Plasmid packaging system and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a plasmid system and application thereof. In particular to a novel three-plasmid packaging system for producing a recombinant adeno-associated virus (rAAV) vector, a packaging plasmid pRC2 in a traditional three-plasmid system is split and modified, a Cap gene is combined with a helper plasmid pHelper, and a Rep gene is independently placed in one plasmid, so that a novel helper packaging plasmid is obtained. The plasmid system not only can improve the yield of rAAV and save high production cost, but also can reduce the content of pollution impurities, namely, replication-competent AAV (Replication-competent AAV, rcAAV), in the vector, control the purity, and improve the economical efficiency and safety of an AAV vector preparation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a plasmid system and application thereof. Background Art

[0002] Recombinant adeno-associated virus (rAAV) 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 six AAV gene therapy drugs have been launched on the market, with very broad commercial prospects. However, in 2017, the first AAV gene therapy drug Glybera was withdrawn from the market because it was priced but not sold. The cost of a single treatment is as high as $1 million, and the high production cost is the fundamental reason for its high pricing. 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.

[0003] Although AAV is considered to be relatively safe in the relevant ratings of biotechnology products, in recent years, the FDA Advisory Committee has paid more and more attention to the results of AAV clinical studies. In the IND application guidelines, the issue of replication-competent adeno-associated virus (rcAAV) is given particular attention. They recommend testing rcAAV and describe the detection method and detection sensitivity. rcAAV refers to replication-competent viral particles containing wtAAV Rep and Cap genes. This AAV can replicate when a helper virus is present. China's "Technical Guidelines for Pharmaceutical Research and Evaluation of In Vivo Gene Therapy Products (Trial)" also clearly pointed out that for the starting raw materials of viral vector products, such as plasmids used for viral vector packaging during transient transfection, the viral vector design should be fully considered and potential risks should be strictly controlled. In order to reduce the risk of recombinant production of replicable or pseudo-wild-type viruses during virus production and use, it is recommended to give priority to virus packaging systems that have been verified to have a higher safety level, appropriately split the essential elements for virus packaging into different plasmids, remove non-essential viral genes as much as possible, and reduce the homology between plasmids, plasmids and packaging cells, and wild virus sequences. In particular, 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.

[0004] Since the replication protein Rep has helicase and endonuclease activities, it may induce DNA damage and cause cytotoxicity, and the capsid protein Cap has also been found to cause severe cellular immune responses. 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.

[0005] The main method for producing rAAV is the AAV Helper-Free System, which contains three plasmids: the first is a vector plasmid containing the target gene (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 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, E4 orf6 and VA RNA separated from adenovirus Ad (adenovirus). 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 need to be cultured with fetal bovine serum, have strict requirements on growth conditions and downstream purification, and need to prepare a large number of high-quality plasmids, so the production cost is very high and difficult to expand. Although 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. However, the disadvantage is that they are affected by the kinetic factors of transfection complex formation, resulting in low transfection efficiency. When the feed amount is equal, the yield of rAAV is not as high as that of adherent culture. In addition, since 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. There are currently a variety of methods to inhibit or avoid packaging of wtAAV genes. The first method to reduce rcAAV formation is to eliminate homologous sequences between the vector plasmid and the RC plasmid. Rep and Cap can be placed under the control of heterologous promoters to replace 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. However, 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.

[0006] Due to the above-mentioned production and safety issues, increasing production and reducing the production cost of rAAV vectors and minimizing the safety impact caused by contaminating impurities rcAAV remain technical issues that need to be resolved. Summary of the invention

[0007] Due to the above-mentioned production and safety issues, it is necessary to increase production and reduce the production cost of rAAV vectors, and minimize the safety impact caused by contaminating impurities rcAAV.

[0008] In one aspect of the present invention, a helper plasmid is provided, 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.

[0009] In some embodiments, 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. In some embodiments, the auxiliary plasmid 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. In some embodiments, 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 spaced nucleotides 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 spaced nucleotides between the promoters.

[0010] 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.

[0011] In some embodiments, 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 position relative to nucleotide 768 of SEQ ID NO: 1.

[0012] In some embodiments, 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.

[0013] In some embodiments, 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.

[0014] In some embodiments, the nucleic acid sequence encoding the Cap gene encodes a polyA sequence, and the polyA sequence is located downstream of the Cap gene.

[0015] 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 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.

[0016] In some embodiments, 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.

[0017] In some embodiments, the helper gene for AAV production of the helper plasmid comprises one or more genes selected from E2A, E4, and VA RNA. In some embodiments, the helper gene comprises E2A, E4, and VA RNA.

[0018] In one aspect of the present invention, a plasmid is provided, characterized in that it contains a nucleic acid sequence encoding a Rep gene, and the plasmid cannot express a functional Cap protein. In some embodiments, the plasmid does not contain a nucleic acid sequence encoding a Cap gene. In some embodiments, the plasmid 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.

[0019] In some embodiments, the Rep gene expresses Rep78, Rep68, Rep52, and 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 321-2300 of SEQ ID NO:1.

[0020] In some embodiments, 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.

[0021] In some embodiments, 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. In some embodiments, 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. 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. 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.

[0022] In some embodiments, the nucleic acid sequence encoding the Rep gene comprises an hGH intron. In some embodiments, the intron is located before the start codon of the Rep gene. In some embodiments, 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. In some embodiments, 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: 3.

