基因的核酸序列，并且所述质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列。
实施方案72.一种质粒，其特征在于，包含编码Rep基因的核酸序列，并且所述质粒不能表达功能性的Cap蛋白。
实施方案73.根据实施方案72所述的质粒，其特征在于，所述质粒不包含编码Cap基因的核酸序列。
实施方案74.根据实施方案72或73所述的质粒，其特征在于，所述质粒不能表达功能性的用于AAV生产的辅助基因的表达产物。
实施方案75.根据实施方案72-74任一项所述的质粒，其特征在于，所述质粒不包含编码用于AAV生产的辅助基因的核酸序列。
实施方案76.根据实施方案1-30,32任一项所述的包含所述Rep基因的重组核酸，或根据实施方案33-34,63-75任一项所述的质粒，其特征在于，所述Rep基因表达Rep78，Rep68，Rep52，和Rep40蛋白。
实施方案77.根据实施方案76所述的重组核酸或质粒，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案78.根据实施方案1-30,32任一项所述的包含所述Rep基因的重组核酸，或根据实施方案33-34,63-75任一项所述的质粒，其特征在于，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案79.根据实施方案78所述的重组核酸或质粒，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2196位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
实施方案80.根据实施方案1-30,32任一项所述的包含所述Rep基因的重组核酸，或根据实施方案33-34,63-75任一项所述的质粒，其特征在于，所述Rep基因表达Rep68和Rep40蛋白；可选地，所述Rep基因不表达Rep78或Rep52蛋白。
实施方案81.根据实施方案80所述的重组核酸或质粒，其特征在于，所述Rep基因包含与SEQ ID NO:69或SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案82.根据实施方案80-81任一项所述的重组核酸或质粒，其特征在于，所述的重组核酸或质粒不包含仅表达Rep52和Rep40蛋白的基因。
实施方案83.根据实施方案1-30,32任一项所述的包含所述Rep基因的重组核酸，根据实施方案33-34,63-75任一项所述的质粒，或根据实施方案76-82任一项所述的重组核酸或质粒，其特征在于，包含一个位于所述Rep基因的上游的P5启动子和/或一个位于所述Rep基因的下游的P5启动子，所述P5启动子与所述Rep基因可操作地连接。
实施方案84.根据实施方案83所述的重组核酸或质粒，其特征在于，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案85.根据实施方案83-84任一项所述的重组核酸或质粒，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸，或与SEQ ID NO:1第191-310位核苷酸，有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案86.根据实施方案1-30,32任一项所述的包含所述Rep基因的重组核酸，根据实施方案33-34,63-75任一项所述的质粒，或根据实施方案76-85任一项所述的重组核酸或质粒，其特征在于，所述Rep基因编码一个功能性的Rep蛋白。
实施方案87.根据实施方案1-30,32任一项所述的包含所述Rep基因的重组核酸，根据实施方案33-34,63-75任一项所述的质粒，或根据实施方案76-86任一项所述的重组核酸或质粒，其特征在于，所述编码Rep基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Rep基因下游。
实施方案88.根据实施方案87所述的重组核酸或质粒，其特征在于，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案89.根据实施方案87-88任一项所述的重组核酸或质粒，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案90.根据实施方案33-34,63-89任一项所述的质粒，其特征在于，所述编码Rep基因的核酸序列包含一个hGH内含子；优选地，所述内含子位于所述Rep基因的起始密码子前。
实施方案91.根据实施方案90所述的质粒，其特征在于，所述P5启动子与所述hGH内含子之间，和/或所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
实施方案92.根据实施方案90-91任一项所述的质粒，其特征在于，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
实施方案93.根据实施方案33-34,63-92任一项所述的质粒，其特征在于，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。
实施方案94.根据实施方案93所述的质粒，其特征在于，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT，或相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
实施方案95.根据实施方案33-94任一项所述的质粒，其特征在于，所述质粒还包含f1 origin位点。
实施方案96.一种三质粒系统，其特征在于，包含:
(i)辅助质粒(pHelper质粒)，
(ii)一个包含腺相关病毒(AAV)的Rep基因和/或Cap基因的质粒，和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游，
其中，(i)和/或(ii)中所述的质粒包含根据实施方案1-32,47-60,76-89任一项所述的编码所述Rep基因和/或所述Cap基因的重组核酸。
实施方案97.根据实施方案96所述的三质粒系统，其特征在于，(ii)中所述质粒包含所述Rep基因和所述Cap基因。
实施方案98.根据实施方案96所述的三质粒系统，其特征在于，所述Rep基因和所述Cap基因在不同的质粒上。
实施方案99.根据实施方案98所述的三质粒系统，其特征在于，(i)所述的辅助质粒包含所述Rep基因，且(ii)中所述质粒包含所述Cap基因。
实施方案100.根据实施方案98所述的三质粒系统，其特征在于，(ii)中所述质粒包含所述Rep基因，且(iii)中所述重组腺相关病毒的质粒包含所述Cap基因。
实施方案101.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，其中一个质粒包含AAV的Rep基因但不包含AAV的Cap基因，另一个不同的质粒包含AAV的Cap基因但不包含AAV的Rep基因。
实施方案102.根据实施方案101所述的三质粒系统，其特征在于，包含一个包含所述Rep基因的辅助质粒(pHelper质粒)，一个包含所述Cap基因的第二质粒，和一个编码包含异源核酸序列的重组腺相关病毒的第三质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，所述第三质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案103.根据实施方案101所述的三质粒系统，其特征在于，包含一个辅助质粒(pHelper质粒)，一个包含所述Rep基因的第二质粒，和一个包含所述Cap基因且编码包含异源核酸序列的重组腺相关病毒的第三质粒；可选的，所述异源核酸序列编码一个异源基因；可选地，所述第三质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案104.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含:
(i)根据实施方案63-70,76-95任一项所述的辅助质粒，其包含所述Rep基因，且所述辅助质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；
(ii)根据实施方案35-36,47-62任一项所述的质粒，其包含所述Cap基因，且该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案105.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Rep基因的重组核酸；可选地，所述辅助质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；
(ii)一个包含Cap基因的质粒；可选地，该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案106.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含:
(i)辅助质粒(pHelper质粒)，
(ii)根据实施方案71-95任一项所述的质粒，其包含所述Rep基因，且该质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列，和
(iii)根据实施方案37-38,47-62任一项所述的包含所述Cap基因和所述异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案107.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)；
(ii)一个包含Rep基因的质粒；可选地，该质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒，且包含Cap基因的质粒；可选地，该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案108.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)根据实施方案39-62任一项所述的辅助质粒，其包含所述Cap基因；
(ii)根据实施方案71-95任一项所述的编码Rep基因的质粒；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
实施方案109.一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
(i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Cap基因的重组核酸；
(ii)一个包含Rep基因的质粒；和
(iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
实施方案110.根据实施方案108-109任一项所述的三质粒系统，其特征在于，所述(i)辅助质粒不能表达功能性的Rep蛋白，和/或所述(ii)编码Rep基因的质粒不能表达功能性的Cap蛋白。
实施方案111.根据实施方案108-110任一项所述的三质粒系统，其特征在于，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案112.根据实施方案108-111任一项所述的三质粒系统，其特征在于，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(i)辅助质粒包含一个表达Rep78和/或Rep52蛋白的基因；可选地，该基因不表达Rep68或Rep40。
实施方案113.根据实施方案108-111任一项所述的三质粒系统，其特征在于，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒包含一个表达Rep78和/或Rep52蛋白的基因；可选地，该基因不表达Rep68或Rep40。
实施方案114.根据实施方案108-113任一项所述的三质粒系统，其特征在于，所述(i)辅助质粒包含E4基因和VA RNA基因；所述(ii)编码Rep基因的质粒包含E2A基因。
实施方案115.根据实施方案108-114任一项所述的三质粒系统，其特征在于，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9；更优选地，所述AAV的血清型是AAV2。
实施方案116.根据实施方案108-115任一项所述的三质粒系统，其特征在于，所述异源核酸序列编码一个异源基因。
实施方案117.一种二质粒系统，其特征在于，包含(i)根据实施方案63-70,76-95任一项所述的含所述Rep基因的第一质粒和(ii)根据实施方案37-38,47-62任一项所述的含所述Cap基因的第二质粒。
实施方案118.一种二质粒系统，其特征在于，包含一个含有AAV的Rep基因的第一质粒和一个含有AAV的Cap基因的第二质粒，所述Rep基因和所述Cap基因分别位于不同质粒上。
实施方案119.根据实施方案117-118任一项所述的二质粒系统，其特征在于，所述Rep和所述Cap基因分别位于不同质粒上。
实施方案120.根据实施方案117-119任一项所述的二质粒系统，其特征在于，含有所述Rep基因的第一质粒为辅助质粒(pHelper质粒)，且含有所述Cap基因的第二质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，所述第二质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案121.根据实施方案117-119任一项所述的二质粒系统，其特征在于，含有所述Cap基因的第二质粒为辅助质粒(pHelper质粒)，且含有所述Rep基因的第一质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选的，所述异源核酸序列编码一个异源基因；可选地，所述第一质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
实施方案122.根据实施方案96-121任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78、Rep68、Rep52和Rep40蛋白。
实施方案123.根据实施方案96-122任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78和Rep52蛋白，而不表达Rep68和Rep40蛋白。
实施方案124.根据实施方案96-122任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep68和Rep40蛋白，而不表达Rep78和Rep52蛋白。
实施方案125.根据实施方案96-124任一项所述的质粒系统，其特征在于，所述质粒系统不包含共表达Rep52蛋白和Rep40蛋白的基因。
实施方案126.根据实施方案96-125任一项所述的质粒系统，其特征在于，所述Rep基因的上游含有一个hGH内含子。
实施方案127.根据实施方案126所述的质粒系统，其特征在于，所述hGH内含子的上游含有一个P5启动子。
实施方案128.根据实施方案96-127任一项所述的质粒系统，其特征在于，所述Rep基因的下游含有polyA序列。
实施方案129.根据实施方案96-128任一项所述的质粒系统，其特征在于，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
实施方案130.根据实施方案96-129任一项所述的质粒系统，其特征在于，所述Cap基因的上游含有三个启动子，从5’至3’方向分别为：P5启动子，P19启动子，和P40启动子，均与所述Cap基因可操作地连接，所述Cap基因的下游含有polyA序列。
