An expression cassette of adeno-associated virus Cap protein

Abstract

The present disclosure relates to the field of genetic engineering, and in particular to an expression frame of an adeno-associated virus Cap protein. The expression frame of the Cap protein comprises a truncated 5'UTR sequence for expressing VP2 / 3 polypeptides. The expression frame of the Cap protein disclosed herein can achieve the split expression of VP1 and VP2 / 3.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to an expression frame of an adeno-associated virus Cap protein. Background Art

[0002] AAV is currently the most commonly used vector for gene therapy and has many advantages. Currently, large-scale production of AAV is usually carried out by transient transfection of three plasmids in suspended 293 cells. However, this method has disadvantages such as high production cost, insufficient yield and difficulty in amplification. These problems seriously limit the cost control and production reliability of AAV vectors in commercial applications. Therefore, the industry is in urgent need of finding ways to improve the efficiency of transient transfection virus packaging and to establish AAV production cell lines. Summary of the invention

[0003] On the one hand, the present disclosure provides an expression frame for expressing VP2 / 3 polypeptide, wherein the 5'UTR sequence of the expression frame for expressing VP2 / 3 polypeptide is a truncated 5'UTR sequence, and the length of the truncated 5'UTR sequence is less than 400 bp, preferably 21 bp to 351 bp, and more preferably 81 bp to 261 bp.

[0004] In some embodiments, the 5' end of the truncated 5' UTR sequence is truncated by more than 90 bp, such as 90-420 bp.

[0005] In some embodiments, the expression cassette is used for the expression of VP2 polypeptide and VP3 polypeptide, but not VP1 polypeptide.

[0006] In some embodiments, the truncated 5'UTR sequence is selected from the nucleotide sequence shown in SEQ ID NO: 2-6, or a nucleotide sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the nucleotide sequence shown in any one of SEQ ID NO: 2-6.

[0007] In another aspect, the present disclosure provides an expression cassette for expressing a VP1 polypeptide, wherein the 5'UTR sequence of the expression cassette is replaced with an SV40 intron or a chimeric intron.

[0008] In some embodiments, the SV40 intron comprises the nucleotide sequence shown in SEQ ID NO:8, or a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:8.

[0009] In some embodiments, the chimeric intron comprises the sequence shown in SEQ ID NO:9, or a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:9.

[0010] In some embodiments, the expression cassette for expressing the VP1 polypeptide is used for the expression of the VP1 polypeptide, but does not express the VP2 polypeptide and the VP3 polypeptide.

[0011] In some embodiments, the splicing site of the intron comprises one or more nucleotide mutations, such as a mutation from TCCA to ACCC 18-21 bp after the start codon of the AAV8 VP1 expression cassette.

[0012] On the other hand, the present disclosure provides a vector comprising the above-mentioned expression cassette for expressing VP2 / 3 polypeptide and / or the expression cassette for expressing VP1 polypeptide of the present disclosure.

[0013] On the other hand, the present disclosure provides a virus packaging system, which comprises an expression frame for expressing a VP1 polypeptide, an expression frame for expressing a VP2 / 3 polypeptide, and other essential element expression frames for packaging viruses, wherein the expression frame for expressing the VP1 polypeptide is the expression frame for expressing the VP1 polypeptide of the present disclosure above; and / or the expression frame for expressing the VP2 / 3 polypeptide is the expression frame for expressing the VP2 / 3 polypeptide of the present disclosure above.

[0014] In some embodiments, the virus is selected from AAV virus, and the serotype of AAV is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17 and AAV serotypes modified based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

[0015] In another aspect, the present disclosure provides a cell line comprising the above-mentioned vector of the present disclosure, or comprising the above-mentioned virus packaging system of the present disclosure.

[0016] AAV Cap protein contains VP1, 2, and 3, which are originally expressed in a nested form. The expression frame cannot be split. Both VP1 and VP2 / 3 are expressed by the endogenous promoter P40, which limits the expression regulation of VP1 and VP2 / 3.