[0023] In some embodiments, the donor site of the original intron of the Rep gene comprises a mutation, and the donor site corresponds to nucleotides 1907-1912 of SEQ ID NO: 7. In some embodiments, the mutation of the donor site comprises mutating CA corresponding to nucleotides 1911-1912 of SEQ ID NO: 7 to GT. In some embodiments, the mutation of the donor site comprises mutating the donor site sequence corresponding to nucleotides 1907-1912 of SEQ ID NO: 7 to GTACGT.

[0024] In some embodiments, the nucleic acid sequence encoding the Rep gene encodes a polyA sequence, and the polyA sequence is located downstream of the Rep gene. In some embodiments, 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.

[0025] In one aspect of the present invention, a three-plasmid system for adeno-associated virus (AAV) expression is provided, characterized in that it comprises: (i) a helper plasmid as described herein; (ii) a plasmid encoding the Rep gene as described herein; and (iii) a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence.

[0026] In one aspect of the present invention, a three-plasmid system for adeno-associated virus (AAV) expression is provided, characterized in that it comprises: (i) a helper plasmid (pHelper plasmid), wherein the helper plasmid comprises a recombinant nucleic acid encoding a Cap gene; (ii) a plasmid encoding a Rep gene of an adeno-associated virus (AAV), and (iii) a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence.

[0027] In some embodiments, (i) the helper plasmid of the three-plasmid system cannot express a functional Rep protein, and / or (ii) the plasmid cannot express a functional Cap protein.

[0028] In some embodiments, (iii) 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.

[0029] In some embodiments, the three-plasmid system is characterized in that the serotype of AAV is AAV2.

[0030] In some embodiments, the heterologous nucleic acid sequence encodes a heterologous gene.

[0031] In one aspect of the present invention, a method for producing a recombinant adeno-associated virus is provided, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0032] In one aspect of the present invention, a recombinant adeno-associated virus is provided, characterized in that it is obtained by the production method described herein. In some embodiments, the host cell is cultured in an adherent culture or a suspension culture.

[0033] In one aspect of the present invention, a host cell is provided, characterized in that the host cell contains all plasmids used in the plasmid system described herein.

[0034] In some embodiments, the host cell is selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.

[0035] In one aspect of the present invention, a pharmaceutical preparation is provided, characterized in that the drug comprises the recombinant adeno-associated virus described herein, and a pharmaceutically acceptable carrier and excipient.

[0036] In one aspect, provided herein is a use of the pharmaceutical preparation described herein in treating a disease caused by gene mutation and / or gene deletion of a target gene in a human body.

[0037] In one aspect, 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.

[0038] In one aspect of the present invention, a kit is provided, comprising the plasmid described herein, the plasmid system described herein, the production method described herein, the recombinant adeno-associated virus described herein, or the host cell described herein.

[0039] The present invention provides the following technical solutions:

[0040] The structure of the traditional three-plasmid system is as follows Figure 1 As shown, by splitting the Rep and Cap genes of the packaging plasmid pRC2, the recombination between its Rep-Cap sequence and the vector plasmid pAAV-GOI is changed to recombination between three plasmids, which greatly reduces the probability of recombination and reduces the generation of contaminating impurities rcAAV. The structure of the novel three-plasmid system is as follows Figure 2 As shown, it is composed of pHelper-Cap plasmid, pRep plasmid and pAAV-GOI plasmid.

[0041] In one aspect of the present invention, a novel three-plasmid system is provided, characterized in that it comprises: (i) an auxiliary plasmid (pHelper plasmid), (ii) a plasmid encoding a Rep gene containing an adeno-associated virus (AAV), and (iii) a plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence, wherein the plasmid described in (i) comprises the recombinant nucleic acid encoding the Cap gene described in the present invention. In some embodiments, the heterologous nucleic acid sequence encodes a heterologous gene. In some embodiments, the heterologous nucleic acid sequence 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.

[0042] In one aspect of the present invention, 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. In some embodiments, the three-plasmid system is characterized in that it comprises an auxiliary plasmid (pHelper plasmid) containing the Cap gene, a second plasmid containing the Rep gene, and a third plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence encodes a heterologous gene. In some embodiments, 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.

[0043] One aspect of the present invention provides a method for producing a recombinant adeno-associated virus, characterized in that the plasmid described in the present invention or all plasmids in the plasmid system described in the present invention are transfected into a host cell, cultured, and isolated to obtain the recombinant adeno-associated virus.

[0044] In one aspect of the present invention, a recombinant adeno-associated virus is provided, characterized in that it is obtained by the production method of the recombinant adeno-associated virus according to the present invention. In some embodiments, the host cell is cultured in an adherent culture or a suspension culture.

[0045] One aspect of the present invention provides a host cell, characterized in that the host cell contains the plasmid described in the present invention or all plasmids in the plasmid system described in the present invention.

[0046] In some embodiments, the host cell is selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.

[0047] One aspect of the present invention provides the use of the plasmid according to the present invention, the plasmid system according to the present invention, the production method according to the present invention, the recombinant adeno-associated virus according to the present invention, or the host cell according to the present invention in the preparation of drugs.

[0048] In one aspect of the present invention, a pharmaceutical preparation is provided, characterized in that the drug comprises the recombinant adeno-associated virus as described in the present invention, and a pharmaceutically acceptable carrier and excipient. In some embodiments, the pharmaceutical preparation is used to treat diseases caused by gene mutation and / or gene deletion of a target gene in the human body.