实施方案131.根据实施方案96-130任一项所述的质粒系统，其特征在于，所述质粒系统包含至少一条与SEQ ID NO:16--28,33-34,40,45-47,68,80-87,96-98任一条有至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
实施方案132.一种质粒系统的改造方法，其特征在于，具有根据下一种或多种修饰方法：
(1)在Rep基因的上游插入P5启动子，与所述Rep基因可操作地连接；
(2)校正位于Cap基因下游的TATA box突变的P5启动子或在Cap基因下游插入P5启动子，与所述Cap基因可操作地连接；
(3)对内含子的donor位点进行突变；
(4)删除掉Cap基因下游包括肝脏特异性增强子-启动子的一段序列；
(5)在Rep基因起始密码子AUG前插入hGH内含子；
(6)在Rep基因起始密码子AUG前插入wtAAV2内含子；
(7)在Rep基因上游或在Cap基因下游插入非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
实施方案133.根据实施方案132所述的质粒系统的改造方法，其特征在于：
(1)中所述在Rep基因的上游插入P5启动子具有如SEQ ID NO:4所示的序列；
(2)中所述突变的P5启动子具有如SEQ ID NO:8所示的序列，在Cap基因下游插入的所述P5启动子具有SEQ ID NO:4的序列；
(3)对内含子的donor位点进行突变是将内含子的donor位点处的GTACCA突变为GTACGT，优选地，突变前的内含子具有如SEQ ID NO:10所示的序列；突变donor后的内含子具有如SEQ ID NO:11所示的序列；
(4)中删除的序列如SEQ ID NO:12所示；
(5)中插入的hGH内含子具有如SEQ ID NO:13所示的序列；
(6)中在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子，优选地，经过改造的wtAAV2内含子具有如SEQ ID NO:14所示的序列；和/或
(7)中非亲本血清型启动子为wtAAV7 P5启动子，其有如SEQ ID NO:15所示的序列。
实施方案134.一种重组腺相关病毒的生产方法，其特征在于，将根据实施方案33-95任一项所述的质粒或根据实施方案96-131任一项所述的质粒系统中的所有质粒转染至宿主细胞，培养，分离得到所述重组腺相关病毒。
实施方案135.根据实施方案134所述的生产方法，其特征在于，包含所述Cap基因的质粒与包含所述Rep基因的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1；优选地，至少3:1。
实施方案136.根据实施方案134-135任一项所述的生产方法，其特征在于，包含所述Cap基因的质粒与包含所述Rep基因的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1；优选地，约3:1。
实施方案137.根据实施方案134-136任一项所述的生产方法，其特征在于，包含所述Rep基因的质粒与包含所述编码包含异源核酸序列的重组腺相关病毒的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1；优选地，至少2.5:1。
实施方案138.根据实施方案134-137任一项所述的生产方法，其特征在于，包含所述Rep基因的质粒与包含所述编码包含异源核酸序列的重组腺相关病毒的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1；优选地，约2.5:1。
实施方案139.根据实施方案134-138任一项所述的生产方法，其特征在于，相比基于传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统的生产方法，所述方法产生的重组腺相关病毒的：
(1)实心率更高；可选地，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少0.70,至少0.75,或至少0.80；
(2)基因组滴度更高；可选地，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍；
(3)Cap编码序列的相关杂质残留更低；可选地，Cap编码序列的相关杂质残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％；和/或
(4)具有复制能力的AAV(rcAAV)残留更低；可选地，rcAAV残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
实施方案140.一种重组腺相关病毒，其特征在于，采用根据实施方案134-139任一项所述的生产方法获得。
实施方案141.根据实施方案140所述的重组腺相关病毒，其特征在于，所述宿主细胞的培养方式包括贴壁培养或悬浮培养。
实施方案142.根据实施方案140-141任一项所述的重组腺相关病毒，其特征在于，所述重组腺相关病毒相比由传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统生产的重组腺相关病毒的：
(1)实心率更高；可选地，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少0.70,至少0.75,或至少0.80；
(2)基因组滴度更高；可选地，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍；
(3)Cap编码序列的相关杂质残留更低；可选地，Cap编码序列的相关杂质残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％；和/或
(4)具有复制能力的AAV(rcAAV)残留更低；可选地，rcAAV残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
实施方案143.一种宿主细胞，其特征在于，所述宿主细胞含有根据实施方案33-95任一项所述的质粒或根据实施方案96-131任一项所述的质粒系统中的所有质粒。
实施方案144.根据实施方案134-139任一项所述的生产方法，或根据实施方案143所述的宿主细胞，其特征在于，所述宿主细胞选自HEK293细胞、HEK293T细胞、HEK293F细胞、VPC2.0细胞、Hela细胞和A549细胞。
实施方案145.根据实施方案1-32任一项所述的重组核酸，实施方案33-95任一项所述的质粒，实施方案96-141任一项所述的质粒系统，实施方案140-142任一项所述的重组腺相关病毒，或实施方案143-144任一项所述的宿主细胞在制备药物中的应用。
实施方案146.一种药物制剂，其特征在于，所述药物包括根据实施方案140-142任一项所述的重组腺相关病毒，以及药学上可接受的载体和赋形剂。
实施方案147.如实施方案146所述的药物制剂在治疗疾病中的应用。
实施方案148.一种治疗疾病的方法，其包括向有需要的患者施用根据实施方案140-142任一项所述的重组腺相关病毒，或实施方案146所述的药物制剂。
实施方案149.如实施方案147所述的应用或者如实施方案148所述的方法，其中，所述疾病是由于人体中的靶基因发生基因突变和/或基因缺失导致的。
实施方案150.一种试剂盒，其包含根据实施方案1-32任一项所述的重组核酸，实施方案33-95任一项所述的质粒，实施方案96-141任一项所述的质粒系统，实施方案140-142任一项所述的重组腺相关病毒，或实施方案143-144任一项所述的宿主细胞。
 具体实施方式
质粒信息
在一些实施例中，本文所述的质粒系统中改造的质粒，具有如下一种或多种修饰(改造方法序号与前述具体改造方法序号保持一致)：
pRC2-1(SEQ ID NO:16)：(1)在Rep基因的上游插入P5启动子，具体为，在pRC2质粒(SEQ ID NO:7)第121nt前插入wtAAV2 191-310nt(SEQ ID NO:4)。
pRC2-4(SEQ ID NO:17)：(1)在Rep基因的上游插入P5启动子，具体为在pRC2质粒(SEQ ID NO:7)第121nt前插入wtAAV2 191-310nt(SEQ ID NO:4)，(2)校正位于Cap基因下游的TATA box突变的P5启动子(SEQ ID NO:8)，具体为把第4469nt T突变为A，把第4477-4483nt GGGGGGG突变为TATTTAA，校正后的P5启动子具有如SEQ ID NO:9所示的序列。
pRC2-5(SEQ ID NO:18)：(3)内含子(SEQ ID NO:10)的donor位点处的GTACCA突变为GTACGT，具体为把pRC2质粒第1721-1722nt CA突变为GT，突变donor后的内含子具有如SEQ ID NO:11所示的序列。
pRC2-8(SEQ ID NO:19)：(1)在Rep基因的上游插入P5启动子，具体为在pRC2质粒(SEQ ID NO:7)第121nt前插入wtAAV2 191-310nt(SEQ ID NO:4)；(2)校正位于Cap基因下游的TATA box突变的P5启动子(SEQ ID NO:8)，具体为第4469nt T突变为A，把第4477-4483nt GGGGGGG突变为TATTTAA，校正后的P5启动子具有如SEQ ID NO:9所示的序列；(3)内含子(SEQ ID NO:10)的donor位点处的GTACCA突变为GTACGT，具体为把第1721-1722nt CA突变为GT，突变donor后的内含子具有如SEQ ID NO:11所示的序列。
pRC2-53(SEQ ID NO:20)：(1)在Rep基因的上游插入P5启动子，具体为在pRC2质粒(SEQ ID NO:7)第121nt前插入wtAAV2 191-310nt(SEQ ID NO:4)；(2)校正位于Cap基因下游的TATA box突变的P5启动子(SEQ ID NO:8)，具体为第4469nt T突变为A，把第4477-4483nt GGGGGGG突变为TATTTAA，校正后的P5启动子具有如SEQ ID NO:9所示的序列；(3)内含子(SEQ ID NO:10)的donor位点处的GTACCA突变为GTACGT，具体为把第1721-1722nt CA突变为GT，突变donor后的内含子具有如SEQ ID NO:11所示的序列；(4)删除掉Cap基因下游的肝脏特异性增强子-启动子序列(LSP，Liver specific enhancer promotor)，具体为删除掉第4271-4344nt(SEQ ID NO:12)。
pRC2-58(SEQ ID NO:21)：(1)在Rep基因的上游插入P5启动子，具体为在pRC2质粒(SEQ ID NO:7)第121nt前插入wtAAV2 191-310nt(SEQ ID NO:4)；(2)校正位于Cap基因下游的TATA box突变的P5启动子(SEQ ID NO:8)，具体为第4469nt T突变为A，把第4477-4483nt GGGGGGG突变为TATTTAA，校正后的P5启动子具有如SEQ ID NO:9所示的序列；(3)内含子(SEQ ID NO:10)的donor位点处的GTACCA突变为GTACGT，具体为把第1721-1722nt CA突变为GT，突变donor后的内含子具有如SEQ ID NO:11所示的序列；(4)删除掉Cap基因下游的肝脏特异性增强子-启动子序列(LSP，Liver specific enhancer promotor)，具体为删除掉第4271-4344nt(SEQ ID NO:12)。(5)在Rep基因(SEQ ID NO:2)起始密码子AUG前插入hGH内含子，具体为在第131nt前插入60nt hGH内含子序列(SEQ ID NO:13)。
pJSD33(SEQ ID NO:22)：(1)在Rep基因的上游插入P5启动子，具体为删除pRC2质粒(SEQ ID NO:7)第121-130nt和第4373-4532nt(Cap后突变P5)，在第131nt前插入wtAAV2 191-310nt(SEQ ID NO:4)；(6)在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子，具体为在第131nt前，P5启动子后插入经过改造的wtAAV2内含子(SEQ ID NO:14)。
pJSD34(SEQ ID NO:23)：(1)在Rep基因的上游插入P5启动子，具体为删除pRC2质粒(SEQ ID NO:7)第121-130nt，在第131nt前先插入wtAAV2 191-310nt(SEQ ID NO:4)；(2)校正位于Cap基因下游的TATA box突变的P5启动子(SEQ ID NO:8)，具体为把第4469ntT突变为A，把第4477-4483nt GGGGGGG突变为TATTTAA，校正后的P5启动子具有如SEQ ID NO:9所示的序列；(6)在Rep基因起始密码子AUG前插入经过改造的wtAAV内含子，具体为在第131nt前，P5启动子后插入经过改造的wtAAV2内含子(SEQ ID NO:14)。
pRC2-64(SEQ ID NO:33)：(2)校正位于Cap基因下游的TATA box突变的P5启动子，具体为把pRC2质粒(SEQ ID NO:7)第4469nt T突变为A，把第4477-4483nt GGGGGGG突变为TATTTAA，校正后的P5启动子具有如SEQ ID NO:9所示的序列；(3)内含子(SEQ ID NO:10)的donor位点处的GTACCA突变为GTACGT，具体为把第1721-1722nt CA突变为GT，突变donor后的内含子具有如SEQ ID NO:11所示的序列；(4)删除掉Cap基因下游的肝脏特异性增强子-启动子序列(LSP，Liver specific enhancer promotor)，具体为删除掉第4271-4344nt(SEQ ID NO:12)。(5)在Rep基因起始密码子AUG前插入hGH内含子，具体为在第131nt前插入60nt hGH内含子序列(SEQ ID NO:13)；(7)在Rep基因上游插入wtAAV7 P5启动子，具体为删除pRC2质粒第121-130nt，在插入的hGH内含子前插入wtAAV7 P5启动子序列(SEQ ID NO:15)。
pRC2-69(SEQ ID NO:34)：(1)在Rep基因的上游插入P5启动子，具体为在pRC2质粒(SEQ ID NO:7)第121nt前插入wtAAV2 191-310nt(SEQ ID NO:4)；(3)内含子(SEQ ID NO:10)的donor位点处的GTACCA突变为GTACGT，具体为把第1721-1722nt CA突变为GT，突变donor后的内含子具有如SEQ ID NO:11所示的序列；(4)删除掉Cap基因下游的肝脏特异性增强子-启动子序列(LSP，Liver specific enhancer promotor)，具体为删除掉第4271-4411nt(SEQ ID NO:35)；(5)在Rep基因起始密码子AUG前插入hGH内含子，具体为在第131nt前插入60nt hGH内含子序列：(SEQ ID NO:13)；(7)在Cap基因下游插入wtAAV7 P5启动子，具体为删除第4368-4532nt(Cap后突变P5，SEQ ID NO:8)，在第4270nt后插入wtAAV7 P5启动子序列(SEQ ID NO:15)。
pHelper-R2(SEQ ID NO:24)：将原RC质粒(SEQ ID NO:7)拆分，把Rep基因放在pHelper上，即在pHelper质粒(SEQ ID NO:44)第2832nt后插入wtAAV2第191-2186nt序列(P5+Rep78/52)(SEQ ID NO:42)；其后加一段SV40polyA序列(SEQ ID NO:36)，维持mRNA作为翻译模板的活性并增加稳定性。其结构示意图如图5所示。