[0017] In response to the above problems, the present disclosure splits the expression frame of the Cap protein and replaces the P40 promoter with an exogenous promoter, so that the VP1 and VP2 / 3 proteins are expressed separately, the expression intensity is improved, and the VP1 expression ratio is increased, thereby increasing the yield of AAV virus packaging and the activity of the virus. The present disclosure further utilizes the split expression frame to achieve AAV packaging, and the AAV generated by the packaging has a high yield and good activity. At the same time, such an expression frame structure can be stably integrated into cells using different methods to construct an AAV production cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present disclosure can be more fully understood with reference to the following drawings.

[0019] Figure 1 The results of Luciferase expression using the split Cap expression plasmid for viral packaging are shown. AAV8 (WT) was used as a control.

[0020] Figure 2 The Western blot results of virus packaging using the split Cap expression plasmid are shown.

[0021] Figure 3 The Western blot results of virus packaging using split expression plasmids for VP1 and VP2 / 3 are shown.

[0022] Figure 4 The results of Luciferase expression using split expression plasmids of VP1 and VP2 / 3 for viral packaging are shown.

[0023] Figure 5 The Western blot results of virus packaging using split expression plasmids for VP1 and VP2 / 3 are shown.

[0024] Figure 6 The Western blot results of virus packaging using split expression plasmids for VP1 and VP2 / 3 are shown.

[0025] Figure 7 The Western blot results of virus packaging using split expression plasmids for VP1 and VP2 / 3 are shown.

[0026] Figure 8The Western blot results of virus packaging using split expression plasmids for VP1 and VP2 / 3 are shown.

[0027] Fig. 9 The results of Luciferase expression using split expression plasmids of VP1 and VP2 / 3 for viral packaging are shown.

[0028] Fig.10 The results of Luciferase expression using split expression plasmids of VP1 and VP2 / 3 for viral packaging are shown.

[0029] Fig.11 The results of Luciferase expression using split expression plasmids of VP1 and VP2 / 3 for viral packaging are shown.

[0030] Fig.12 The Western blot results of virus packaging using split expression plasmids for VP1 and VP2 / 3 are shown. DETAILED DESCRIPTION

[0031] The following description of the present disclosure is intended only to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0032] In one aspect, the present disclosure provides an expression cassette for expressing a VP2 / 3 polypeptide, wherein the 5'UTR sequence of the expression cassette is a truncated 5'UTR sequence, and the length of the truncated 5'UTR sequence is less than 400 bp.

[0033] Preferably, the length of the truncated 5'UTR sequence is less than 351 bp, preferably 21 bp to 351 bp, and more preferably 81 bp to 261 bp. In some embodiments, the length of the truncated 5'UTR sequence can be, for example, 20 bp, 60 bp, 80 bp, 100 bp, 150 bp, 200 bp, 250 bp, 270 bp, 300 bp, 350 bp, 400 bp, or any range consisting of the above lengths.

[0034] In some embodiments, the 5' end of the truncated 5' UTR sequence is truncated by 90-420 bp, for example, it can be truncated by 90 bp, 180 bp, 270 bp, 360 bp, 420 bp or any range consisting of the above lengths.

[0035] By including the above-mentioned truncated 5'UTR sequence, the expression frame for expressing VP2 / 3 polypeptides disclosed in the present invention can achieve the split expression of VP1 and VP2 / 3 proteins. That is, the expression frame is used for the expression of VP2 polypeptide and VP3 polypeptide, but does not express VP1 polypeptide.

[0036] In some embodiments, the nucleotide sequence of the 5'UTR before truncation is as shown in SEQ ID NO: 1.

[0037] In some embodiments, the truncated 5'UTR sequence is selected from the nucleotide sequence shown in SEQ ID NO: 2-6, or a nucleotide sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the nucleotide sequence shown in any one of SEQ ID NO: 2-6.