[0049] One aspect of the present invention provides a method for treating a disease, which comprises administering the recombinant adeno-associated virus according to the present invention or the pharmaceutical preparation according to the present invention to a patient in need thereof.

[0050] In one aspect of the present invention, a kit is provided, which comprises the plasmid according to the present invention, the plasmid system according to the present invention, the production method according to the present invention, the recombinant adeno-associated virus according to the present invention, or the host cell according to the present invention. Adeno-associated virus

[0051] The genome of adeno-associated virus AAV 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 (e.g., 145bp). ITR consists of an approximately 125bp palindromic sequence that forms a T-shaped hairpin structure through complementary base pairing. The remaining approximately 20 bases are not paired 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-deficient parvovirus that requires an auxiliary virus to help it replicate and amplify in vivo. Rep gene

[0052] 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. The expression of Rep78 / 68 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 the same as 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. Cap Gene

[0053] 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 approximately 60 copies of VP1, VP2 and VP3 are assembled into an icosahedral particle with a diameter of about 26 nm, with a molar ratio of 1:1:10. Different capsid proteins determine the serotype of AAV, and these serotypes constitute different tissue preferences. In some embodiments, the serotype of the capsid protein encoded by the Cap gene is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, or a derivative capsid protein based on one of the serotypes.

[0054] 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.

[0055] 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).

[0056] Shuffled or chimeric capsid proteins can also be produced by DNA shuffling or by error-prone PCR. 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.

[0057] 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. In particular, 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.

[0058] 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 that helps to purify the viral particles as part of the production process, i.e., an epitope or affinity tag. 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. The present invention further encompasses providing sequences of the AAV genome in an order and configuration different from the natural AAV genome. The present invention also encompasses replacing one or more AAV sequences or genes with a sequence from another virus or with a chimeric gene consisting of a sequence from more than one virus. Such chimeric genes may consist of sequences of two or more related viral proteins from different viral species.

[0059] Novel three-plasmid system: Rep or Cap is placed on an auxiliary plasmid or a vector plasmid, and another plasmid expresses Rep or Cap alone. The present invention separates Cap into an auxiliary plasmid, and Rep is a separate plasmid.

[0060] In some embodiments, no single plasmid in the system contains both a Rep gene and a Cap gene.

[0061] Preferably, the novel three-plasmid system consists of pHelper-Cap plasmid, pRep plasmid and pAAV-GOI plasmid.

[0062] In some embodiments, the helper plasmid (pHelper plasmid) comprises a polynucleotide sequence encoding the helper genes E2a, E4 and VA RNA derived from adenovirus Ad (adenovirus) or a derivative sequence thereof.

[0063] Put the Cap gene on the auxiliary plasmid pHelper, and another plasmid expresses the Rep plasmid alone, forming two new auxiliary plasmids, which can become public plasmids. When carrying out other projects, it will not increase the cost of plasmid production, nor is it necessary to add Rep or Cap sequences to the vector plasmid where the target gene is located, reducing the risk of packaging these two elements. The two plasmids can also be modified separately to regulate the expression of Rep and Cap genes respectively, so that the yield is doubled. In addition, 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. Placing them on one plasmid makes it easy to control the feeding, so the new three-plasmid system structure is more suitable for rAAV packaging.

[0064] In some embodiments, the plasmid expressing Rep may also include one or more auxiliary genes. In some embodiments, in the plasmid system described herein, one or more of all the necessary auxiliary genes required for expressing AAV are included on the plasmid expressing Rep, and the remaining necessary auxiliary genes are included on the plasmid expressing Cap.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] The present invention provides several methods for transforming the pRC2 plasmid in the traditional three-plasmid system. The novel three-plasmid system composed of the transformed plasmids 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 an 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 requirements for rAAV vector materials, and is quickly and easily applied to the production system, greatly reducing the production cost, and can reduce a lot of manpower and material input, and improve production efficiency. Formulations, routes of administration and effective doses

[0067] Another aspect of the present disclosure relates to the formulation, administration route and effective dosage of a pharmaceutical composition comprising the pharmaceutical agent or pharmaceutical combination of the present disclosure. Such pharmaceutical formulations can be used to treat diseases (such as eye diseases).

[0068] The compounds of the present disclosure can be administered as pharmaceutical formulations, including those suitable for oral (including buccal and sublingual), rectal, nasal, topical, transdermal patch, 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 atomization, inhalation or insufflation. 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).

[0069] In various embodiments, 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. Examples of excipients 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. In some embodiments, pharmaceutical preparations are substantially free of preservatives. In other embodiments, 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.

[0070] The compounds may also be encapsulated in liposomes using well-known techniques. Biodegradable microspheres may 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.

[0071] The compound can be administered in the form of liposomes or microspheres (or microparticles). The preparation methods of liposomes and microspheres for administration to subjects 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, if necessary, together with a surfactant, and the substance is dialyzed or sonicated if necessary. G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, Page 2, Supplement 87-341 (Academic Press, 1979) provides a review of known methods.

[0072] 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, for example, U.S. Patents 4,906,474, 4,925,673, and 3,625,214 and Jein, TIPS 19: 155-157 (1998), which are hereby incorporated by reference.

[0073] 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.