pHelper-R5(SEQ ID NO:68)：总体设计类似pHelper-R2,把Rep基因放在pHelper上，与pHelper-R2的区别在于包含两个Rep基因，一个表达Rep68/40，另一个表达Rep52/40。将原RC质粒(SEQ ID NO:7)拆分，把表达Rep68/40和Rep52/40的序列放在pHelper上，即在pHelper质粒(SEQ ID NO:44)第2832nt后插入wtAAV2第191-1906nt+2228-2309nt序列(P5+Rep68/40，其中1823T-C，1826-1828AAG-CTC失活P40)(SEQ ID NO:69)，再在其后加上wtAAV2第741-2300nt序列(P5+Rep52/40，其中1823T-C，1826-1828AAG-CTC失活P40)(SEQ ID NO:70)，最后加上polyA序列(SEQ ID NO:37)。其结构示意图如图41所示。
pHelper-R10(SEQ ID NO:25)：将原RC质粒(SEQ ID NO:7)拆分，把Rep基因放在pHelper上，即在pHelper质粒(SEQ ID NO:44)第2832nt后插入wtAAV2第191-2300nt序列(P5+Rep78/68/52/40)(SEQ ID NO:41)，后加一段wtAAV2第4411-4460 polyA序列(SEQ ID NO:37)，再在Rep基因起始密码子AUG前插入hGH内含子序列(SEQ ID NO:13)，最后再插入一段P5启动子序列(SEQ ID NO:4)。其结构示意图如图6所示。
pRC-2A(SEQ ID NO:26)：将原RC质粒(SEQ ID NO:7)拆分，把Cap2基因单独放在一个质粒上，质粒序列排布依次为P5启动子(wtAAV2第191-320nt)(SEQ ID NO:38)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)(SEQ ID NO:5)，P40启动子(wtAAV2第1701-2202nt)(SEQ ID NO:6)，其后加上Cap2基因(SEQ ID NO:3)及polyA序列(SEQ ID NO:37)。其结构示意图如图7所示。
pRC-5A(SEQ ID NO:80)：将原pRC5质粒(SEQ ID NO:77)拆分，把Cap5基因单独放在一个质粒上，质粒序列排布依次为P5启动子(wtAAV2第191-320nt)(SEQ ID NO:38)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)(SEQ ID NO:5)，P40启动子(wtAAV2第1701-2202nt)(SEQ ID NO:6)，其后加上Cap5基因(SEQ ID NO:62)和polyA及其邻近序列(SEQ ID NO:101)，最后有一个突变的P5启动子(SEQ ID NO:8)。其结构示意图如图42所示。
pRC-8A(SEQ ID NO:81)：将原pRC8质粒(SEQ ID NO:78)拆分，把Cap8基因单独放在一个质粒上，质粒序列排布依次为P5启动子(wtAAV2第191-320nt)(SEQ ID NO:38)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)(SEQ ID NO:5)，P40启动子(wtAAV2第1701-2202nt)(SEQ ID NO:6)，其后加上Cap8基因(SEQ ID NO:65)及polyA序列(SEQ ID NO:37)。其结构示意图如图43所示。
pRC-9A(SEQ ID NO:27)：将原RC质粒拆分，把Cap9基因单独放在一个质粒上，质粒序列排布依次为P5启动子(wtAAV2第191-320nt)(SEQ ID NO:38)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)(SEQ ID NO:5)，P40启动子(wtAAV2第1701-2202nt)(SEQ ID NO:6)，其后加上Cap9基因(SEQ ID NO:39)及polyA序列(SEQ ID NO:37)。其结构示意图如图8所示。
pRep1(SEQ ID NO:28)：将原pRC2(SEQ ID NO:7)质粒拆分，把wtAAV2(SEQ ID NO:1)中的Rep2基因单独放在一个质粒上，质粒序列排布为P5启动子和Rep基因(wtAAV2第191-2300nt，表达Rep78/68/52/40四种蛋白，SEQ ID NO:41)，其后再加上P5启动子序列(wtAAV2第191-310nt，SEQ ID NO:7)。其结构示意图如图9所示。
pRep3(SEQ ID NO:40)：将原pRC2(SEQ ID NO:7)质粒拆分，把wtAAV2(SEQ ID NO:1)中的Rep2基因单独放在一个质粒上，质粒序列排布为P5启动子和Rep基因(wtAAV2第191-2196nt，表达Rep78/52两种蛋白，SEQ ID NO:43)，其后再加上P5启动子序列(wtAAV2第191-310nt，SEQ ID NO:4)。其结构示意图如图10所示。
pRep5(SEQ ID NO:45)：将原pRC2(SEQ ID NO:7)质粒拆分，把wtAAV2(SEQ ID NO:1)中的Rep2基因单独放在一个质粒上，质粒序列排布为P5启动子和Rep基因(wtAAV2第191-2300nt，表达Rep78/68/52/40四种蛋白，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，再在Rep基因起始密码子AUG前(wtAAV2第321nt)插入hGH内含子序列(SEQ ID NO:13)，最后在第2300nt后加上P5启动子序列(wtAAV2第191-310nt)。其结构示意图如图27所示。
pRep6(SEQ ID NO:46)：将原pRC2(SEQ ID NO:7)质粒拆分，把wtAAV2(SEQ ID NO:1)中的Rep2基因单独放在一个质粒上，质粒序列排布为P5启动子和Rep基因(wtAAV2第191-2300nt，表达Rep78/68/52/40四种蛋白，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，再在Rep基因起始密码子AUG前(wtAAV2第321nt)插入hGH内含子序列(SEQ ID NO:13)，最后在第2300nt后加上polyA序列(wtAAV2第4411-4460nt)。其结构示意图如图28所示。
pHelper-Cap2(SEQ ID NO:47)：把Cap2基因放在pHelper质粒上(SEQ ID NO:44)，在Cap序列排布依次为P5启动子(wtAAV2第191-320nt)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)，P40启动子(wtAAV2第1701-2202nt，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，其后加上Cap2基因(wtAAV2第2203-4410nt)及polyA序列(wtAAV2第4411-4460nt)。其结构示意图如图26所示。
pHelper-Cap5(SEQ ID NO:96)：把Cap5基因放在pHelper质粒上(SEQ ID NO:44)，排布依次为P5启动子(wtAAV2第191-320nt)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)，P40启动子(wtAAV2第1701-2202nt，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，其后加上Cap5基因(SEQ ID NO:62)及polyA序列(SEQ ID NO:37)。其结构示意图如图44所示。
pHelper-Cap8(SEQ ID NO:97)：把Cap8基因放在pHelper质粒上(SEQ ID NO:44)，排布依次为P5启动子(wtAAV2第191-320nt)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)，P40启动子(wtAAV2第1701-2202nt，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，其后加上Cap8基因(SEQ ID NO:65)及polyA序列(SEQ ID NO:37)。其结构示意图如图45所示。
pHelper-Cap9(SEQ ID NO:98)：把Cap9基因放在pHelper质粒上(SEQ ID NO:44)，排布依次为P5启动子(wtAAV2第191-320nt)，P19启动子(wtAAV2第741-890nt，其中第768G突变为T)，P40启动子(wtAAV2第1701-2202nt，其中内含子donor位点处的GTACCA突变为GTACGT，具体为把第1911-1912nt CA突变为GT)，其后加上Cap9基因(SEQ ID NO:66)及polyA序列(SEQ ID NO:37)。其结构示意图如图46所示。
本文所述的质粒还包含以下质粒。
pRC5质粒(SEQ ID NO:77)：编码Rep基因和Cap5基因的传统pRC质粒。
pRC8质粒(SEQ ID NO:78)：编码Rep基因和Cap8基因的传统pRC质粒。
pRC9质粒(SEQ ID NO:79)：编码Rep基因和Cap9基因的传统pRC质粒。
GOI1-C1质粒：把Cap基因放在pAAV-GOI1质粒上，构成pAAV-GOI1-C1质粒，如图11所示(在Cap基因下游插入P5启动子(SEQ ID NO:4)的序列)。
GOI1-C6质粒：设计与GOI1-C1类似，区别为GOI1-C6的Cap基因下游不含有P5启动子。
GOI1-CF质粒：设计与GOI1-C1类似，区别为GOI1-CF的Cap基因下游不含有P5启动子，且不含有f1 ori。
pHelper-C2R4(SEQ ID NO:82)：拆分pRep6质粒(SEQ ID NO:46)上的Rep蛋白，将只表达Rep78/52蛋白的序列(SEQ ID NO:43)放在pHelper-Cap2质粒(SEQ ID NO:47)上，其后加上polyA序列(SEQ ID NO:37)。另外更换了kana抗性序列，由原kana抗性及其邻近序列(SEQ ID NO:99)替换为新kana抗性及其邻近序列(SEQ ID NO:100)。其结构示意图如图47所示。
VA-Cap2(SEQ ID NO:84)：将pHelper-Cap2质粒(SEQ ID NO:47)上的两个Helper辅助基因E4(SEQ ID NO:94)和E2A(SEQ ID NO:93)拆分出去，只保留一个辅助基因VA RNA(SEQ ID NO:95)。其结构示意图如图48所示。
VAE4-Cap2(SEQ ID NO:85)：将pHelper-Cap2质粒(SEQ ID NO:47)上的一个Helper辅助基因E2A(SEQ ID NO:93)拆分出去，只保留两个辅助基因VA RNA(SEQ ID NO:95)和E4(SEQ ID NO:94)。其结构示意图如图49所示。
pRep6-E2A(SEQ ID NO:86)：在pRep6质粒(SEQ ID NO:46)上增加一个Helper辅助基因E2A(SEQ ID NO:93)，其结构示意图如图50所示。
pRep6-E4E2A(SEQ ID NO:87)：在pRep6质粒(SEQ ID NO:46)上增加两个Helper辅助基因E4(SEQ ID NO:94)和E2A(SEQ ID NO:93)，其结构示意图如图51所示。
pRep4(SEQ ID NO:88)：拆分pRep6质粒(SEQ ID NO:46)上的Rep蛋白，只保留表达Rep68/40蛋白的序列(SEQ ID NO:103)，其后再加一个P5启动子(SEQ ID NO:4)。其结构示意图如图52所示。
实施例
实施例1：传统三质粒系统中改造的RC质粒生产的rAAV2-ND4产量比较
此实施例为用T175培养瓶贴壁培养HEK293细胞，经三质粒共转染生产，贴壁细胞37℃，6％CO2条件下培养，转染前按适当密度接种，当细胞汇合度达到80～90％时，即可转染。转染前换新鲜DMEM培养基(无血清)，将转染复合物加入到细胞培养瓶中，放回培养箱。转染72h后，倒掉培养基，往培养瓶中加入细胞裂解缓冲液，在37℃条件下处理2h，待反应结束后，取出培养瓶，向培养瓶中加入高浓度NaCl溶液，然后3000×g离心10min，收集上清。采用荧光定量PCR(qPCR)方法测定细胞裂解上清液中rAAV2物理滴度。
pRC2质粒为安捷伦三质粒转染系统中经过抗性改造(氨苄-卡那)的pAAV-RC质粒，适用于临床生产的血清型2型rAAV的RC质粒。对pRC2质粒进行改造，采用不同的改造方法分别得到pRC2-1、pRC2-4、pRC2-5质粒，与辅助质粒pHelper和载体质粒pAAV-ND4质粒(目的基因ND4)共转染包装rAAV2-ND4(SEQ ID NO:29)。如图12所示，从倍数变化上可以看出pRC2-1、pRC2-4、pRC2-5质粒相对于pRC2质粒，rAAV2-ND4(目的基因ND4)病毒基因组滴度分别提高了1.5、2.6、1.6倍左右，说明第(1)(2)(3)种改造方法均是有效的，且在图13中也得到了验证，将此3种方法组合构造出质粒pRC2-8，发现不管是在T175培养瓶，还是进行规模放大后的10层细胞工厂，产量都得到了进一步提升，且具有倍数叠加效果。当规模为T175培养瓶时，pRC2-4质粒提高了2.8倍，pRC2-8质粒提高了4.4倍。当规模放大到10层细胞工厂时，pRC2-8质粒包装出来的rAAV2-ND4病毒基因组滴度提高了4.8倍。
分别对细胞工厂生产出的rAAV2-ND4细胞裂解上清液进行亲和层析，使用Cytiva填料填充重力柱纯化。用不少于10mL的平衡缓冲液平衡层析柱。平衡完成后，加入10mL澄清过滤收集液，在重力作用下，流经层析柱。上样完成后，用不少于10mL的平衡液冲洗层析柱。然后在重力柱下方放置样品收集容器。先用1mL亲和洗脱液洗脱样品，弃废液，再用2mL亲和洗脱液洗脱，收集层析洗脱液，用中和缓冲液调节pH至7.5左右。将收集的亲和层析洗脱液用SEC-MALS法检测实心率。检测结果发现在包装过程中，pRC2-4和pRC2-8均比pRC2包装效率高，各自实心率都有所提高。如图14所示，对于rAAV2-ND4，pRC2-4和pRC2-8质粒包装出的病毒实心率分别是0.42和0.36，而原pRC2质粒包装出的病毒实心率为0.25。
实施例2：传统三质粒系统中改造的RC质粒生产的rAAV2-OPA1产量比较
选择pRC2-4和pRC2-8质粒贴壁生产另一种病毒载体rAAV2-OPA1(所用载体质粒为pAAV-OPA1(SEQ ID NO:30)，目的基因OPA1)，实验方法同实施例1，检测方法为数字微滴PCR(ddPCR)。如图15所示，相较于原来的pRC2质粒，病毒基因组滴度分别约提高了2.8倍和4.2倍(在细胞工厂中)，由此可见经过改造的pRC2-4和pRC2-8质粒也适用于其他外源基因的病毒包装。而且不管在培养瓶(pRC2-8质粒的病毒基因组滴度提高了4.0倍)，还是在细胞工厂上均具有同样的提高效果，说明规模放大没有问题。同样的，对于病毒包装效率，如图16所示，原始RC质粒、pRC2-4质粒和pRC2-8质粒包装出的病毒实心率分别是0.28、0.34和0.31，也都有所提升。
实施例3：传统三质粒系统中改造的RC质粒悬浮生产rAAV2-GOI1
方案一：pRC2-4和pRC2-8不仅可以应用于贴壁生产，还可以应用在悬浮培养系统中。质粒用量为0.5～1.5μg/106cells，按一定比例再加入转染试剂配制，形成转染复合物后加入到悬浮无血清培养的293F细胞中，在摇床中37℃，8％CO2，80％湿度，110rpm条件下培养。如图17所示，在摇瓶中三质粒共转染生产rAAV2-GOI1(GOI1是目的基因代码)，经ddPCR检测pRC2-4和pRC2-8生产的rAAV2-GOI1基因组滴度也均比pRC2高，分别提高了2倍和2.6倍。
方案二：在质粒改造过程中，发现原pRC2质粒上有可能导致安全性问题的元件-肝脏特异性增强子-启动子LSP，我们采用了方法(4)在pRC2-8基础上删除了Cap基因下游的包括LSP在内的74nt序列，构造了质粒pRC2-53。又以pRC2-53质粒为模板，采用方法(5)在Rep基因起始密码子AUG前插入hGH内含子，构造了质粒pRC2-58。分别用这2个质粒包装rAAV2-GOI1，细胞裂解液的病毒基因组滴度各自提高了3.7和4.3倍，如图18所示。再将细胞裂解液经过亲和层析纯化和除菌过滤后的样品与Ad5wt共同感染293T细胞，经过三轮感染后qPCR法检测rAAV原液中存在的rcAAV数量(CT值代表循环次数，CT值越低，表示感染滴度越高，即rcAAV含量越高)。