[0038] In another aspect, the present disclosure provides an expression cassette for expressing a VP1 polypeptide, wherein a 5'UTR sequence of the expression cassette is replaced with an SV40 intron or a chimeric intron.

[0039] In some embodiments, the SV40 intron comprises the nucleotide sequence set forth in SEQ ID NO: 8, or a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleotide sequence of the SV40 intron is set forth in SEQ ID NO: 8.

[0040] In some embodiments, the chimeric intron comprises the nucleotide sequence set forth in SEQ ID NO: 9, or a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleotide sequence of the chimeric intron is set forth in SEQ ID NO: 9.

[0041] By using the above-mentioned intron containing the truncated 5'UTR sequence, the expression frame for expressing the VP1 polypeptide disclosed in the present invention can achieve the split expression of VP1 and VP2 / 3 proteins. That is, the expression frame is used for the expression of the VP1 polypeptide, but does not express the VP2 polypeptide and the VP3 polypeptide.

[0042] In some embodiments, the splicing site of the intron may include one or more nucleotide mutations. In the present disclosure, the splicing site may be an intron splicing acceptor site (such as intron acceptor splicing site 2). Specifically, the nucleotide mutation may include: a mutation from TCCA to ACCC 18-21 bp after the start codon of the AAV8 VP1 expression frame.

[0043] On the other hand, the present disclosure provides a vector comprising the above-mentioned expression cassette for expressing VP2 / 3 polypeptide and / or the expression cassette for expressing VP1 polypeptide of the present disclosure.

[0044] In the present disclosure, the vector can be specifically selected according to the AAV serotype to be expressed. The vector can be obtained by introducing the expression frame of the present disclosure through genetic engineering means on the basis of a commercially available or self-prepared plasmid.

[0045] On the other hand, the present disclosure provides a virus packaging system, which comprises an expression frame for expressing a VP1 polypeptide, an expression frame for expressing a VP2 / 3 polypeptide, and other essential element expression frames for packaging viruses, wherein the expression frame for expressing the VP1 polypeptide is the expression frame for expressing the VP1 polypeptide of the present disclosure above; and / or the expression frame for expressing the VP2 / 3 polypeptide is the expression frame for expressing the VP2 / 3 polypeptide of the present disclosure above.

[0046] In some embodiments, the virus is selected from AAV virus. The serotype of AAV is selected from AAV9, 8, 1, 2, 3, 3B, 4, 5, 6, 7, 10, 11, 12, 13, rh10 or hu37 and any AAV serotype isolated from human and non-human mammals or variants thereof.

[0047] Genes required for packaging AAV viruses can generally include: (a) a nucleic acid template comprising at least one AAV ITR sequence, (b) an AAV sequence sufficient to replicate the nucleic acid template and encapsulate in an AAV capsid (e.g., an AAV rep sequence and an AAV cap sequence encoding an AAV capsid), and (c) a coding sequence comprising an auxiliary gene. Optionally, the nucleic acid template may further comprise at least one heterologous nucleic acid sequence. In some embodiments, the nucleic acid template comprises two AAV ITR sequences located at the 5' and 3' ends of the heterologous nucleic acid sequence, respectively.

[0048] In the present disclosure, as the virus packaging system, for example, it can be a single plasmid, two plasmids, three plasmids or multi-plasmid adeno-associated virus packaging system composed of multiple plasmids containing AAV Rep protein coding region sequences, AAV Cap protein VP1, VP2, VP3 coding region sequences, ITR sequences, and target gene coding sequences and auxiliary gene coding sequences as required. Among them, the Cap protein VP1 polypeptide is expressed by the expression frame expressing the VP1 polypeptide of the present disclosure, and / or the Cap protein VP2 / 3 polypeptide is expressed by the expression frame expressing the VP2 / 3 polypeptide of the present disclosure.

