An expression cassette for inducible expression of Rep polypeptide

Abstract

The present disclosure relates to the field of genetic engineering, and in particular to an inducible P19 promoter and an expression frame for inducible expression of Rep polypeptides. The inducible P19 promoter comprises an intron located downstream of a TATA frame, and two copies of a TetO inducible element are inserted into the intron. The expression frame for inducible expression of Rep polypeptides comprises the inducible P19 promoter, which is used to induce the expression of Rep52 polypeptides and Rep40 polypeptides. The expression frame for inducible expression of Rep polypeptides disclosed in the present disclosure can achieve the induced expression of each Rep protein, and the expression amount after induced expression is high, and the background leakage before induction is low.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to an expression frame for inducibly expressing a Rep polypeptide. 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] In one aspect, the present disclosure provides an inducible P19 promoter, wherein the inducible P19 promoter comprises an intron located downstream of the TATA box, and two copies of TetO inducible elements are inserted into the intron. Preferably, the two copies of TetO inducible elements are inserted after the 5' end common motif or before the 3' end polypyrimidine region of the intron.

[0004] In some embodiments, the intron is located downstream of the TATA box, such as 1-10 bp after the TATA box, preferably 1 bp after the TATA box.

[0005] In some embodiments, the inducible P19 promoter comprises a nucleotide sequence selected from SEQ ID NOs: 1-18.

[0006] On the other hand, the present disclosure provides an expression cassette for inducibly expressing a Rep polypeptide, wherein the expression cassette comprises the inducible P19 promoter of the present disclosure described above, and the inducible P19 promoter is used to induce the expression of a Rep52 polypeptide and a Rep40 polypeptide.

[0007] In some embodiments, the P5 promoter that expresses the Rep78 polypeptide and the Rep68 polypeptide is replaced with an inducible promoter.

[0008] In some embodiments, the inducible promoter is derived from P5, EF1A, RSV, CMV, MNDU3, hCEF1, or SV40 promoter, preferably from EF1A, RSV or CMV promoter.

[0009] In another aspect, the present disclosure provides a vector comprising the inducible P19 promoter or the inducible expression cassette for expressing the Rep polypeptide of the present disclosure.

[0010] In another aspect, the present disclosure provides a virus packaging system, which comprises the above-mentioned vector of the present disclosure and an expression plasmid of other necessary elements for packaging viruses.

[0011] 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 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, and AAVHSC17.

[0012] 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.

[0013] In order to achieve the induced expression of each Rep protein (including Rep78 / 68, Rep52 / 40), it is usually necessary to replace the P5 and P19 promoters that initiate Rep expression with inducible promoters. However, since the P19 promoter used to express Rep52 / 40 is nested in the expression frame of Rep78 / 68, direct replacement will destroy the expression frame and affect the expression of Rep protein. In response to the above problems, the inventors designed the inducible control element in the intron and inserted the intron into the P19 promoter to achieve the effect of induced expression without affecting the expression of the overall expression frame; and further replaced the P5 promoter with an inducible promoter, thereby achieving the induced expression of each Rep protein.

[0014] By adopting the inducible expression frame of the Rep polypeptide disclosed in the present invention, the expression amount of the Rep protein after induction is high, and the background leakage before induction is low. The inducible AAV virus packaging can be achieved by using the inducible plasmid containing the expression frame. At the same time, this structure can also be stably integrated into cells by various means to construct an inducible expression AAV production cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present disclosure can be more fully understood with reference to the following drawings.

[0016] Figure 1 Shown are the results of Western blot using inducible plasmids to express each Rep protein.

[0017] Figure 2 The expression levels of the Luciferase reporter gene in Huh-7 cells infected with cell lysates of AAV packaged with each inducible plasmid are shown.

[0018] Figure 3 The expression levels of the Luciferase reporter gene in Huh-7 cells infected with cell lysates of AAV packaged with each inducible plasmid are shown.

[0019] Figure 4 Shown are the results of Western blot using inducible plasmids to express each Rep protein.