[0074] The compounds of the present disclosure can be formulated as sterile solutions or suspensions in suitable vehicles. 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).

[0075] 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. For example, the preparation may contain 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.

[0076] The term "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.

[0077] 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. In addition, 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. Examples of suitable alkalis include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, etc.

[0078] Pharmaceutically acceptable 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.

[0079] In some embodiments, the agent can be administered in combination with one or more other compounds, 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. For example, the molar ratio of the first active agent to the other active agents can be about 99:1 to about 1:99. In some subsets of the embodiments, 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 compounds may be formulated together in the same dosage unit, for example in one cream, suppository, tablet, capsule, or powder packet to be dissolved in a beverage; or each dose, form and / or compound may be formulated in a separate unit, for example, 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.

[0080] The 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.

[0081] 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. As used herein, 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.

[0082] In some embodiments, due to, for example, the presence of a large lipophilic portion, an oil or non-aqueous solvent can be used to bring the agent into solution. Alternatively, an emulsion, suspension or other formulation, such as a liposomal formulation, can be used. With regard to liposomal formulations, any known method can 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 can also be attached to liposomes to direct these compositions to specific sites of action. The agents of the present disclosure can 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.

[0083] 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.

[0084] For injectable preparations, 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.

[0085] In some embodiments, the composition is formulated into a pharmaceutical preparation suitable for intravenous administration to a human being according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. Where necessary, the composition or preparation may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Typically, 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 the active agent. 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. In the case where the composition or preparation is administered by injection, an ampoule of sterile water for injection or saline may be provided so that each component may be mixed before administration.

[0086] 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 a preparatant, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the active compound may be in powder form and reconstituted with a suitable vehicle, such as sterile pyrogen-free water, before use. In some embodiments, the pharmaceutical preparation does not include an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the pharmaceutical preparation includes a substance that suppresses the immune response to the peptide. Preparation methods are known in the art, for example, as disclosed in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton P.

[0087] In some embodiments, ophthalmic solutions, suspensions, ointments or inserts 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. 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, hydroxypropyl cellulose, or other agents known to those skilled in the art).

[0088] The solubility of the components of the compositions or formulations of the present invention may be increased by surfactants or other suitable co-solvents in the compositions or formulations. Such 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. Such co-solvents may be used at a level of about 0.01% to 2% by weight.

[0089] Compositions or preparations of the present disclosure can be packaged in multiple doses. Preservatives can 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 certain embodiments, the use level of this type of preservative can be 0.004% to 0.02%. In the composition or preparation of the present application, a preservative, preferably benzalkonium chloride, can 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 the benzalkonium chloride of 0.005% concentration can be enough to protect the composition or preparation of the present disclosure from microbial attack.

[0090] In some embodiments, 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. In general, formulations such as gels, creams, lotions, suppositories, and ointments can 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.

[0091] In addition, it is foreseeable that 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 in preparations suitable for intraocular implantation or injection for sustained release administration, and any suitable biodegradable and biocompatible polymers can also be used. definition

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or considered herein are standard methods. The materials, methods and examples are illustrative only and not restrictive.

[0093] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0094] As used herein, unless otherwise indicated, the term "or" may be a conjunction or a disjunctive. As used herein, unless otherwise indicated, any embodiment may be combined with any other embodiment.

[0095] As used herein, unless otherwise indicated, certain application embodiments herein contemplate numerical ranges. Where a range is present, the range includes the range endpoints. In addition, each subrange and value within the range is present as if explicitly written out.

[0096] 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%. For example, the amount "about 10" includes amounts of 9 to 11.

[0097] The term "comprising" (and related terms, such as "including" or "having" or "including") is not intended to exclude certain other embodiments, for example, any embodiment of the composition of the materials, compositions, methods or processes described herein may "consist of" or "consist essentially of the described features."

[0098] The term "subject" refers to a mammal that has been or will be the object of treatment, observation or experiment. The term "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. In some embodiments, the subject is a human.

[0099] The term "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.

[0100] The term "therapeutically 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.

[0101] As used herein, unless otherwise indicated, the terms "nucleic acid" and "polynucleotide" are used interchangeably.

[0102] As used herein, unless otherwise indicated, when describing a gene or nucleic acid site, the term "upstream" refers to the 5' end of the gene or nucleic acid site, and the term "downstream" refers to the 3' end of the gene or nucleic acid site.

[0103] The term "promoter" refers to a DNA sequence located in the 5'-upstream region of a structural gene, which can activate RNA polymerase, allowing it to bind accurately to the template DNA and have the specificity to initiate transcription.

[0104] The term "liver specific enhancer promotor" (LSP) refers to a promoter that can enhance the expression of foreign genes in the liver and is located between the stop codon of the cap gene and the right terminal inverted repeat sequence (see Logan et al., Identification of liver-specific enhancer-promoter activity in the 3'untranslated region of the wild-type AAV2 genome). In some cases, due to the natural tropism of AAV for the liver, AAV expression systems containing liver-specific promoters are prone to cause AAV accumulation in the liver, which may affect the safety of AAV vectors.

[0105] The term "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. In certain embodiments, the term "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.