[0049] In some embodiments, the virus packaging system comprises an expression plasmid for expressing VP1 polypeptide, VP2 polypeptide, VP3 polypeptide and other necessary elements for packaging virus, wherein the expression plasmid for expressing VP1 polypeptide, VP2 polypeptide, VP3 polypeptide and other necessary elements for packaging virus comprises the above-mentioned expression frame for expressing VP1 polypeptide and / or expression frame for expressing VP2 / 3 polypeptide of the present invention.

[0050] In some embodiments, the viral packaging system comprises a first expression plasmid for expressing VP1 polypeptide, VP2 polypeptide and VP3 polypeptide, and a second expression plasmid for expressing other necessary elements for packaging virus, wherein the expression plasmid for expressing VP1 polypeptide, VP2 polypeptide and VP3 polypeptide comprises the above-mentioned expression frame for expressing VP1 polypeptide and / or the expression frame for expressing VP2 / 3 polypeptide of the present invention.

[0051] In some embodiments, the viral packaging system comprises a first expression plasmid for expressing a VP1 polypeptide, a second expression plasmid for expressing a VP2 polypeptide and a VP3 polypeptide, and a third expression plasmid for expressing other necessary elements for packaging the virus; wherein the first expression plasmid comprises the expression frame for expressing the VP1 polypeptide of the present invention as described above; and / or the second expression plasmid comprises the expression frame for expressing the VP2 / 3 polypeptide of the present invention as described above.

[0052] In another aspect, the present disclosure provides a cell line comprising the above-mentioned vector of the present disclosure, or comprising the above-mentioned virus packaging system of the present disclosure.

[0053] The host cells suitable for the cell lines of the present disclosure are not particularly limited and are known in the art. Preferably, the host cells show constitutive E1A expression. In a preferred embodiment, the cell line is selected from HEK293 cells (including cells obtained by gene editing, transformation, domestication, and / or screening of HEK293 cells such as HEK293T, HEK293F, or HEK293FT cells), CAP cells, Per.C6 cells, etc.

[0054] The method for producing the cell line of the present invention, i.e., the method for introducing the expression cassette, vector or viral packaging system of the present invention into a suitable host cell, is not particularly limited and is known in the art. For example, the cell line can be obtained by transfecting cells with the above-mentioned vector or viral packaging system.

[0055] As used herein, the terms "nucleotide" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0056] The term "promoter" as used herein means a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence, such as a gene or a transgene. Promoters can be constitutive, inducible, repressible or tissue-specific.

[0057] As used herein, the term "intron" refers to an intervening sequence within a gene that does not appear in the mature RNA molecule and is removed by processing after transcription.

[0058] The term "splicing site" or "acceptor splicing site" as used herein means the boundary between the right end of an intron and the left end of an adjacent exon. The exons and introns of eukaryotic genes are transcribed in a primary transcript RNA molecule, and the intron is excised to produce a mature RNA molecule. This process is called RNA splicing. When an intron is spliced, a splicing point in the upstream direction is called the 5' splicing point or the left splicing point, and a splicing point in the downstream direction of the intron is called the 3' splicing point or the right splicing point. In the disclosure, VP2 / 3 intron acceptor splicingsite 2 refers to the 3' splicing point of the intron.

[0059] The term "UTR" or "untranslated region" as used herein refers to any fragment located at both ends of the coding sequence of the mRNA chain or the corresponding DNA chain. If it is located at the 5' end, it is called the 5' untranslated region (5'UTR), whereas if it is located at the 3' end, it is called the 3' untranslated region (3'UTR).

[0060] The term "expression cassette" as used herein refers to the complete elements required to express a gene, including an operably linked promoter and gene coding sequence.

[0061] The term "vector" as used herein refers to a nucleic acid comprising, consisting essentially of, or consisting of a complete replicon, such that the vector can be replicated when placed into a cell by, for example, a transfection, infection, or transformation process. It is understood in the art that once inside a cell, the vector can replicate as an extrachromosomal (episomal) element, or can be integrated into a host cell chromosome. The vector may include nucleic acids derived from retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papillomaviruses, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like, preferably, the vector described herein is selected from an AAV vector, an adenoviral vector, or a lentiviral vector.