将细胞接种到T75/T175培养瓶中，在＞80％汇合度下，消化细胞后接种细胞于12孔板中。第二天，以5×103的感染复数(MOI)和待测试的rAAV2产品和辅助野生型腺病毒5型(“Ad5wt”)共转染细胞，每个rAAV2待测样品设置3个重复孔。孵育2天后，通过机械分离收获细胞。将第一轮每孔中的细胞通过3个冻融循环(-80℃冷冻和37℃解冻)进行裂解，并通过在56℃下孵育30分钟以灭活Ad5wt，以2500g离心10分钟去除细胞碎片。用每孔中第一次收获的三分之一裂解物在有Ad5wt的情况下共感染新的12孔板，作为第二轮感染，剩余裂解物-80℃保存备用(储存以备需要调查分析)，重复整个过程以收集来自第二次收获的样品。用来自第二次收获的裂解物在有Ad5wt的情况下转导接种用于第三次转导的细胞，并重复整个过程，以收集来自第三次收获的样品。
使用Proteinase K处理第二次扩增收获的样品以从裂解液中包装的AAV颗粒中分离DNA，随后在90℃孵育裂解物10分钟，使Proteinase K失活，然后以EASY Dilution稀释获得qPCR待测样品。
将体积为5μL的qPCR待测样品添加到20μLqPCR反应中，使用特异性针对Rep编码序列(“Rep”)的引物扩增qPCR待测样品，以确认在rAAV2/Ad5wt感染的孔中是否存在含有Rep序列的AAV。同时检测含Rep序列的标准品和qPCR待测样品，反应结束后，设定阈值，比较标准品的检测CT值与qPCR待测样品CT值。若qPCR待测样品CT值小于标准品CT值，则表明样品中有rcAAV检出(CT值越小表明检出的rcAAV水平越高)。
阳性对照使用的是野生型AAV2(wtAAV2)，其结构示意图如图4所示；阴性对照由未感染的细胞(有腺病毒存在)组成，它们与rAAV2产品按同样方式进行测试。
如表1所示，每个样品做了三个复孔的感染，pRC2-53质粒在1E+08水平检测出的CT值分别是25.306、30.363、30.062，pRC2-58质粒在1E+07水平检测出的CT值分别是32.727、37.535、28.820，UND代表未检测到。表明rcAAV残留不多，此种改造方法不仅可以提高产量，还可以降低rcAAV。
表1：pRC2-53和pRC2-58质粒包装系统rcAAV检测结果
方案三：pJSD33质粒删除掉原pRC2质粒上Cap基因后突变的P5启动子且采用方法(1)在Rep基因上游插入wtAAV2P5启动子，然后采用方法(6)在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子，pJSD34质粒则在pJSD33质粒基础上又在Cap基因后插入P5启动子(相当于方法(2))构建而成，分别三质粒共转染悬浮培养的VPC2.0细胞，规模为125mL培养瓶，细胞裂解液滴度经ddPCR检测，结果如图19所示，较原pRC2质粒分别提高了1.6和2.6倍。
方案四：pRC2-64质粒采用方法(7)把pRC2-58质粒Rep基因前的wtAAV2P5启动子替换为wtAAV7 P5启动子，pRC2-69质粒则是把Cap基因后的P5启动子替换为wtAAV7 P5启动子，实验方法和检测方法同实施例3，发现这2个质粒包装出来的rAAV2-anti-VEGF，病毒基因组滴度分别提高了4.5和4.6倍，如图20所示。说明第(7)种改造方法也是有效的。
实施例4：新型三质粒系统I悬浮生产rAAV-GOI1和rAAV2-EGFP
将病毒包装必需元件适当拆分至不同的质粒中，降低质粒之间、质粒与包装细胞及野生病毒序列之间的同源性。所以把包装质粒RC拆分开，把Rep基因放在pHelper质粒上，构建出pHelper-Rep质粒，Cap基因单独一个质粒，由此和目的基因所在载体质粒组成新型的三质粒系统，此种系统命名为新型三质粒系统I(如图2A所示)。本文提供了2种pHelper-Rep质粒，pHelper-R2和pHelper-R10，另外提供了2种Cap质粒，一种是血清型2型的pRC-2A，另一种是血清型9型的pRC-9A。如表2所示，分别用pHelper-R2和pRC-9A转染293F细胞包装rAAV9-GOI1，pHelper-R10和pRC-2A转染VPC2.0细胞包装rAAV2-GOI1，细胞裂解液滴度分别为1.80E+11vg/mL和1.90E+11vg/mL，经亲和层析纯化后检测rcAAV残留，CT值均较高，说明rcAAV含量很少，此种新型的三质粒系统I既可以提高产量，又可以减少rcAAV。
在另一组实验中，如图34A和图34B显示，基于pHelper-R2和pHelper-R10的新型三质粒系统I生产的AAV的载体产量和病毒实心率要显著优于基于pHelper-R5的对照三质粒系统以及传统三质粒系统。
用pHelper-R10和pRC-2A转染VPC2.0细胞包装报告基因EGFP(所用载体质粒为pAAV-EGFP，SEQ ID NO:32)，同样可以提高病毒基因组滴度，如图21所示，与原传统三质粒系统相比，新型的三质粒系统I可以提高产量至9.6倍。在另一组实验中，将基于pHelper-R10的编码不同AAV血清型的三质粒系统I(分别使用pRC-2A，pRC-5A，pRC-8A，和pRC-9A)与相应的编码不同血清型的传统三质粒系统生产编码EGFP的AAV2、AAV5、AAV8、和AAV9，各个系统产生的AAV滴度如图34C所示，证明新型三质粒系统I在生产各种不同血清型的AAV时的产量都比传统三质粒系统要好。另外，在对实心率的检测中，也显示新型三质粒系统I生产的AAV2的实心率(0.265)和AAV8的实心率(0.307)要显著高于传统三质粒系统生产的相应AAV2的实心率(0.147)和AAV8的实心率(0.143)。因此，新型三质粒系统I相较于传统三质粒系统更适用于包装多种血清型病毒。
表2：新型三质粒系统I包装rAAV-GOI1的rcAAV检测结果
实施例5：新型三质粒系统I悬浮生产rAAV2-hNRF2及三质粒比例优化
用pHelper-R10和pRC-2A转染293F细胞包装另一种病毒rAAV2-hNRF2(所用载体质粒为pAAV-hNRF2,SEQ ID NO:31)，并进行三质粒比例优化测试，当pRC-2A：pHelper-R10：pAAV-hNRF2的比例分别为1:1:1，1:1:0.4和3:1:0.3时，病毒产量分别是2.30E+11vg/mL、3.40E+11vg/mL和4.70E+11vg/mL，可见经过三质粒比例优化，病毒基因组滴度可以进一步得到提高。当用细胞裂解液滴度最高的样品(4.70E+11vg/mL)，经亲和层析洗脱后，结果见下表3和图22，检测在1.00E+08水平的CT值复孔一为38.02，复孔二和复孔三则未检测到，说明新型三质粒系统I也适用于包装其他外源基因，提高AAV病毒产量，rcAAV含量也极少。
表3：新型三质粒系统I包装rAAV2-hNRF2的rcAAV检测结果
实施例6：新型三质粒系统II悬浮生产rAAV2-GOI1
拆分包装质粒RC，也可以把Cap基因放在目的基因所在载体质粒上，构建出pVector-Cap质粒，Rep基因单独一个质粒，由此和pHelper质粒组成新型的三质粒系统，此种系统命名为新型三质粒系统II(如图2B所示)。本文提供两种Rep质粒pRep1和pRep3，另外把Cap基因放在pAAV-GOI1质粒上，构成pAAV-GOI1-C1质粒，如图11所示(在Cap基因下游插入SEQ ID NO:4的序列)，其中GOI1为目的基因1，编码一个IgG抗体Fab片段。与pHelper质粒共转染293F细胞，如图23所示，包出的rAAV2-GOI1病毒基因组滴度分别较原三质粒系统提高了3.7和2.3倍。
在另一组实验中，比较由pHelper+各种pRep+GOI1-C1组成的新型三质粒系统II与pHelper+各种pRep+GOI1-CF组成的三质粒系统生产的AAV载体相对产量，所用的pRep质粒分别为pRep1，pRep5，或pRep6。根据图35A所显示的结果，使用GOI1-C1的新型三质粒系统II的AAV载体产量要优于使用GOI1-CF的三质粒系统。
将不同比例的pHelper质粒、pRep1质粒、pEGFP-Cap2质粒，在各种不同的质粒与细胞相对比例、以及不同的转染试剂与质粒相对比例的条件下表达编码EGFP的AAV。具体地，用pHelper、pRep6和pEGFP-Cap2质粒进行转染条件优化来得到更高滴度的rAAV2-EGFP，质粒用量设置两个条件，分别是0.5μg/106cells和1μg/106cells，转染试剂和质粒的比例(V/W)也是两个参数0.75和1.5，另外三质粒pHelper:pRep6:pEGFP-Cap2比例有四组，分别是1：1：0.4、1：3：0.4、1：1：1和1：3：1，共16个转染条件，每个条件2个重复，转染前48h稀释细胞密度到0.9E+106cells/mL，转染三天后裂解收获病毒。其结果(图35B)显示，当pHelper质粒、pRep1质粒、pEGFP-Cap质粒的相对比率为1:1:0.4或1:1:1时，AAV载体的产量较高；而当这三个质粒的相对比率为1:3:1时，AAV载体的产量相对低一些。
在另一组实验中，将基于pHelper、pRep6、以及编码不同血清型Cap蛋白的pEGFP-Cap质粒与相应的传统三质粒系统生产编码EGFP的AAV2、AAV5、AAV8、和AAV9，所使用的各个pEGFP-Cap质粒分别编码EGFP和Cap2(pEGFP-Cap2；SEQ ID NO:72)，EGFP和Cap5(pEGFP-Cap5；SEQ ID NO:74)，EGFP和Cap8(pEGFP-Cap8；SEQ ID NO:75)，EGFP和Cap9(pEGFP-Cap9；SEQ ID NO:76)。各个系统产生的AAV滴度如图35C所示，证明新型三质粒系统II在生产各种不同血清型的AAV时的产量都比传统三质粒系统要好。另外，在对实心率的检测中，也显示新型三质粒系统II生产的AAV2、AAV8和AAV9的实心率达到了0.23-0.34之间，远高于传统三质粒系统的相应实心率。因此新型三质粒系统II相较于传统三质粒系统更适用于包装多种血清型病毒。
实施例7：二质粒系统生产rAAV2-GOI1
把Cap基因放在目的基因所在载体质粒上，构建出pVector-Cap质粒，Rep基因放在pHelper上构成pHelper-Rep质粒，这样拆分RC质粒后就组成更为简易的二质粒系统，如图3所示，此种排列更为经济，能够节约不少质粒生产成本。还可将提高滴度的改造方法分别使用在两个质粒上，产量可以得到进一步提升。本实施例使用pHelper-R2和pAAV-GOI1-C1两种质粒转染293F细胞，包装出的rAAV2-GOI1病毒基因组滴度为5.50E+10vg/mL，经过rcAAV检测，如表4A所示，CT值分别为36.299、36.995、36.659。说明此二质粒系统可以提高rAAV载体的安全性和质量，还降低了rAAV制造的成本。
表4A：二质粒系统包装rAAV2-GOI1的rcAAV检测结果
进一步的，检测了多种不同设计的二质粒系统的包装rAAV2-GOI1的各项生产指标。除了上述的pHelper-R2+GOI1-C1二质粒系统，还检测了pHelper-R5+GOI1-CF、pHelper-R2+GOI1-C6、pHelper-R10+GOI1-C1和pHelper-R10+GOI1-C6这四个二质粒系统。两质粒摩尔比均为1：1，转染悬浮培养的VPC2.0细胞。除了检测病毒基因组滴度，另外将样品经过重力柱亲和纯化后按一定比例稀释，添加到20μL qPCR反应中，使用特异性探针引物对质粒上存在的Kana抗性序列扩增，测出来的浓度相对于纯化后的病毒基因组滴度来确认在包装过程中产生的含有kana抗性序列的质粒残留量，其结果显示，以pHelper-R2或pHelper-R10以及GOI1-C1或GOI1-C6组成的各个二质粒系统，其在病毒基因组滴度(图32A)，Cap残留(图32B)，质粒残留(图32C)等各项指标上都显著优于以pHelper-R5+GOI1-CF组成的二质粒系统。rcAAV检测结果如表4B所示，对照系统三个复孔的CT值分别为18.247、15.047、18.195，远低于其他二质粒系统，说明其含量很高，超过1rcAAV/1×108vg，而其他二质粒系统的CT值很高，说明rcAAV残留很少。结果表明，本文所述的多个二质粒系统不仅可以提高rAAV2-GOI1病毒基因组滴度，而且产品相关杂质均少于对照二质粒系统，安全性显著提高。
表4B：不同二质粒系统包装rAAV2-GOI1的rcAAV检测结果
*UND表示无法检出
另外进行了二质粒系统悬浮生产rAAV2-EGFP工艺优化的对比。将不同比例的表达Rep的pHelper质粒、表达Cap的GOI质粒，在各种不同的质粒与细胞相对比例，以及不同的转染试剂与质粒相对比例的条件下进行AAV表达。具体地，分别用二质粒系统pHelper-R5+pEGFP-Cap2F(对照；SEQ ID NO:73；不包含f1 ori)和pHelper-R10+pEGFP-Cap2包装rAAV2-EGFP，通过不同转染条件优化来得到更高滴度。质粒用量设置两个条件，分别是0.5μg/106cells和1μg/106cells，转染试剂和质粒的比例(V/W)也是两个参数0.75和1.5，质粒摩尔比有3种为1:1、1:3和3:1，由此形成12个工艺条件，如图33所示。可以发现，不管在哪个工艺参数下，二质粒系统pHelper-R10+pEGFP-Cap2包装出的病毒基因组滴度都比对照二质粒系统pHelper-R5+pEGFP-Cap2F高，特别是当质粒用量为0.5μg/106cells，转染试剂/质粒＝1.5，pHelper-R10：pEGFP-Cap2＝1：1或1：3时，滴度较高，能达到8E+11vg/mL。而对照二质粒系统需要质粒用量为1μg/106cells时，滴度才能达到2E+11vg/mL，可见本文设计的二质粒系统是优于对照二质粒系统的。另外，当表达Rep的pHelper质粒与表达Cap的GOI质粒的数量比率为1:1至1:3时，所得的AAV的表达量要高于两者的比率为3:1时的表达量。
实施例8：新型三质粒系统III和传统三质粒系统包装的rAAV病毒基因组滴度和实心率的比较
用125mL摇瓶悬浮培养HEK293细胞，培养条件为37℃，8％CO2，80％湿度，110rpm。转染前按适当密度接种，稀释48h后转染。转染时的质粒用量为0.5～1μg/106cells，三质粒按一定比例添加，再加入转染试剂配制，形成转染复合物后加入到无血清培养的VPC2.0细胞中。转染72h后，加入高倍数的细胞裂解缓冲液，在37℃条件下处理2h，待反应结束后，取出摇瓶，向摇瓶中加入高浓度NaCl溶液，然后3000×g离心10min，收集上清。采用数字PCR(dPCR)方法测定细胞裂解上清液中rAAV2物理滴度。
如图29所示，pRC2质粒为安捷伦传统三质粒转染系统中经过抗性改造(氨苄-卡那)的pRC2质粒，适用于临床生产的血清型2型rAAV的RC质粒。对pRC2质粒进行拆分，将Cap基因放在pHelper质粒上组合成pHelper-Cap2质粒，而Rep基因置于一个单独的质粒上，采用不同的修饰方法分别得到pRep1、pRep3、pRep5、pRep6质粒。pHelper-Cap2质粒和不同的pRep质粒包装rAAV2-GOI1(目的基因GOI1)，从病毒基因组滴度上可以看出新型三质粒系统III中pRep1、pRep3、pRep5、pRep6质粒包装出的病毒基因组滴度有所差别，分别是1.1E+11、6.85E+10、1.4E+11、2.4E+11vg/mL，但都比传统三质粒系统包装出的滴度(4.30E+10vg/mL)高，说明新型三质粒系统III较传统三质粒系统更优，可以得到更高的病毒产量。