[0106] 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. When the plasmid replicates in the host cell, the host cell provides energy, materials, enzymes and other conditions. For example, the host cell can be selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 This is a schematic diagram of the structure of the traditional three-plasmid system;

[0108] Figure 2 It is a schematic diagram of the structure of the new three-plasmid system;

[0109] Figure 3 is a schematic diagram of the wtAAV2 genome structure;

[0110] Figure 4 is a schematic diagram of the pHelper-Cap2 plasmid structure;

[0111] Figure 5 is a schematic diagram of the pRep1 plasmid structure;

[0112] Figure 6 is a schematic diagram of the pRep3 plasmid structure;

[0113] Figure 7 is a schematic diagram of the pRep5 plasmid structure;

[0114] Figure 8 is a schematic diagram of the pRep6 plasmid structure;

[0115] Fig. 9 The titer graphs of rAAV2-GOI1 vector genomes packaged with pHelper-Cap2 plasmid and pRep1, pRep3, pRep5, and pRep6 plasmids, respectively;

[0116] Fig.10 This is the viral genome titer graph of the optimized process of pHelper-Cap2 plasmid and pRep5 packaging rAAV2-GOI1;

[0117] Fig.11 This is a solid diagram of the new three-plasmid system and the traditional three-plasmid system for packaging rAAV2-GOI1 vector. DETAILED DESCRIPTION Plasmid information

[0118] The modified plasmids in the novel three-plasmid system have the following modifications:

[0119] pRep1 (SEQ ID NO: 4): The original pRC2 (SEQ ID NO: 2) plasmid was split, and the Rep2 gene in wtAAV2 (SEQ ID NO: 1) was placed on a separate plasmid. The plasmid sequence was arranged as P5 promoter and Rep gene (wtAAV2 191-2300 nt, expressing four proteins Rep78 / 68 / 52 / 40), followed by P5 promoter sequence (wtAAV2 191-310 nt). Its structural diagram is shown below: Figure 5 shown.

[0120] pRep3 (SEQ ID NO: 5): The original pRC2 (SEQ ID NO: 2) plasmid was split, and the Rep2 gene in wtAAV2 (SEQ ID NO: 1) was placed on a separate plasmid. The plasmid sequence was arranged as P5 promoter and Rep gene (wtAAV2 191-2196 nt, expressing Rep78 / 52 proteins), followed by P5 promoter sequence (wtAAV2 191-310 nt). Its structural diagram is shown below: Figure 6 shown.

[0121] pRep5 (SEQ ID NO: 6): The original pRC2 (SEQ ID NO: 2) plasmid was split, and the Rep2 gene in wtAAV2 (SEQ ID NO: 1) was placed on a plasmid alone. The plasmid sequence was arranged as P5 promoter and Rep gene (wtAAV2 191-2300 nt, expressing four proteins Rep78 / 68 / 52 / 40, wherein the GTACCA at the intron donor site was mutated to GTACGT, specifically, the CA at 1911-1912 nt was mutated to GT), and then the hGH intron sequence (SEQ ID NO: 3) was inserted before the start codon AUG of the Rep gene (wtAAV2 321 nt), and finally the P5 promoter sequence (wtAAV2 191-310 nt) was added after 2300 nt. Its structural schematic diagram is shown as follows Figure 7 shown.

[0122] pRep6 (SEQ ID NO: 7): The original pRC2 (SEQ ID NO: 2) plasmid was split, and the Rep2 gene in wtAAV2 (SEQ ID NO: 1) was placed on a plasmid alone. The plasmid sequence was arranged as P5 promoter and Rep gene (wtAAV2 191-2300 nt, expressing four proteins Rep78 / 68 / 52 / 40, wherein the GTACCA at the intron donor site was mutated to GTACGT, specifically, the CA at 1911-1912 nt was mutated to GT), and then the hGH intron sequence (SEQ ID NO: 3) was inserted before the AUG start codon of the Rep gene (wtAAV2 321 nt), and finally the polyA sequence was added after 2300 nt (wtAAV2 4411-4460 nt). Its structural schematic diagram is shown as follows Figure 8 shown.

[0123] pHelper-Cap2 (SEQ ID NO: 9): The Cap2 gene is placed on the pHelper plasmid (SEQ ID NO: 8), and the Cap sequence is arranged in the order of P5 promoter (wtAAV2 191-320nt), P19 promoter (wtAAV2 741-890nt, wherein the 768G is mutated to T), P40 promoter (wtAAV2 1701-2202nt, wherein the GTACCA at the intron donor site is mutated to GTACGT, specifically, the 1911-1912nt CA is mutated to GT), followed by the Cap2 gene (wtAAV2 2203-4410nt) and polyA sequence (wtAAV2 4411-4460nt). Its structural schematic diagram is shown as follows: Figure 4 shown. Example Example 1. Comparison of rAAV2-GOI1 viral genome titers packaged in the novel three-plasmid system and the traditional three-plasmid system

[0124] HEK293 cells were cultured in suspension in a 125 mL shake flask at 37°C, 8% CO2, 80% humidity, and 110 rpm. Inoculate at an appropriate density before transfection and dilute for 48 hours before transfection. The amount of plasmid used during transfection was 0.5-1 μg / 106 cells. The three plasmids were added in a certain proportion, and then the transfection reagent was added to form a transfection complex and added to the serum-free cultured VPC2.0 cells. After 72 hours of transfection, a high-multiple cell lysis buffer was added and treated at 37°C for 2 hours. After the reaction was completed, the shake flask was removed, a high-concentration NaCl solution was added to the shake flask, and then centrifuged at 3000×g for 10 minutes to collect the supernatant. The physical titer of rAAV2 in the cell lysis supernatant was determined by digital PCR (dPCR).