[0062] As used herein, the term "adeno-associated virus" or "AAV" refers to members of the class of viruses associated with that name and belonging to the genus Dependoviridae of the family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows exclusively in cells, where certain functions are provided by a co-infecting helper virus. All AAV serotypes clearly display a homologous rep Gene-mediated very similar replication characteristics; and all carry three related capsid proteins (Cap). At least 13 naturally occurring AAV serotypes numbered in sequence are known in the art. Non-limiting exemplary serotypes used in the methods disclosed herein include any of these 13 serotypes, such as AAV2, AAV8, AAV9, or variant serotypes such as AAV-DJ and AAV PHP.B. AAV particles contain three major viral proteins VP1, VP2 and VP3, and are essentially composed of or composed of the proteins. In an embodiment, the AAV comprises an AAV capsid protein selected from the group consisting of AAVPHP.B, AAVrh74, AAV 110, AAV 204, AAV 214, AAV214A, AAV 214e, AAV 214e8, AAV 214e9, AAV 214e10, AAV ITB102_45 and AAV 214AB. In an embodiment, AAV refers to serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and novel AAV serotypes modified based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, or any one of the AAV serotypes isolated from human and non-human mammals, or variants thereof.

[0063] Example

[0064] The following examples are illustrative only and are not intended to limit the scope or content of the present disclosure in any way.

[0065] Western Blot Detection of Cap Protein

[0066] FectoVIR-AAV transfection reagent was used to transfect the Cap protein expression plasmid or co-transfect multiple plasmids on suspended HEK293 cells. 72 h after transfection, a total of about 3M cells were collected and washed by centrifugation with dPBS, and repeated 3 times. After washing, the cells were resuspended in RIPA lysis buffer and lysed by ultrasonication. After lysis, the supernatant was taken as the sample to be tested for Western Blot (WB), and the sample was quantified by BCA protein. For Western Blot, equal amounts of protein were loaded on SDS-PAGE according to the BCA protein quantification results and electrophoresed. After electrophoresis, the cells were transferred to the membrane, and the expression of VP1 / 2 / 3 was detected using anti-AAV VP1 / VP2 / VP3 mouse monoclonal, B1 (Progen 65158).

[0067] AAV virus packaging

[0068] FectoVIR-AAV transfection reagent was used to package the co-transfection virus of multiple plasmids in suspended HEK293 cells. 72 h after transfection, the cells were collected by centrifugation, resuspended in lysis buffer, and lysed by freeze-thaw in liquid nitrogen three times. After lysis, 80 IU / mL nuclease was added to the lysate and digested at 37℃ for 1 h. After digestion, the cell debris was removed by centrifugation at 10000 g for 10 min, and the supernatant was the crude AAV virus extract.

[0069] AAV virus crude extract titer detection

[0070] After the crude virus extract was diluted, it was treated with DNase. After the treatment, the sample was heated at 95°C to release the AAV viral genome. After the lysate was graded diluted, the AAV viral crude extract genome titer was detected using the BioRad QX200 ddPCR system.

[0071] AAV virus activity detection

[0072] One day before infection, Huh-7 cells were plated in 96-well plates at a density of 20,000 cells / well. On the day of infection, 5 μL of crude AAV virus extract was added directly to the wells for infection. After 72 h of infection incubation, the 96-well plate was removed and 100 μL of Luciferase detection reagent (Biyuntian RG056M) was added directly to each well. After incubation at room temperature for more than 5 minutes, the signal reading of each well was detected using the chemiluminescence mode of the microplate reader to represent the activity of the AAV virus.

[0073] Sequence Listing

[0074] Table A

[0075]

[0076] Table B

[0077]

[0078] Table C

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] In the table, the uppercase part indicates the 5'UTR, and the uppercase bold part indicates the endogenous intron.