对rAAV2-GOI1细胞裂解上清液进行亲和层析，使用Cytiva填料填装重力柱。用不少于10mL的平衡缓冲液平衡层析柱。平衡完成后，加入10mL澄清过滤收集液，在重力作用下，流经层析柱。上样完成后，用不少于10mL的平衡液冲洗层析柱。然后在重力柱下方放置样品收集容器。先用1mL亲和洗脱液洗脱样品，弃废液，再用2mL亲和洗脱液洗脱，收集层析洗脱液(NAE)，用中和缓冲液调节pH至7.5左右。将收集的亲和层析洗脱液用SEC-MALS法检测实心率。检测结果发现在包装过程中，使用pRep6质粒的新型三质粒系统III中包装效率比使用pRC2质粒的传统三质粒系统包装效率高。如图31所示，对于rAAV2-GOI1，新型三质粒系统III包装出的病毒实心率是0.116，而传统三质粒系统包装出的病毒实心率仅为0.051，实心率得到了较高的提升。
在另一组实验中，以编码不同血清型Cap蛋白的pHelper-Cap质粒，pRep6质粒、以及编码EGFP的重组AAV质粒组成的新型三质粒系统III，以及相应的传统三质粒系统生产编码EGFP的AAV2、AAV5、AAV8、和AAV9，各个系统产生的不同血清型的AAV基因组滴度如图36A所示，实心率如图36B所示。其结果证明新型三质粒系统III在生产各种不同血清型的AAV时的产量与实心率基本都比传统三质粒系统要好。因此，新型三质粒系统III相较于传统三质粒系统更适用于包装多种血清型病毒。
实施例9：新型三质粒系统III包装的rAAV2-GOI1工艺优化数据
用pHelper-Cap2、pRep5和pAAV-GOI1质粒进行转染条件优化来得到更高滴度的rAAV2-GOI1，质粒用量设置两个条件，分别是0.5μg/106cells和1μg/106cells，转染试剂和质粒的比例(V/W)也是两个参数0.75和1.5，由此形成四个条件，如图30右侧图例所示，前一个数值表示质粒量，后一数值表示转染试剂量。另外三质粒pHelper-Cap2:pRep5:pAAV-GOI1比例也有四组，分别是1:1:0.4、3:1:0.4、1:1:1和3:1:1，而转染时的细胞密度设置低密度(2.7E+06cells/mL)和高密度(3.8E+06cells/mL)组，如横坐标所示，有8个大组，共32组转染条件。从病毒基因组滴度来看，质粒量为0.5μg/106cells，转染试剂和质粒的比例(V/W)为1.5时，产量更高。三质粒比例为3:1:0.4和3:1:1时比1:1:0.4和1:1:1时滴度更高，表明pHelper-Cap2投入量较多时，效果较好。
在另一组实验中，以不同比例的pHelper-Cap2质粒、pRep6质粒、pAAV-EGFP质粒，在各种不同的质粒与细胞相对比例、以及不同的转染试剂与质粒相对比例的条件下表达编码EGFP的AAV，进行转染条件优化来得到更高滴度的rAAV2-EGFP。具体地，质粒用量设置两个条件，分别是0.5μg/106cells和1μg/106cells，转染试剂和质粒的比例(V/W)也是两个参数0.75和1.5，另外三质粒pHelper-Cap2:pRep6:pAAV-EGFP比例有五组，分别是1：1：0.4、3：1：0.4、1：1：1、3：1：1、和1：0.6：0.3，共20个转染条件，每个条件2个重复，转染前48h稀释细胞密度到0.9E+06cells/mL，转染三天后裂解收获病毒。其结果(图37)显示，当pRep6质粒和编码重组基因的pAAV-EGFP的比例为1:0.4或0.6:0.3时，新型三质粒系统III的AAV表达量相对较高，而pRep6质粒和编码重组基因的pAAV-EGFP的比例1:1时，AAV表达量相对较低。在各个测试条件下，pHelper-Cap2:pRep6:pAAV-EGFP质粒比率为约1:0.6:0.3时，AAV载体基因组滴度最高。三质粒比率为1：1：0.4和3：1：0.4时滴度也很高。
实施例10：新型三质粒系统III和传统三质粒系统包装的rAAV2-GOI1检测rcAAV残留比较
将细胞裂解液经过亲和层析纯化和除菌过滤后的样品与Ad5wt共同感染293T细胞，经过三轮感染后dPCR法检测rAAV原液中存在的rcAAV数量(CT值代表循环次数，CT值越低，表示感染滴度越高，即rcAAV含量越高)。
将细胞接种到T75/T175培养瓶中，在＞80％汇合度下，消化细胞后接种细胞于12孔板中。第二天，以5×103的感染复数(MOI)和待测试的rAAV2产品和辅助野生型腺病毒5型(“Ad5wt”)共转染细胞，每个rAAV2待测样品设置3个重复孔。孵育2天后，通过机械分离收获细胞。将第一轮每孔中的细胞通过3个冻融循环(-80℃冷冻和37℃解冻)进行裂解，并通过在56℃下孵育30分钟以灭活Ad5wt，以2500g离心10分钟去除细胞碎片。用每孔中第一次收获的三分之一裂解物在有Ad5wt的情况下共感染新的12孔板，作为第二轮感染，剩余裂解物-80℃保存备用(储存以备需要调查分析)，重复整个过程以收集来自第二次收获的样品。用来自第二次收获的裂解物在有Ad5wt的情况下转导接种用于第三次转导的细胞，并重复整个过程，以收集来自第三次收获的样品。
使用Proteinase K处理第二次扩增收获的样品以从裂解液中包装的AAV颗粒中分离DNA，随后在90℃孵育裂解物10分钟，使Proteinase K失活，然后以EASY Dilution稀释获得qPCR待测样品。
将体积为5μL的dPCR待测样品添加到20μL qPCR反应中，使用特异性针对Rep编码序列(“Rep”)的引物扩增qPCR待测样品，以确认在rAAV2/Ad5wt感染的孔中是否存在含有Rep序列的AAV。同时检测含Rep序列的标准品和qPCR待测样品，反应结束后，设定阈值，比较标准品的检测CT值与dPCR待测样品CT值。若qPCR待测样品CT值小于标准品CT值，则表明样品中有rcAAV检出(CT值越小表明检出的rcAAV水平越高)。
阳性对照使用的是野生型AAV2(wtAAV2)，其结构示意图如图25所示；阴性对照由未感染的细胞(有腺病毒存在)组成，它们与rAAV2产品按同样方式进行测试。
如表5所示，每个样品做了三个复孔的感染，新型三质粒系统III中的pRep5质粒包装出的样品在1E+08水平检测出的CT值分别是32.899、34.507、UND，UND.代表未检测到。表6中pRep6质粒包装出的样品检测出的CT值分别是38.631、36.039、37.554，表明rcAAV残留很少，<1rcAAV/1×108vg。而传统三质粒系统包装出的病毒中，如表7所示，复孔二检测出的CT值为29.632，表明残留量>1rcAAV/1×108vg。所以新型三质粒系统III较传统三质粒系统包装出的rAAV2-GOI1中rcAAV量较少，纯度更高。
表5：新型三质粒系统III包装rAAV2-GOI1的rcAAV检测结果1
表6：新型三质粒系统III包装rAAV2-GOI1的rcAAV检测结果2
表7：传统三质粒系统包装rAAV2-GOI1的rcAAV检测结果
实施例11：新型三质粒系统III和传统三质粒系统包装的rAAV2-GOI1检测Cap残留比较
将待测样品按一定比例稀释后，添加到20μL qPCR反应中，使用特异性探针对Cap编码序列(“Cap”)的引物扩增q样品，以确认在包装过程中是否存在含有Cap序列的相关杂质残留。如表8所示，分别用pRep5质粒和pRep6质粒与pHelper-Cap2包装的rAAV2-GOI1病毒载体中Cap残留结果和传统三质粒系统相比相当甚至更低，由此表明新型三质粒包装系统较为安全。
表8：新型三质粒系统III和传统三质粒系统包装rAAV2-GOI1的Cap残留检测结果
实施例12：对新型三质粒系统III的Rep基因和辅助基因部分的改造
在一组实验中，对Rep基因进行拆分。具体的，将pRep6质粒中的Rep基因改造为仅编码Rep68/40蛋白(命名为pRep4,SEQ ID NO:88)，而将编码Rep78/52蛋白的基因置于pHelper-Cap2中(形成pHelper-C2R4,SEQ ID NO:82)、或置于编码EGFP的重组AAV质粒中(形成pAAV-EGFP-R2,SEQ ID NO:83)，并比较了两个改造后的质粒系统(一个是pHelper-C2R4+pRep4+pAAV-EGFP；另一个是pHelper-Cap2+pRep4+pAAV-EGFP-R2)与改造前的新型三质粒系统III体系(pHelper-Cap2+pRep6+pAAV-EGFP)的AAV产量。如图38A的结果显示，改造后的新型三质粒系统III的AAV产量并未降低，将编码Rep78/52蛋白的基因置于编码目的基因的重组AAV质粒中的三质粒系统的AAV产量甚至进一步上升。另外实心率结果如图38B所示，也有部分提高，Cap残留和rcAAV检测结果如表9和表10所示，含量都十分低，所以将Rep基因拆分后形成的新型三质粒系统III扩展系统I作为rAAV包装体系效果也很好。
表9：新型三质粒系统III扩展系统I包装rAAV2-EGFP的Cap残留检测结果
表10：新型三质粒系统III扩展系统I包装rAAV2-EGFP的rcAAV检测结果
在另一组实验中，将pHelper-Cap2上的一个或两个辅助基因拆分到pRep6上。具体的，将原pHelper-Cap2上的E4、E2A辅助基因移至pRep6(形成pRep6-E4E2A,SEQ ID NO:87)，改造为VA-Cap2(SEQ ID NO:84)+pRep6-E4E2A+pAAV-EGFP三质粒系统；或将pHelper-Cap2上的E2A辅助基因移至pRep6(形成pRep6-E2A,SEQ ID NO:86)，改造为VAE4-Cap2(SEQ ID NO:85)+pRep6-E2A+pAAV-EGFP三质粒系统。如图39A和图39B结果显示，改造后的新型三质粒系统III的AAV产量并未降低，将E2A辅助基因移至pRep6质粒后的三质粒系统的AAV产量和实心率甚至有显著的提升，滴度提高了1.5倍，实心率近2倍。另外Cap残留和rcAAV检测结果如表11和表12所示，含量都十分低，因此将辅助基因拆分后形成的新型三质粒系统III扩展系统也是一种有效的rAAV包装系统。
表11：新型三质粒系统III扩展系统II包装rAAV2-EGFP的Cap残留检测结果
表12：新型三质粒系统III扩展系统II包装rAAV2-EGFP的rcAAV检测结果
实施例13：新型三质粒系统III和传统三质粒系统包装rAAV-scEGFP多血清型比较
本实施例检测了新型三质粒系统III，以及传统三质粒系统，在表达不同血清型的包装自互补型EGFP基因(scEGFP)的AAV的效果。检测的AAV血清型包含AAV2、AAV5、AAV8、以及AAV9。具体的，对于各个AAV血清型所使用的新型三质粒系统III以及传统三质粒系统包括：pHelper-Cap2+pRep6+pAAV-scEGFP对比pHelper+pRC2+pAAV-scEGFP包装rAAV2-scEGFP，pHelper-Cap5+pRep6+pAAV-scEGFP对比pHelper+pRC5+pAAV-scEGFP包装rAAV5-sc EGFP，pHelper-Cap8+pRep6+pAAV-scEGFP对比pHelper+pRC8+pAAV-scEGFP包装rAAV8-scEGFP，pHelper-Cap9+pRep6+pAAV-scEGFP对比pHelper+pRC9+pAAV-scEGFP包装rAAV9-scEGFP，
[根据细则91更正 23.04.2024]
如图40A所示，对于病毒基因组滴度，新型三质粒系统III相对于传统三质粒系统分别提高了3.3、3.2、3.4和4.2倍。对于实心率，图40B显示新型三质粒系统III包装出的2型、5型、8型、9型rAAV-sc EGFP的实心率是传统三质粒系统的对应血清型的4到8倍左右。另外，对于新型三质粒系统III包装的rcAAV残留的检测结果(表13)显示，在4E+08水平并未检测到。因此，新型三质粒系统III相较于传统三质粒系统更适用于包装多种血清型和目的基因病毒，残留杂质少，可扩展性强。
表13：新型三质粒系统III包装rAAV-sc EGFP的rcAAV检测结果

Claims (150)

  1. 一种重组核酸，包含腺相关病毒(AAV)的Rep基因和/或Cap基因，且有如下的一种或多种特征：
    (1)在所述Rep基因的上游和/或所述Cap基因的下游含有一个P5启动子；
    (2)位于所述Cap基因下游的原有P5启动子中的TATA box突变被修正，或在所述Cap基因下游插入一个所述P5启动子，与所述Cap基因可操作地连接；
    (3)所述腺相关病毒的病毒基因组原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:7的第1717-1722核苷酸位点；
    (4)所述Cap基因下游原有的肝脏特异性增强子-启动子序列被删除；
    (5)在所述Rep基因起始密码子前含有一个hGH内含子；
    (6)在所述Rep基因起始密码子前含有一个腺相关病毒基因组的内含子；
    和/或
    (7)在所述Rep基因上游或在所述Cap基因下游含有一个非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
  2. 根据权利要求1所述的重组核酸，其特征在于，包含特征(1)，且位于所述Rep基因上游的所述P5启动子的插入位置紧接在相当于SEQ ID NO:7的腺相关病毒基因组的120bp的核苷酸位点后，与所述Rep基因可操作地连接。
  3. 根据权利要求1-2任一项所述的重组核酸，其特征在于，在所述Cap基因下游插入一个P5启动子，与所述Cap基因可操作地连接。
  4. 根据权利要求2-3任一项所述的重组核酸，其特征在于，所述P5启动子与所述Rep基因或所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
  5. 根据权利要求1-4任一项所述的重组核酸，其特征在于，所述P5启动子的序列与SEQ ID NO:4有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  6. 根据权利要求1-5任一项所述的重组核酸，其特征在于，包含特征(2)，且所述原有P5启动子的TATA box突变的修正包含将相当于SEQ ID NO:7第4469位核苷酸的T突变为A，和/或将相当于SEQ ID NO:7第4477-4483位核苷酸的GGGGGGG突变为TATTTAA。
  7. 根据权利要求6所述的重组核酸，其特征在于，所述原有P5启动子修正后的序列与SEQ ID NO:9有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  8. 根据权利要求1-7任一项所述的重组核酸，其特征在于，包含特征(3)，且所述donor位点的突变包含将相当于SEQ ID NO:7第1721-1722位核苷酸的CA突变为GT，或相当于SEQ ID NO:7第1717-1722位核苷酸的donor位点序列突变为GTACGT。
  9. 根据权利要求8所述的重组核酸，其特征在于，突变后的内含子序列与SEQ ID NO:11有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  10. 根据权利要求1-9任一项所述的重组核酸，其特征在于，包含特征(4)，且所述肝脏特异性增强子-启动子序列被删除。
  11. 根据权利要求1-10任一项所述的重组核酸，其特征在于，所述肝脏特异性增强子-启动子序列与SEQ ID NO:12或SEQ ID NO:35有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  12. 根据权利要求1-11任一项所述的重组核酸，其特征在于，包含特征(5)，且所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  13. 根据权利要求12任一项所述的重组核酸，其特征在于，所述hGH内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。
  14. 根据权利要求13所述的重组核酸，其特征在于，所述hGH内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
  15. 根据权利要求1-14任一项所述的重组核酸，其特征在于，所述hGH内含子与所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
  16. 根据权利要求15所述的重组核酸，其特征在于，所述hGH内含子与所述Rep基因起始密码子之间没有间隔的核苷酸。