[0125] as follows Fig. 9 As shown, the pRC2 plasmid is a pRC2 plasmid that has undergone resistance modification (ampicillin-cannabinoid) in Agilent's traditional three-plasmid transfection system, and is suitable for the RC plasmid of serotype 2 rAAV produced clinically. The pRC2 plasmid was split, and the Cap gene was placed on the pHelper plasmid to form the pHelper-Cap2 plasmid, while the Rep gene was placed on a separate plasmid. Different modification methods were used to obtain the pRep1, pRep3, pRep5, and pRep6 plasmids. pHelper-Cap2 plasmid and different pRep plasmids were used to package rAAV2-GOI1 (target gene GOI1). From the viral genome titers, it can be seen that the viral genome titers packaged by pRep1, pRep3, pRep5, and pRep6 plasmids in the new three-plasmid system are different, which are 1.1E+11, 6.85E+10, 1.4E+11, and 2.4E+11 vg / mL, respectively. However, they are all higher than the titer packaged by the traditional three-plasmid system (4.30E+10 vg / mL), indicating that the new three-plasmid system is superior to the traditional three-plasmid system and can obtain a higher virus yield. Example 2: Process optimization data of rAAV2-GOI1 packaged with a novel three-plasmid system

[0126] The transfection conditions were optimized using pHelper-Cap2, pRep5, and pAAV-GOI1 plasmids to obtain higher titer rAAV2-GOI1. Two conditions were set for the amount of plasmid, 0.5 μg / 106 cells and 1 μg / 106 cells, respectively. The ratio of transfection reagent to plasmid (V / W) was also two parameters, 0.75 and 1.5, thus forming four conditions, such as Fig.10 As shown in the legend on the right, the first value indicates the amount of plasmid, and the second value indicates the amount of transfection reagent. In addition, there are four groups of ratios of the three plasmids pHelper-Cap2:pRep5:pAAV-GOI1, namely 1:1:0.4, 3:1:0.4, 1:1:1 and 3:1:1, and the cell density during transfection is set to low density (2.7E+06cells / mL) and high density (3.8E+06cells / mL) groups. As shown on the horizontal axis, there are 8 large groups, a total of 32 transfection conditions. From the perspective of viral genome titer, the yield is higher when the plasmid amount is 0.5μg / 106cells and the ratio of transfection reagent to plasmid (V / W) is 1.5. The titer of the three plasmid ratios of 3:1:0.4 and 3:1:1 is higher than that of 1:1:0.4 and 1:1:1, indicating that the effect is better when the amount of pHelper-Cap2 is larger. Example 3: Comparison of the solid rate of rAAV2-GOI1 packaged by the novel three-plasmid system and the traditional three-plasmid system

[0127] The rAAV2-GOI1 cell lysate supernatant was subjected to affinity chromatography using a Cytiva chromatography gravity column. The chromatography column was equilibrated with no less than 10 mL of equilibration buffer. After equilibration, 10 mL of clarified filtered collection liquid was added and allowed to flow through the chromatography column under gravity. After loading, the chromatography column was rinsed with no less than 10 mL of equilibration liquid. A sample collection container was then placed under the gravity column. The sample was first eluted with 1 mL of affinity eluent, the waste liquid was discarded, and then eluted with 2 mL of affinity eluent, the chromatography eluate (NAE) was collected, and the pH was adjusted to about 7.5 with a neutralization buffer. The collected affinity chromatography eluate was used to detect the solid rate using the SEC-MALS method. The test results showed that during the packaging process, the packaging efficiency in the new three-plasmid system using the pRep6 plasmid was higher than that in the traditional three-plasmid system using the pRC2 plasmid. Fig.11 As shown, for rAAV2-GOI1, the virus solidity rate packaged by the new three-plasmid system is 0.116, while the virus solidity rate packaged by the traditional three-plasmid system is only 0.051, and the solidity rate has been greatly improved. Example 4: Comparison of rcAAV residue detection in rAAV2-GOI1 packaged with the novel three-plasmid system and the traditional three-plasmid system

[0128] The cell lysate was purified by affinity chromatography and sterilized and filtered, and then co-infected with Ad5wt to infect 293T cells. After three rounds of infection, the dPCR method was used to detect the amount of rcAAV in the rAAV stock solution (the CT value represents the number of cycles, and the lower the CT value, the higher the infection titer, that is, the higher the rcAAV content).

[0129] The cells were seeded into T75 / T175 culture flasks, and at a confluence of >80%, the cells were digested and inoculated into 12-well plates. The next day, the cells were co-transfected with the rAAV2 product to be tested and the auxiliary wild-type adenovirus type 5 ("Ad5wt") at a multiplicity of infection (MOI) of 5×103, and 3 replicate wells were set for each rAAV2 sample to be tested. After 2 days of incubation, the cells were harvested by mechanical separation. The cells in each well in the first round were lysed by 3 freeze-thaw cycles (freezing at -80°C and thawing at 37°C), and Ad5wt was inactivated by incubation at 56°C for 30 minutes, and cell debris was removed by centrifugation at 2500g for 10 minutes. A new 12-well plate was co-infected with one-third of the lysate harvested for the first time in each well in the presence of Ad5wt as the second round of infection, and the remaining lysate was stored at -80°C for standby use (stored for investigation and analysis), and the whole process was repeated to collect samples from the second harvest. Cells seeded for the third transduction were transduced with lysate from the second harvest in the presence of Ad5wt, and the entire process was repeated to collect samples from the third harvest.