[0086] Example 1: Split expression of Cap protein

[0087] (1) Construction of Cap protein expression plasmid

[0088] The Cap protein is expressed by the P40 promoter, which is nested in the Rep protein expression frame. In order to express the Cap protein alone, the Cap protein expression part in RC2 / 8 (Addgene 112864, containing SEQ ID NO: 14) was cloned and the P40 promoter was replaced with an exogenous promoter to construct the plasmid shown in Table 1 below to express the Cap protein alone.

[0089] Table 1

[0090]

[0091] (2) Use the split Cap expression plasmid for viral packaging

[0092] The Rep expression part of the RC2 / 8 plasmid was cloned separately to construct the Rep protein expression plasmid. The Cap protein expression plasmid separated in Table 1 was co-transfected with the Rep protein expression plasmid, Helper plasmid (Cell Biolabs 340202) and AAV010 (ITR plasmid, obtained by constructing the Luciferase and Zsgreen expression frames into Addgene 105922) in suspended HEK293 cells for virus packaging. Cell lysate was collected 72 hours after transfection and used to infect Huh-7 cells. Luciferase expression was detected 72 hours after infection to represent the virus packaging yield and activity, and compared with the virus packaged by the conventional three-plasmid transfection system. The normalized Luciferase expression results are shown in Figure 2. Figure 1 At the same time, the cell lysate was subjected to Western blot to detect the expression of Cap protein VP1 / 2 / 3. The WB results are shown in Figure 2 shown.

[0093] From the above results, it can be seen that after the Cap protein is split, VP1 / 2 / 3 can be expressed normally, the expression intensity is close to the original structure, and the VP1 / 2 / 3 ratio is close to the original structure. At the same time, the split Cap plasmid can also support AAV transient virus packaging.

[0094] Example 2: Split expression of VP1 and VP2 / 3

[0095] (1) Construction of VP1 and VP2 / 3 split expression plasmids

[0096] VP1 and VP2 / 3 are expressed from different start codons under a unified expression frame. The split expression of VP1 and VP2 / 3 can be achieved by deleting part of the intron sequence before the VP1 start codon (58-351 bp of SEQ ID NO: 14) and connecting the expression frames to exogenous promoters respectively, and constructing the plasmids shown in Table 2 below.

[0097] Table 2

[0098]

[0099] (2) Split expression of VP1 and VP2 / 3

[0100] The plasmids in Table 2 were used to transfect suspended HEK293 cells, and the unsplit VP1 / 2 / 3 expression plasmids were transfected as controls. 72 h after transfection, the cells were collected and lysed for Western Blot detection of the expression of each Cap protein. The WB results are shown in Figure 3As shown in the WB results, VP1 and VP2 / 3 can be expressed separately.

[0101] (3) Use the split expression VP1 and VP2 / 3 plasmids for virus packaging

[0102] The VP1 and VP2 / 3 protein expression plasmids separated in Table 2 were co-transfected with the Rep protein expression plasmid, Helper plasmid (Cell Biolabs 340202) and AAV010 (ITR plasmid) in suspension HEK293 cells for virus packaging. Cell lysate was collected 72 hours after transfection and used to infect Huh-7 cells. Luciferase expression was detected 72 hours after infection to represent the virus packaging yield and activity, and compared with the virus transfected and packaged with the unsplit VP1 / 2 / 3 expression plasmid RC2 / 8 control plasmid (Addgene 112864). The Luciferase results after normalization are shown in Figure 2. Figure 4 At the same time, the cell lysate was subjected to Western blot to detect the expression of Cap protein VP1 / 2 / 3. The WB results are shown in Figure 5 shown.

[0103] From the above results, it can be seen that after VP1 / 2 / 3 were split and expressed, VP1 / 2 / 3 could be expressed normally, with expression intensity close to the original structure, and the proportion of VP1 was increased. At the same time, the split Cap plasmid could also support AAV transient virus packaging; compared with the unsplit version 410, the virus activity titer after packaging was higher.