  17. 根据权利要求1-16任一项所述的重组核酸，其特征在于，包含特征(6)，且所述腺相关病毒基因组的内含子的序列与SEQ ID NO:14有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  18. 根据权利要求1-17任一项所述的重组核酸，其特征在于，所述腺相关病毒基因组的内含子位于特征(1)所述P5启动子的下游，并位于特征(1)所述Rep基因的上游。
  19. 根据权利要求18所述的重组核酸，其特征在于，所述腺相关病毒基因组的内含子和特征(1)所述P5启动子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
  20. 根据权利要求1-19任一项所述的重组核酸，其特征在于，所述腺相关病毒基因组的内含子和所述Rep基因起始密码子之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
  21. 根据权利要求1-20任一项所述的重组核酸，其特征在于，包含特征(7)，且所述非亲本血清型启动子的序列与SEQ ID NO:15有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  22. 根据权利要求1-21任一项所述的重组核酸，其特征在于，(i)所述非亲本血清型启动子位于所述Cap基因的下游，或(ii)所述非亲本血清型启动子位于所述Rep基因上游，且所述所述非亲本血清型启动子位于所述hGH内含子的上游。
  23. 根据权利要求22所述的重组核酸，其特征在于，所述非亲本血清型启动子位于相当于SEQ ID NO:7第4270-4271位核苷酸之间的位置。
  24. 根据权利要求1-23任一项所述的重组核酸，其特征在于，所述重组核酸不含有相当于SEQ ID NO:7第4368-4532位核苷酸的序列，或不含有相当于SEQ ID NO:7第4373-4532位核苷酸的序列。
  25. 根据权利要求1-24任一项所述的重组核酸，其特征在于，所述重组核酸不含有相当于SEQ ID NO:7第121-130位核苷酸的序列。
  26. 根据权利要求1-25任一项所述的重组核酸，其特征在于：
    (i)所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9；更优选地，所述AAV的血清型是AAV2；
    (ii)所述Rep基因的序列与SEQ ID NO:2有至少90％，至少95％，至少98％，至少99％，或100％的相同性；和/或
    (iii)所述Cap基因的序列与SEQ ID NO:3有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  27. 根据权利要求1-26任一项所述的重组核酸，其特征在于：所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
  28. 根据权利要求1-27任一项所述的重组核酸，其特征在于,所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  29. 根据权利要求1-28任一项所述的重组核酸，其特征在于，包括所述特征：
    (1)；或
    (1)和(2)；或
    (3)；或
    (1)(2)和(3)；或
    (1)(2)(3)和(4)；或
    (1)(2)(3)(4)和(5)；或
    (1)和(6)；或
    (1)(2)和(6)；或
    (2)(3)(4)(5)和(7)；或
    (1)(3)(4)(5)和(7)。
  30. 根据权利要求1-29任一项所述的重组核酸，其特征在于，包含所述Rep基因和所述Cap基因。
  31. 根据权利要求1,3-7,10-11,21-29任一项所述的重组核酸，其特征在于，包含所述Cap基因，但不包含编码Rep基因的核酸序列。
  32. 根据权利要求1-2,4-5,8-9,12-29任一项所述的重组核酸，其特征在于，包含所述Rep基因，但不包含编码Cap基因的核酸序列。
  33. 一种质粒，包含根据权利要求1-32任一项所述的重组核酸。
  34. 根据权利要求33所述的质粒，其特征在于，所述质粒为重组腺相关病毒无辅助病毒系统中的包装质粒(RC质粒)，或辅助质粒(pHelper质粒)，或编码包含异源核酸序列的重组腺相关病毒的质粒。
  35. 根据权利要求33-34所述的质粒，其特征在于，所述质粒包含所述Cap基因，但不包含编码Rep基因的核酸序列，也不包含编码用于AAV生产的辅助基因的核酸序列，也不包含AAV反向重复序列(ITR)。
  36. 一种质粒，其特征在于，包含Cap基因，但不包含编码Rep基因的核酸序列，也不包含编码用于AAV生产的辅助基因的核酸序列，也不包含AAV反向重复序列(ITR)。
  37. 根据权利要求33-34任一项所述的质粒，其特征在于，所述质粒为编码包含异源核酸序列 的重组腺相关病毒的质粒，且包含所述Cap基因。
  38. 根据权利要求37所述的质粒，其特征在于，所述目的基因质粒包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  39. 一种辅助质粒，其特征在于，包含根据权利要求1-31任一项所述的包含所述Cap基因的重组核酸，且所述辅助质粒包含编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。
  40. 一种辅助质粒，其特征在于，包含编码Cap基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列，所述Cap基因编码一个功能性的Cap蛋白。
  41. 根据权利要求39-40任一项所述的辅助质粒，其特征在于，所述辅助质粒包含Rep基因；可选地，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  42. 根据权利要求39-41任一项所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。
  43. 根据权利要求42所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的两个基因；可选地，所述辅助质粒包含E4基因和VA RNA基因。
  44. 根据权利要求42所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含E2A，E4，和VA RNA。
  45. 根据权利要求42-44任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)；可选地，所述E2A基因包含与SEQ ID NO:93有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列；可选地，所述E4基因包含与SEQ ID NO:94有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核 苷酸序列；可选地，所述VA RNA基因包含与SEQ ID NO:95有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  46. 根据权利要求42-44任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)。
  47. 根据权利要求1-31任一项所述的重组核酸，或根据权利要求31-41任一项所述的质粒，其特征在于，包含选自P5启动子，P19启动子，和P40启动子的至少一个启动子，且所述启动子与所述Cap基因可操作地连接。
  48. 根据权利要求47所述的重组核酸或质粒，其特征在于，包含P5启动子，P19启动子，和P40启动子；优选地，所述编码Cap基因的核酸序列包含，由5’端至3’端方向，编码所述P5启动子，所述P19启动子，所述P40启动子，和所述Cap基因的序列。
  49. 根据权利要求47-48任一项所述的重组核酸或质粒，其特征在于，所述启动子之间，和/或所述启动子和所述Cap基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
  50. 根据权利要求47-49任一项所述的重组核酸或质粒，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  51. 根据权利要求47-50任一项所述的重组核酸或质粒，其特征在于，所述P19启动子的序列与SEQ ID NO:1第741-890位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P19启动子的序列在相对于SEQ ID NO:1第768位核苷酸的位置是T。
  52. 根据权利要求47-51任一项所述的重组核酸或质粒，其特征在于，所述P40启动子的序列与SEQ ID NO:1第1701-2202位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性；优选地，所述P40启动子的序列在相对于SEQ ID NO:1第1911-1912位核 苷酸的位置是GT。
  53. 根据权利要求1-31任一项所述的重组核酸，根据权利要求33-46任一项所述的质粒，或根据权利要求47-52任一项所述的重组核酸或质粒，其特征在于，所述Cap基因的序列包含与SEQ ID NO:1第2203-4410位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
  54. 根据权利要求1-31任一项所述的重组核酸，根据权利要求33-46任一项所述的质粒，或根据权利要求47-52任一项所述的重组核酸或质粒，其特征在于，所述Cap基因编码的Cap蛋白包含与选自SEQ ID NO:48-57的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的蛋白序列。
  55. 根据权利要求54所述的重组核酸或质粒，其特征在于，所述Cap基因包含与选自SEQ ID NO:58-67的一条序列有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  56. 根据权利要求1-31任一项所述的重组核酸，根据权利要求33-46任一项所述的质粒，或根据权利要求47-55任一项所述的重组核酸或质粒，其特征在于，所述编码Cap基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Cap基因下游。
  57. 根据权利要求56所述的重组核酸或质粒，其特征在于，所述Cap基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
  58. 根据权利要求56-57任一项所述的重组核酸或质粒，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  59. 根据权利要求1-31任一项所述的重组核酸，根据权利要求33-46任一项所述的质粒，或根据权利要求47-58任一项所述的重组核酸或质粒，其特征在于，在所述Cap基因下游还包含一个P5启动子，与所述Cap基因可操作地连接；可选地，所述P5启动子位于Cap基因下 游的polyA序列的下游；可选地，所述P5启动子与所述Cap基因之间，或所述P5启动子与所述polyA序列之间，间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸，或没有间隔的核苷酸。
  60. 根据权利要求1-31任一项所述的重组核酸，根据权利要求33-46任一项所述的质粒，或根据权利要求47-59任一项所述的重组核酸或质粒，其特征在于，所述Cap基因编码一个功能性的Cap蛋白。
  61. 根据权利要求33-60任一项所述的质粒，其特征在于，所述质粒不能表达功能性的Rep蛋白。
  62. 根据权利要求33-61任一项所述的质粒，其特征在于，所述质粒不包含编码Rep基因的核酸序列。
  63. 一种辅助质粒，其特征在于，包含根据权利要求1-30,32任一项所述的包含所述Rep基因的重组核酸，且所述辅助质粒包含编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列。
  64. 一种辅助质粒，其特征在于，包含编码Rep基因的核酸序列和编码至少一个用于腺相关病毒(AAV)生产的辅助基因的核酸序列，
  65. 根据权利要求63-64任一项所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的一个或多个基因。
  66. 根据权利要求65所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含选自E2A，E4，和VA RNA的两个基因；可选地，所述辅助质粒包含E4基因和VA RNA基因。
  67. 根据权利要求65所述的辅助质粒，其特征在于，所述用于AAV生产的辅助基因包含E2A，E4，和VA RNA。
  68. 根据权利要求65-67任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA 来源于腺病毒2型(Ad2)；可选地，所述E2A基因包含与SEQ ID NO:93有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列；可选地，所述E4基因包含与SEQ ID NO:94有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列；可选地，所述VA RNA基因包含与SEQ ID NO:95有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  69. 根据权利要求65-67任一项所述的辅助质粒，其特征在于，所述E2A，E4，和/或VA RNA来源于腺病毒2型(Ad2)。
  70. 根据权利要求63-69任一项所述的辅助质粒，其特征在于，包含编码所述Rep基因的核酸序列，并且所述质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列。
  71. 根据权利要求33-34任一项所述的质粒，其特征在于，包含编码所述Rep基因的核酸序列，并且所述质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列。
  72. 一种质粒，其特征在于，包含编码Rep基因的核酸序列，并且所述质粒不能表达功能性的Cap蛋白。
  73. 根据权利要求72所述的质粒，其特征在于，所述质粒不包含编码Cap基因的核酸序列。
  74. 根据权利要求72或73所述的质粒，其特征在于，所述质粒不能表达功能性的用于AAV生产的辅助基因的表达产物。
  75. 根据权利要求72-74任一项所述的质粒，其特征在于，所述质粒不包含编码用于AAV生产的辅助基因的核酸序列。
  76. 根据权利要求1-30,32任一项所述的包含所述Rep基因的重组核酸，或根据权利要求33- 34,63-75任一项所述的质粒，其特征在于，所述Rep基因表达Rep78，Rep68，Rep52，和Rep40蛋白。
  77. 根据权利要求76所述的重组核酸或质粒，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2300位核苷酸有有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
  78. 根据权利要求1-30,32任一项所述的包含所述Rep基因的重组核酸，或根据权利要求33-34,63-75任一项所述的质粒，其特征在于，所述Rep基因表达Rep78和Rep52蛋白；可选地，所述Rep基因不表达Rep68或Rep40蛋白；可选地，所述Rep基因包含与SEQ ID NO:71有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  79. 根据权利要求78所述的重组核酸或质粒，其特征在于，所述Rep基因的序列包含与SEQ ID NO:1第321-2196位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性的序列。