[0130] The samples harvested from the second amplification were treated with Proteinase K to separate DNA from the AAV particles packaged in the lysate. The lysates were then incubated at 90°C for 10 minutes to inactivate Proteinase K, and then diluted with EASY Dilution to obtain samples for qPCR testing.

[0131] A volume of 5 μL of the dPCR test sample was added to a 20 μL qPCR reaction, and the qPCR test sample was amplified using primers specific for the Rep coding sequence ("Rep") to confirm whether AAV containing the Rep sequence is present in the rAAV2 / Ad5wt-infected wells. The standard and qPCR test sample containing the Rep sequence were detected at the same time. After the reaction was completed, a threshold was set to compare the detection CT value of the standard with the CT value of the dPCR test sample. If the CT value of the qPCR test sample is less than the CT value of the standard, it indicates that rcAAV is detected in the sample (the smaller the CT value, the higher the level of rcAAV detected).

[0132] The positive control used wild-type AAV2 (wtAAV2), the structure of which is shown in the figure below. Figure 3 Negative controls consisted of uninfected cells (in the presence of adenovirus), which were tested in the same manner as the rAAV2 products.

[0133] As shown in Table 1, each sample was infected with three replicates. The CT values ​​of the samples packaged with the pRep5 plasmid in the new three-plasmid system at the 1E+08 level were 32.899, 34.507, and UND, respectively, where UND. stands for undetected. The CT values ​​of the samples packaged with the pRep6 plasmid in Table 2 were 38.631, 36.039, and 37.554, respectively, indicating that there were very few rcAAV residues, <1rcAAV / 1×108vg. As shown in Table 3, the CT value of the virus packaged by the traditional three-plasmid system was 29.632 in the replicate two, indicating that the residual amount was >1rcAAV / 1×108vg. Therefore, the amount of rcAAV in the rAAV2-GOI packaged by the new three-plasmid system is less than that of the traditional three-plasmid system, and the purity is higher. Table 1 Detection results of rcAAV with the novel three-plasmid system packaging rAAV2-GOI1 Table 2 rcAAV detection results of the new three-plasmid system packaging rAAV2-GOI1 Table 3 Detection results of rcAAV packaged with rAAV2-GOI1 in the traditional three-plasmid system Example 5. Comparison of Cap Residue Detection in rAAV2-GOI1 Packaged by the New Three-plasmid System and the Traditional Three-plasmid System

[0134] After the sample to be tested was diluted in a certain ratio, it was added to a 20 μL qPCR reaction, and the q sample was amplified using a specific probe for the primer of the Cap coding sequence ("Cap") to confirm whether there were any impurities containing the Cap sequence remaining during the packaging process. As shown in Table 4, the Cap residue results in the rAAV2-GOI1 viral vector packaged with pRep5 plasmid and pRep6 plasmid and pHelper-Cap2 were comparable to or even lower than those of the traditional three-plasmid system, indicating that the new three-plasmid packaging system is relatively safe. Table 4 Cap residue detection results of the novel three-plasmid system and the traditional three-plasmid system for packaging rAAV2-GOI1

Claims

1. A helper plasmid, characterized in that The invention comprises a nucleic acid sequence encoding a Cap gene and a nucleic acid sequence encoding at least one auxiliary gene for adeno-associated virus (AAV) production, wherein the Cap gene encodes a functional Cap protein.

2. The helper plasmid according to claim 1, characterized in that The method 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.

3. The helper plasmid according to claim 2, characterized in that It comprises P5 promoter, P19 promoter, and P40 promoter; preferably, 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.

4. The helper plasmid according to claim 2 or 3, 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.

5. The helper plasmid according to any one of claims 2 to 4, 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.

6. The helper plasmid according to any one of claims 2 to 5, 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.

7. The helper plasmid according to any one of claims 2 to 6, 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.

8. The helper plasmid according to any one of claims 1 to 7, 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.

9. The helper plasmid according to any one of claims 1 to 8, 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.

10. The helper plasmid according to claim 9, 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 is no intervening nucleotide.

11. The helper plasmid according to any one of claims 9 to 10, 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.

12. The helper plasmid according to any one of claims 1 to 11, characterized in that The helper plasmid cannot express functional Rep protein.

13. The helper plasmid according to any one of claims 1 to 12, characterized in that The helper plasmid does not contain the nucleic acid sequence encoding the Rep gene.

14. The helper plasmid according to any one of claims 1 to 13, characterized in that The auxiliary genes for AAV production include one or more genes selected from E2A, E4, and VA RNA.

15. The helper plasmid according to claim 14, characterized in that The auxiliary genes include E2A, E4, and VARNA.

16. A plasmid, characterized in that Contains a nucleic acid sequence encoding the Rep gene, and the plasmid cannot express a functional Cap protein.

17. The plasmid according to claim 16, characterized in that The plasmid does not contain the nucleic acid sequence encoding the Cap gene.

18. The plasmid according to claim 16 or 17, characterized in that The plasmid is unable to express functional expression products of auxiliary genes for AAV production.

19. The plasmid according to any one of claims 16 to 18, characterized in that The plasmid does not contain nucleic acid sequences encoding auxiliary genes for AAV production.