[0104] Example 3: Optimization of VP2 / 3 expression cassette

[0105] (1) Construction of VP2 / 3 expression plasmids containing UTRs of different lengths

[0106] By gradually shortening the length of the 5'UTR of VP2 / 3, plasmids with 5'UTR lengths as shown in Table 3 were constructed.

[0107] Table 3

[0108]

[0109] (2) Expression of VP2 / 3 proteins containing different lengths of UTR

[0110] Each plasmid in Table 3 was used to transfect suspended HEK293 cells, and plasmid 471 containing full-length UTR was transfected as a control. After 72 hours of transfection, the cells were collected and lysed for Western Blot detection of the expression of each Cap protein. The WB results are shown in Figure 6 The grayscale analysis of the images was performed using ImageJ software, and the results are shown in Table 4.

[0111] Table 4

[0112]

[0113] *“9.4E+04” in the table means 9.4×10 4 , and so on.

[0114] From the above results, it can be seen that when the length of UTR is reduced to 21 bp or less, the expression intensity of VP2 will be significantly reduced; while when the UTR is between 81 bp and 261 bp, the expression intensity of VP2 and VP3 is high.

[0115] Example 4: Optimization of VP1 expression cassette

[0116] (1) Construction of VP1 expression plasmids containing different introns

[0117] The plasmids shown in Table 5 were constructed by deleting the endogenous UTR of the VP1 expression cassette (corresponding to 1-351 bp of SEQ ID NO: 14) and inserting different exogenous introns.

[0118] Table 5

[0119]

[0120] (2) Expression of VP1 using different exogenous introns

[0121] The plasmids in Table 5 were used to transfect suspended HEK293 cells, and the 472 plasmid containing UTR but no exogenous intron was transfected as a control. After 72 h of transfection, the cells were collected and lysed for Western Blot detection of the expression of each Cap protein. The WB results are shown in Figure 7 The grayscale analysis of the images was performed using ImageJ software, and the results are shown in Table 6.

[0122] Table 6

[0123]

[0124] From the above results, it can be seen that the expression ratio of VP1 protein was significantly increased after replacing SV40 intron or chimeric intron.

[0125] (3) Construction of VP1 expression plasmid with splice acceptor site mutation

[0126] Based on plasmids 472 and 497, the plasmids shown in Table 7 were constructed by mutating TCCA (corresponding to 651-654 bp of SEQ ID NO: 13) of VP2 / 3 intron acceptor splicing site 2 to ACCC.

[0127] Table 7

[0128]

[0129] (4) Expression of VP1 plasmid containing splice acceptor site mutation

[0130] The plasmids in Table 7 were used to transfect suspended HEK293 cells, and 472 and 497 without splice site mutations were transfected as controls. 72 h after transfection, the cells were collected and lysed for Western Blot detection of the expression of each Cap protein. The WB results are shown in Figure 8 The grayscale analysis of the images was performed using ImageJ software, and the results are shown in Table 8.

[0131] Table 8

[0132]

[0133] From the above results, it can be seen that after the splicing site mutation, the expression intensity of VP1 is improved, while the VP1 expression ratio remains unchanged or further increases.

[0134] Example 5: Serotype expansion

[0135] (1) Construction of plasmids for split expression of VP1 and VP2 / 3 of different serotypes

[0136] According to the splitting and transformation methods of plasmids 497 and 479, the RC2 / 2 control plasmid (Addgene 104963) and the RC2 / 9 control plasmid (Addgene 112865) were transformed respectively to obtain the plasmids shown in Table 9 below.