  80. 根据权利要求1-30,32任一项所述的包含所述Rep基因的重组核酸，或根据权利要求33-34,63-75任一项所述的质粒，其特征在于，所述Rep基因表达Rep68和Rep40蛋白；可选地，所述Rep基因不表达Rep78或Rep52蛋白。
  81. 根据权利要求80所述的重组核酸或质粒，其特征在于，所述Rep基因包含与SEQ ID NO:69或SEQ ID NO:103有至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  82. 根据权利要求80-81任一项所述的重组核酸或质粒，其特征在于，所述的重组核酸或质粒不包含仅表达Rep52和Rep40蛋白的基因。
  83. 根据权利要求1-30,32任一项所述的包含所述Rep基因的重组核酸，根据权利要求33-34,63-75任一项所述的质粒，或根据权利要求76-82任一项所述的重组核酸或质粒，其特征在于，包含一个位于所述Rep基因的上游的P5启动子和/或一个位于所述Rep基因的下游的P5启动 子，所述P5启动子与所述Rep基因可操作地连接。
  84. 根据权利要求83所述的重组核酸或质粒，其特征在于，所述启动子和所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
  85. 根据权利要求83-84任一项所述的重组核酸或质粒，其特征在于，所述P5启动子的序列与SEQ ID NO:1第191-320位核苷酸，或与SEQ ID NO:1第191-310位核苷酸，有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  86. 根据权利要求1-30,32任一项所述的包含所述Rep基因的重组核酸，根据权利要求33-34,63-75任一项所述的质粒，或根据权利要求76-85任一项所述的重组核酸或质粒，其特征在于，所述Rep基因编码一个功能性的Rep蛋白。
  87. 根据权利要求1-30,32任一项所述的包含所述Rep基因的重组核酸，根据权利要求33-34,63-75任一项所述的质粒，或根据权利要求76-86任一项所述的重组核酸或质粒，其特征在于，所述编码Rep基因的核酸序列编码一个polyA序列，所述polyA序列位于所述Rep基因下游。
  88. 根据权利要求87所述的重组核酸或质粒，其特征在于，所述Rep基因与所述polyA序列之间间隔不多于30bp，不多于25bp，不多于20bp，不多于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
  89. 根据权利要求87-88任一项所述的重组核酸或质粒，其特征在于，所述polyA序列与SEQ ID NO:1第4411-4460位核苷酸有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  90. 根据权利要求33-34,63-89任一项所述的质粒，其特征在于，所述编码Rep基因的核酸序列包含一个hGH内含子；优选地，所述内含子位于所述Rep基因的起始密码子前。
  91. 根据权利要求90所述的质粒，其特征在于，所述P5启动子与所述hGH内含子之间，和/或所述hGH内含子与所述Rep基因之间间隔不多于30bp，不多于25bp，不多于20bp，不多 于15bp，不多于10bp，或不多于5bp的核苷酸；优选地，没有间隔的核苷酸。
  92. 根据权利要求90-91任一项所述的质粒，其特征在于，所述hGH内含子的序列与SEQ ID NO:13有至少90％，至少95％，至少98％，至少99％，或100％的相同性。
  93. 根据权利要求33-34,63-92任一项所述的质粒，其特征在于，所述Rep基因原有内含子的donor位点包含突变，所述donor位点相当于SEQ ID NO:1的第1907-1912核苷酸位点。
  94. 根据权利要求93所述的质粒，其特征在于，所述donor位点的突变包含将相当于SEQ ID NO:1第1911-1912位核苷酸的CA突变为GT，或相当于SEQ ID NO:1第1907-1912位核苷酸的donor位点序列突变为GTACGT。
  95. 根据权利要求33-94任一项所述的质粒，其特征在于，所述质粒还包含f1origin位点。
  96. 一种三质粒系统，其特征在于，包含:
    (i)辅助质粒(pHelper质粒)，
    (ii)一个包含腺相关病毒(AAV)的Rep基因和/或Cap基因的质粒，和
    (iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游，
    其中，(i)和/或(ii)中所述的质粒包含根据权利要求1-32,47-60,76-89任一项所述的编码所述Rep基因和/或所述Cap基因的重组核酸。
  97. 根据权利要求96所述的三质粒系统，其特征在于，(ii)中所述质粒包含所述Rep基因和所述Cap基因。
  98. 根据权利要求96所述的三质粒系统，其特征在于，所述Rep基因和所述Cap基因在不同的质粒上。
  99. 根据权利要求98所述的三质粒系统，其特征在于，(i)所述的辅助质粒包含所述Rep基因，且(ii)中所述质粒包含所述Cap基因。
  100. 根据权利要求98所述的三质粒系统，其特征在于，(ii)中所述质粒包含所述Rep基因，且(iii)中所述重组腺相关病毒的质粒包含所述Cap基因。
  101. 一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，其中一个质粒包含AAV的Rep基因但不包含AAV的Cap基因，另一个不同的质粒包含AAV的Cap基因但不包含AAV的Rep基因。
  102. 根据权利要求101所述的三质粒系统，其特征在于，包含一个包含所述Rep基因的辅助质粒(pHelper质粒)，一个包含所述Cap基因的第二质粒，和一个编码包含异源核酸序列的重组腺相关病毒的第三质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，所述第三质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  103. 根据权利要求101所述的三质粒系统，其特征在于，包含一个辅助质粒(pHelper质粒)，一个包含所述Rep基因的第二质粒，和一个包含所述Cap基因且编码包含异源核酸序列的重组腺相关病毒的第三质粒；可选的，所述异源核酸序列编码一个异源基因；可选地，所述第三质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  104. 一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含:
    (i)根据权利要求63-70,76-95任一项所述的辅助质粒，其包含所述Rep基因，且所述辅助质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；
    (ii)根据权利要求35-36,47-62任一项所述的质粒，其包含所述Cap基因，且该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；和
    (iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  105. 一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
    (i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Rep基因的重组核酸；可选地，所述辅 助质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；
    (ii)一个包含Cap基因的质粒；可选地，该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；和
    (iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  106. 一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含:
    (i)辅助质粒(pHelper质粒)，
    (ii)根据权利要求71-95任一项所述的质粒，其包含所述Rep基因，且该质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列，和
    (iii)根据权利要求37-38,47-62任一项所述的包含所述Cap基因和所述异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  107. 一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
    (i)辅助质粒(pHelper质粒)；
    (ii)一个包含Rep基因的质粒；可选地，该质粒(a)不能表达功能性的Cap蛋白，和/或(b)不包含编码Cap基因的核酸序列；和
    (iii)一个编码包含异源核酸序列的重组腺相关病毒，且包含Cap基因的质粒；可选地，该质粒(a)不能表达功能性的Rep蛋白，和/或(b)不包含编码Rep基因的核酸序列；可选地，所述异源核酸序列编码一个异源基因；可选地，还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  108. 一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
    (i)根据权利要求39-62任一项所述的辅助质粒，其包含所述Cap基因；
    (ii)根据权利要求71-95任一项所述的编码Rep基因的质粒；和
    (iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
  109. 一种用于腺相关病毒(AAV)表达的三质粒系统，其特征在于，包含：
    (i)辅助质粒(pHelper质粒)，所述辅助质粒包含编码Cap基因的重组核酸；
    (ii)一个包含Rep基因的质粒；和
    (iii)一个编码包含异源核酸序列的重组腺相关病毒的质粒。
  110. 根据权利要求108-109任一项所述的三质粒系统，其特征在于，所述(i)辅助质粒不能表达功能性的Rep蛋白，和/或所述(ii)编码Rep基因的质粒不能表达功能性的Cap蛋白。
  111. 根据权利要求108-110任一项所述的三质粒系统，其特征在于，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  112. 根据权利要求108-111任一项所述的三质粒系统，其特征在于，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(i)辅助质粒包含一个表达Rep78和/或Rep52蛋白的基因；可选地，该基因不表达Rep68或Rep40。
  113. 根据权利要求108-111任一项所述的三质粒系统，其特征在于，所述(ii)编码Rep基因的质粒表达Rep68和Rep40蛋白但不表达Rep78或Rep52蛋白，所述(iii)编码包含异源核酸序列的重组腺相关病毒的质粒包含一个表达Rep78和/或Rep52蛋白的基因；可选地，该基因不表达Rep68或Rep40。
  114. 根据权利要求108-113任一项所述的三质粒系统，其特征在于，所述(i)辅助质粒包含E4基因和VA RNA基因；所述(ii)编码Rep基因的质粒包含E2A基因。
  115. 根据权利要求108-114任一项所述的三质粒系统，其特征在于，所述AAV的血清型是AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、或AAV-DJ；优选地，所述AAV血清型为AAV2、AAV5、AAV8、或AAV9；更优选地，所述AAV的血清型是AAV2。
  116. 根据权利要求108-115任一项所述的三质粒系统，其特征在于，所述异源核酸序列编码一个异源基因。
  117. 一种二质粒系统，其特征在于，包含(i)根据权利要求63-70,76-95任一项所述的含所述Rep基因的第一质粒和(ii)根据权利要求37-38,47-62任一项所述的含所述Cap基因的第二质粒。
  118. 一种二质粒系统，其特征在于，包含一个含有AAV的Rep基因的第一质粒和一个含有AAV的Cap基因的第二质粒，所述Rep基因和所述Cap基因分别位于不同质粒上。
  119. 根据权利要求117-118任一项所述的二质粒系统，其特征在于，所述Rep和所述Cap基因分别位于不同质粒上。
  120. 根据权利要求117-119任一项所述的二质粒系统，其特征在于，含有所述Rep基因的第一质粒为辅助质粒(pHelper质粒)，且含有所述Cap基因的第二质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选地，所述异源核酸序列编码一个异源基因；可选地，所述第二质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  121. 根据权利要求117-119任一项所述的二质粒系统，其特征在于，含有所述Cap基因的第二质粒为辅助质粒(pHelper质粒)，且含有所述Rep基因的第一质粒是一个编码包含异源核酸序列的重组腺相关病毒的质粒；可选的，所述异源核酸序列编码一个异源基因；可选地，所述第一质粒还包含至少两个AAV反向重复序列(ITR)，所述两个AAV反向重复序列分别位于所述异源核酸序列的上游和下游。
  122. 根据权利要求96-121任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78、Rep68、Rep52和Rep40蛋白。
  123. 根据权利要求96-122任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep78和Rep52蛋白，而不表达Rep68和Rep40蛋白。
  124. 根据权利要求96-122任一项所述的质粒系统，其特征在于，所述Rep基因表达Rep68和Rep40蛋白，而不表达Rep78和Rep52蛋白。
  125. 根据权利要求96-124任一项所述的质粒系统，其特征在于，所述质粒系统不包含共表达Rep52蛋白和Rep40蛋白的基因。
  126. 根据权利要求96-125任一项所述的质粒系统，其特征在于，所述Rep基因的上游含有一个hGH内含子。
  127. 根据权利要求126所述的质粒系统，其特征在于，所述hGH内含子的上游含有一个P5启动子。
  128. 根据权利要求96-127任一项所述的质粒系统，其特征在于，所述Rep基因的下游含有polyA序列。
  129. 根据权利要求96-128任一项所述的质粒系统，其特征在于，所述Rep基因的上游和/或下游含有一个P5启动子，与所述Rep基因可操作地连接。
  130. 根据权利要求96-129任一项所述的质粒系统，其特征在于，所述Cap基因的上游含有三个启动子，从5’至3’方向分别为：P5启动子，P19启动子，和P40启动子，均与所述Cap基因可操作地连接，所述Cap基因的下游含有polyA序列。
  131. 根据权利要求96-130任一项所述的质粒系统，其特征在于，所述质粒系统包含至少一条与SEQ ID NO:16--28,33-34,40,45-47,68,80-87,96-98任一条有至少90％，至少95％，至少98％，至少99％，或100％的相同性的多核苷酸序列。
  132. 一种质粒系统的改造方法，其特征在于，具有根据下一种或多种修饰方法：
    (1)在Rep基因的上游插入P5启动子，与所述Rep基因可操作地连接；