20. The plasmid according to any one of claims 16 to 19, characterized in that The Rep gene expresses Rep78, Rep68, Rep52, and Rep40 proteins.

21. The plasmid according to claim 20, characterized in that 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.

22. The plasmid according to any one of claims 16 to 19, characterized in that The Rep gene expresses Rep78 and Rep52 proteins; optionally, the Rep gene does not express Rep68 or Rep40 proteins.

23. The plasmid according to claim 22, characterized in that 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.

24. The plasmid according to any one of claims 16 to 23, characterized in that The plasmid 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.

25. The plasmid according to claim 24, 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 promoter and the Rep gene; preferably, there is no intervening nucleotide.

26. The plasmid according to any one of claims 24-25, 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.

27. The plasmid according to any one of claims 16 to 26, characterized in that The nucleic acid sequence encoding the Rep gene comprises an hGH intron; preferably, the intron is located before the start codon of the Rep gene.

28. The plasmid according to claim 27, 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 P5 promoter and the hGH intron, and / or between the hGH intron and the Rep gene; preferably, there are no intervening nucleotides.

29. The plasmid according to any one of claims 27-28, 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:

3.

30. The plasmid according to any one of claims 16 to 29, characterized in that The donor site of the original intron of the Rep gene contains a mutation, and the donor site corresponds to the nucleotide sites 1907-1912 of SEQ ID NO:

7.

31. The plasmid according to claim 30, characterized in that The mutation of the donor site comprises mutating CA corresponding to nucleotides 1911-1912 of SEQ ID NO:7 to GT, or mutating the donor site sequence corresponding to nucleotides 1907-1912 of SEQ ID NO:7 to GTACGT.

32. The plasmid according to any one of claims 16 to 31, 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.

33. The plasmid according to claim 32, 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 is no intervening nucleotide.

34. A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that: Include: (i) the helper plasmid according to any one of claims 1 to 15; (ii) a plasmid encoding the Rep gene according to any one of claims 16 to 33; and (iii) a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence.

35. A three-plasmid system for adeno-associated virus (AAV) expression, characterized in that: Include: (i) a helper plasmid (pHelper plasmid), wherein the helper plasmid comprises a recombinant nucleic acid encoding a Cap gene; (ii) a plasmid encoding the Rep gene of an adeno-associated virus (AAV); and (iii) a plasmid encoding a recombinant adeno-associated virus comprising a heterologous nucleic acid sequence.

36. The three-plasmid system according to claim 35, characterized in that (i) the helper plasmid is unable to express a functional Rep protein, and / or (ii) the plasmid is unable to express a functional Cap protein.

37. The three-plasmid system according to any one of claims 34 to 36, characterized in that: The (iii) plasmid encoding a recombinant adeno-associated virus containing a heterologous nucleic acid sequence further comprises at least two AAV inverted repeat sequences (ITR), wherein the two AAV inverted repeat sequences are located upstream and downstream of the heterologous nucleic acid sequence, respectively.

38. The three-plasmid system according to any one of claims 34 to 37, characterized in that: The serotype of the AAV is AAV2.

39. The three-plasmid system according to any one of claims 34 to 38, characterized in that The heterologous nucleic acid sequence encodes a heterologous gene.

40. A method for producing a recombinant adeno-associated virus, characterized in that: The plasmid described in any one of claims 1 to 33 or all plasmids in the plasmid system described in any one of claims 34 to 39 are transfected into a host cell, cultured, and isolated to obtain the recombinant adeno-associated virus.

41. The production method according to claim 40, characterized in that The ratio of the (i) helper 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.

42. The production method according to claim 40 or 41, characterized in that The ratio of the (i) helper 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.

43. The production method according to any one of claims 40 to 42, characterized in that: The ratio of (ii) the plasmid encoding the Rep gene to (iii) the plasmid encoding the recombinant adeno-associated virus containing 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.

44. The production method according to any one of claims 40 to 43, characterized in that: The ratio of (ii) the plasmid encoding the Rep gene to (iii) the plasmid encoding the recombinant adeno-associated virus containing 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.

45. A recombinant adeno-associated virus, characterized in that Obtained by the production method according to any one of claims 40-44.

46. ​​The recombinant adeno-associated virus according to claim 45, characterized in that The host cell culture method includes adherent culture or suspension culture.

47. A host cell, characterized in that The host cell contains all plasmids in the plasmid system described in any one of claims 34-39.

48. The host cell according to claim 47, characterized in that The host cell is selected from HEK293 cells, HEK293T cells, HEK293F cells, VPC2.0 cells, Hela cells and A549 cells.

49. A pharmaceutical preparation, characterized in that The drug comprises the recombinant adeno-associated virus according to claim 45 or 46, and a pharmaceutically acceptable carrier and excipient.

50. Use of the pharmaceutical preparation according to claim 49 in treating diseases caused by gene mutation and / or gene deletion of a target gene in a human body.

51. A method for treating a disease, comprising administering the recombinant adeno-associated virus according to claim 45 or 46, or the pharmaceutical preparation according to claim 49 to a patient in need thereof.

52. A kit comprising the plasmid of any one of claims 1-33, the plasmid system of any one of claims 34-39, the production method of any one of claims 40-44, the recombinant adeno-associated virus of claim 45 or 46, or the host cell of any one of claims 47-48.

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