[0137] Table 9

[0138]

[0139] (2) Use the split expression VP1 and VP2 / 3 plasmids to package each serotype virus

[0140] AAV2, AAV8 and AAV9 were packaged using AAV8 split plasmids 497 and 479 and the expression plasmids in Table 9, respectively. Virus packaging was performed by co-transfection with Rep protein expression plasmid, Helper plasmid (Cell Biolabs 340202) and AAV010 (ITR plasmid) in suspension HEK293 cells. Cell lysate was collected 72 h after transfection and used to infect Huh-7 cells. Luciferase expression was detected 72 h after infection to represent the virus packaging yield and activity, and compared with the virus transfected with the respective serotype control packaging plasmids without split VP1 / 2 / 3. The normalized Luciferase expression results are shown in Figure 2. Figure 9-11 At the same time, the cell lysate was subjected to Western blot to detect the expression of Cap protein VP1 / 2 / 3. The WB results are shown in Fig.12 shown.

[0141] From the above results, it can be seen that after VP1 / 2 / 3 are split and expressed, VP1 / 2 / 3 can be expressed normally, the total expression amount is increased, and the VP1 ratio is increased. At the same time, the split Cap plasmid can also support AAV transient virus packaging. Compared with the conventional virus packaging method, the virus activity titer after packaging is higher.

[0142] Incorporated by Reference

[0143] Each patent and scientific article mentioned herein is incorporated by reference in its entirety for all purposes.

[0144] Equivalence

[0145] The present disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present disclosure is indicated by the appended claims rather than by the above description, and all changes within the equivalent meaning and scope of the claims are intended to be covered therein.

Claims

1. An expression cassette for expressing the VP2 / 3 polypeptide of AAV virus, characterized in that: The 5'UTR sequence of the expression frame for expressing the VP2 / 3 polypeptide of the AAV virus is a truncated 5'UTR sequence, the length of the truncated 5'UTR sequence is 81 bp to 351 bp, the truncated 5'UTR sequence is selected from the nucleotide sequence shown in SEQ ID NO: 2-5, and the expression frame for expressing the VP2 / 3 polypeptide of the AAV virus is used for the expression of the VP2 polypeptide and VP3 polypeptide of the AAV virus, but does not express the VP1 polypeptide of the AAV virus.

2. The expression cassette according to claim 1, wherein: The length of the truncated 5'UTR sequence is 81 bp to 261 bp.

3. A vector comprising the expression frame for expressing the VP2 / 3 polypeptide of the AAV virus as described in claim 1 or 2.

4. A virus packaging system, comprising an expression cassette for expressing the VP1 polypeptide of the AAV virus, an expression cassette for expressing the VP2 / 3 polypeptide, and other expression cassettes of necessary elements for packaging the virus; in, The expression frame for expressing the VP2 / 3 polypeptide is the expression frame for expressing the VP2 / 3 polypeptide of the AAV virus described in any one of claims 1-3.

5. The virus packaging system according to claim 4, wherein: The 5'UTR sequence of the expression frame expressing the VP1 polypeptide of the AAV virus is replaced by an SV40 intron or a chimeric intron, the nucleotide sequence of the SV40 intron is shown in SEQ ID NO: 8, and the nucleotide sequence of the chimeric intron is shown in SEQ ID NO:

9.

6. The virus packaging system according to claim 4, wherein: The expression frame for expressing the VP1 polypeptide of the AAV virus is used for the expression of the VP1 polypeptide, but does not express the VP2 polypeptide and the VP3 polypeptide.

7. The virus packaging system according to claim 5, wherein: The splicing site of the SV40 intron or the chimeric intron comprises one or more nucleotide mutations.

8. The virus packaging system according to claim 7, wherein: The splicing site of the SV40 intron or the chimeric intron comprises a nucleotide mutation corresponding to 651-654 bp of SEQ ID NO: 13, from TCCA to ACCC.

9. The virus packaging system according to any one of claims 4 to 8, wherein: The virus is selected from AAV virus, and the serotype of AAV is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17 and AAV serotypes modified based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

10. A cell line comprising the vector of claim 3, or the viral packaging system of any one of claims 4 to 9.

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