    (2)校正位于Cap基因下游的TATA box突变的P5启动子或在Cap基因下游插入P5启动子，与所述Cap基因可操作地连接；
    (3)对内含子的donor位点进行突变；
    (4)删除掉Cap基因下游包括肝脏特异性增强子-启动子的一段序列；
    (5)在Rep基因起始密码子AUG前插入hGH内含子；
    (6)在Rep基因起始密码子AUG前插入wtAAV2内含子；
    (7)在Rep基因上游或在Cap基因下游插入非亲本血清型启动子，与所述Rep基因和/或所述Cap基因可操作地连接。
  133. 根据权利要求132所述的质粒系统的改造方法，其特征在于：
    (1)中所述在Rep基因的上游插入P5启动子具有如SEQ ID NO:4所示的序列；
    (2)中所述突变的P5启动子具有如SEQ ID NO:8所示的序列，在Cap基因下游插入的所述P5启动子具有SEQ ID NO:4的序列；
    (3)对内含子的donor位点进行突变是将内含子的donor位点处的GTACCA突变为GTACGT，优选地，突变前的内含子具有如SEQ ID NO:10所示的序列；突变donor后的内含子具有如SEQ ID NO:11所示的序列；
    (4)中删除的序列如SEQ ID NO:12所示；
    (5)中插入的hGH内含子具有如SEQ ID NO:13所示的序列；
    (6)中在Rep基因起始密码子AUG前插入经过改造的wtAAV2内含子，优选地，经过改造的wtAAV2内含子具有如SEQ ID NO:14所示的序列；和/或
    (7)中非亲本血清型启动子为wtAAV7P5启动子，其有如SEQ ID NO:15所示的序列。
  134. 一种重组腺相关病毒的生产方法，其特征在于，将根据权利要求33-95任一项所述的质粒或根据权利要求96-131任一项所述的质粒系统中的所有质粒转染至宿主细胞，培养，分离得到所述重组腺相关病毒。
  135. 根据权利要求134所述的生产方法，其特征在于，包含所述Cap基因的质粒与包含所述Rep基因的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1；优选地，至少3:1。
  136. 根据权利要求134-135任一项所述的生产方法，其特征在于，包含所述Cap基因的质粒与包含所述Rep基因的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1；优选地，约3:1。
  137. 根据权利要求134-136任一项所述的生产方法，其特征在于，包含所述Rep基因的质粒与包含所述编码包含异源核酸序列的重组腺相关病毒的质粒的比率为至少1:1，至少1.5:1，至少2:1，至少2.5:1，至少3:1，至少3.5:1，或至少4:1；优选地，至少2.5:1。
  138. 根据权利要求134-137任一项所述的生产方法，其特征在于，包含所述Rep基因的质粒与包含所述编码包含异源核酸序列的重组腺相关病毒的质粒的比率为约1:1，约1.5:1，约2:1，约2.5:1，约3:1，约3.5:1，或约4:1；优选地，约2.5:1。
  139. 根据权利要求134-138任一项所述的生产方法，其特征在于，相比基于传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统的生产方法，所述方法产生的重组腺相关病毒的：
    (1)实心率更高；可选地，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少0.70,至少0.75,或至少0.80；
    (2)基因组滴度更高；可选地，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍；
    (3)Cap编码序列的相关杂质残留更低；可选地，Cap编码序列的相关杂质残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％；和/或
    (4)具有复制能力的AAV(rcAAV)残留更低；可选地，rcAAV残留比传统方法减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
  140. 一种重组腺相关病毒，其特征在于，采用根据权利要求134-139任一项所述的生产方法获得。
  141. 根据权利要求140所述的重组腺相关病毒，其特征在于，所述宿主细胞的培养方式包括贴壁培养或悬浮培养。
  142. 根据权利要求140-141任一项所述的重组腺相关病毒，其特征在于，所述重组腺相关病毒相比由传统的辅助质粒+Rep/Cap质粒+目的基因质粒的三质粒系统生产的重组腺相关病毒的：
    (1)实心率更高；可选地，实心率数值高至少0.05,至少0.10,至少0.15,至少0.20,至少0.25,至少0.30,至少0.35,至少0.40,至少0.45,至少0.50,至少0.55,至少0.60,至少0.65,至少 0.70,至少0.75,或至少0.80；
    (2)基因组滴度更高；可选地，基因组滴度高至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少70％,至少1倍，至少2倍，至少3倍，至少4倍，至少5倍,至少7倍，至少10倍，至少20倍，至少50倍，或至少100倍；
    (3)Cap编码序列的相关杂质残留更低；可选地，Cap编码序列的相关杂质残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％；和/或
    (4)具有复制能力的AAV(rcAAV)残留更低；可选地，rcAAV残留比传统生产的重组腺相关病毒减少至少5％,至少10％,至少20％,至少30％,至少40％,至少50％,至少60％,至少70％,至少80％,至少90％,至少95％,至少98％,至少99％,至少99.9％,或至少99.99％。
  143. 一种宿主细胞，其特征在于，所述宿主细胞含有根据权利要求33-95任一项所述的质粒或根据权利要求96-131任一项所述的质粒系统中的所有质粒。
  144. 根据权利要求134-139任一项所述的生产方法，或根据权利要求143所述的宿主细胞，其特征在于，所述宿主细胞选自HEK293细胞、HEK293T细胞、HEK293F细胞、VPC2.0细胞、Hela细胞和A549细胞。
  145. 根据权利要求1-32任一项所述的重组核酸，权利要求33-95任一项所述的质粒，权利要求96-141任一项所述的质粒系统，权利要求140-142任一项所述的重组腺相关病毒，或权利要求143-144任一项所述的宿主细胞在制备药物中的应用。
  146. 一种药物制剂，其特征在于，所述药物包括根据权利要求140-142任一项所述的重组腺相关病毒，以及药学上可接受的载体和赋形剂。
  147. 如权利要求146所述的药物制剂在治疗疾病中的应用。
  148. 一种治疗疾病的方法，其包括向有需要的患者施用根据权利要求140-142任一项所述的重组腺相关病毒，或权利要求146所述的药物制剂。
  149. 如权利要求147所述的应用或者如权利要求148所述的方法，其中，所述疾病是由于人体中的靶基因发生基因突变和/或基因缺失导致的。
  150. 一种试剂盒，其包含根据权利要求1-32任一项所述的重组核酸，权利要求33-95任一项所述的质粒，权利要求96-141任一项所述的质粒系统，权利要求140-142任一项所述的重组腺相关病毒，或权利要求143-144任一项所述的宿主细胞。
PCT/CN2024/074973 2023-02-01 2024-01-31 质粒包装系统及其应用 WO2024160232A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202310049101.7 2023-02-01
CN202310049101.7A CN118460617A (zh) 2023-02-01 2023-02-01 质粒系统及其应用
CN202310651836 2023-06-02
CN202310651836.7 2023-06-02

Publications (1)

Publication Number Publication Date
WO2024160232A1 true WO2024160232A1 (zh) 2024-08-08

Family

ID=92145845

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/074973 WO2024160232A1 (zh) 2023-02-01 2024-01-31 质粒包装系统及其应用

Country Status (1)

Country Link
WO (1) WO2024160232A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110785495A (zh) * 2017-04-18 2020-02-11 葛兰素史克知识产权开发有限公司 腺相关病毒载体的生产方法
CN111511922A (zh) * 2018-01-19 2020-08-07 牛津遗传学有限公司 用于生产aav颗粒的载体
WO2020235543A1 (ja) * 2019-05-20 2020-11-26 Ｊｃｒファーマ株式会社 組換えａａｖビリオンの製造に用いる核酸分子
CN112888426A (zh) * 2018-10-25 2021-06-01 百特奥尔塔公司 Aav三质粒系统
CN113891942A (zh) * 2019-04-12 2022-01-04 自由行疗法有限公司 质粒系统
CN114450393A (zh) * 2019-07-15 2022-05-06 查尔斯河实验室股份有限公司 重组修饰腺相关病毒辅助载体及其在提高重组修饰腺相关病毒的包装效率的用途
CN115197967A (zh) * 2021-04-08 2022-10-18 广州派真生物技术有限公司 制备重组腺相关病毒的辅助质粒及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110785495A (zh) * 2017-04-18 2020-02-11 葛兰素史克知识产权开发有限公司 腺相关病毒载体的生产方法
CN111511922A (zh) * 2018-01-19 2020-08-07 牛津遗传学有限公司 用于生产aav颗粒的载体
CN112888426A (zh) * 2018-10-25 2021-06-01 百特奥尔塔公司 Aav三质粒系统
CN113891942A (zh) * 2019-04-12 2022-01-04 自由行疗法有限公司 质粒系统
WO2020235543A1 (ja) * 2019-05-20 2020-11-26 Ｊｃｒファーマ株式会社 組換えａａｖビリオンの製造に用いる核酸分子
CN114450393A (zh) * 2019-07-15 2022-05-06 查尔斯河实验室股份有限公司 重组修饰腺相关病毒辅助载体及其在提高重组修饰腺相关病毒的包装效率的用途
CN115197967A (zh) * 2021-04-08 2022-10-18 广州派真生物技术有限公司 制备重组腺相关病毒的辅助质粒及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WU, HONGYU; LU, YING-HUI; XU, RUI-AN; WANG, QI-ZHAO: "Improvement of Recombination Adeno-Associated Virus Production", LETTERS IN BIOTECHNOLOGY, JUNSHI YIXUE KEXUEYUAN, CN, vol. 23, no. 4, 31 July 2012 (2012-07-31), CN , pages 595 - 600, XP009556719, ISSN: 1009-0002, DOI: 10.3969/j.issn.1009-0002.2012.04.030 *

Similar Documents

Publication Publication Date Title
EP3113787B1 (en) Improved raav vectors and methods for transduction of photoreceptors and rpe cells
JP2021121638A (ja) 変異体キャプシドを有するアデノ関連ウイルスビリオンおよびその使用方法
JP6174318B2 (ja) アデノ随伴ウイルス構築物を含んでなるコンダクティング気道細胞を標的とするための組成物
JP4573437B2 (ja) アデノ随伴ウイルス血清型１核酸配列、ベクターおよび同一物を含有する宿主細胞
JP2022010141A (ja) アデノ随伴ウイルス第ｖｉｉｉ因子ベクター、関連ウイルス粒子、及びそれらを含む治療的製剤
AU2022200273A1 (en) High-transduction-efficiency raav vectors, compositions, and methods of use
EP4269579A2 (en) Gene therapy constructs for treating wilson disease
JP2001514845A (ja) 組換えａａｖベクターの高力価ヘルパーなし調製物を生成するための方法
EP4349852A1 (en) Recombinant adeno-associated virus having variant capsid, and application thereof
EP2037892A2 (en) Modified factor viii and factor ix genes and vectors for gene therapy
WO2008021140A2 (en) Recombinant aav production in mammalian cells
CN111876432B (zh) 一组肝靶向新型腺相关病毒的获得及其应用
WO2017201121A1 (en) Recombinant aav for gene therapy in lungs
JP2023540085A (ja) ＳＡＲＳ－ＣｏＶ－２に対するＡＡＶ５に基づくワクチン
JP2023504735A (ja) ヒトｍｅｃｐ２遺伝子を発現するように設計されたトランスジーンカセット
WO2022222869A1 (zh) 重组腺相关病毒及其应用
CN114981444A (zh) Aav5的分离的修饰的vp1衣壳蛋白
WO2022226289A2 (en) Aavrh74 vectors for gene therapy of muscular dystrophies
WO2024160232A1 (zh) 质粒包装系统及其应用
WO2005035743A1 (fr) Procede de production, d&#39;isolation, de purification et utilisations de vecteurs virus adeno-associes recombinants multi-types
CN118460617A (zh) 质粒系统及其应用
WO2023035687A1 (zh) 用于治疗庞贝氏病的基因治疗构建体、药物组合物和方法
WO2023131345A1 (zh) 用于x染色体连锁肾上腺脑白质营养不良的基因治疗药物和方法
WO2024160047A1 (zh) 一种组织嗜性的腺相关病毒衣壳及其用途
US20240076691A1 (en) Codon-optimized nucleic acid encoding the fix protein

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24749711

Country of ref document: EP

Kind code of ref document: A1