[0020] Figure 5 The expression levels of the Luciferase reporter gene in HEK293T cells infected with cell lysates of AAV packaged with each inducible plasmid are shown.

[0021] Figure 6 The expression levels of the Luciferase reporter gene in HEK293T cells infected with cell lysates of AAV packaged with each inducible plasmid are shown. DETAILED DESCRIPTION

[0022] 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.

[0023] In one aspect, the present disclosure provides an inducible P19 (also referred to as iP19) promoter, wherein the inducible P19 promoter comprises an intron located downstream of a TATA box, and two copies of a TetO inducible element are inserted into the intron. Preferably, the two copies of the TetO inducible element are inserted after the 5' end common motif of the intron or before the 3' end polypyrimidine region.

[0024] The term "TetO inducible element" as used herein refers to a tetracycline regulated gene (TetO), which can change the conformation of the regulatory protein by inducing drugs (such as tetracycline and doxycycline (DOX) etc.), thereby achieving the purpose of regulating the expression of the target protein. In the inducible P19 promoter disclosed in the present invention, the target proteins are Rep52 and Rep40 polypeptides.

[0025] In some embodiments, the nucleotide sequence of the TetO inducible element may be, for example, TCCCTATCAGTGATAGAGA (SEQ ID NO: 24).

[0026] In some embodiments, there is at least a 2-5 bp interval, preferably a 2 bp interval, between the two copies of the TetO operator, and the spacer sequence is preferably TC.

[0027] As used herein, the term "5' common motif" refers to the site recognized by U1 snRNP pairing; and the term "3' polypyrimidine region" refers to the site recognized by U2 snRNP.

[0028] In some embodiments, the two copies of the TetO inducible element are inserted 0-5 bp (preferably 0-2 bp or 0-1 bp) after the 5' common motif of the intron and / or 0-5 bp (preferably 0-2 bp or 0-1 bp) before the 3' polypyrimidine region. In some embodiments, the two copies of the TetO inducible element are inserted at a position adjacent to the 5' common motif and / or the 3' polypyrimidine region.

[0029] In some embodiments, the intron is located downstream of the TATA box, such as 1-10 bp after the TATA box, preferably 1 bp after the TATA box.

[0030] In some embodiments, the intron sequence in the inducible P19 promoter is selected from SEQ ID NO: 1-18. Wherein, in the introns shown in SEQ ID NO: 1-14, the TetO inducible element is inserted after the 5' common motif. The 5' common motif as shown in these sequences is a sequence starting with gt before TetO, which may be different between each intron. In the introns shown in SEQ ID NO: 15-18, the TetO inducible element is inserted before the 3' polypyrimidine region. The 3' polypyrimidine region as shown in these sequences is a sequence starting with ct / tt after the TetO sequence, which may also be different between each intron. Preferably, two copies of the TetO inducible element are inserted after the 5' common motif of the intron.

[0031] The inducible P19 promoter of the present disclosure can be used for the expression of Rep52 polypeptides and Rep40 polypeptides from various AAV serotypes.

[0032] On the other hand, the present disclosure provides an expression cassette for inducible expression of Rep polypeptide, wherein the expression cassette comprises the inducible P19 promoter of the present disclosure, and the inducible P19 promoter is used to induce expression of Rep52 polypeptide and Rep40 polypeptide.

[0033] In order to achieve the inducible expression of each Rep polypeptide, preferably, the P5 promoter for expressing the Rep78 polypeptide and the Rep68 polypeptide is replaced with an inducible promoter.

[0034] The inducible promoter used can be "endogenous," "exogenous," or "heterologous" relative to the gene to which it is operably linked. An "endogenous" promoter is one that is naturally associated with a given gene in the genome. An "exogenous" or "heterologous" promoter is one that has been placed in juxtaposition to a gene by genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is directed by the linked promoter.

[0035] In some embodiments, the inducible promoter may be derived from, for example, P5, EF1A, RSV, CMV, MNDU3, hCEF1, or SV40 promoter, wherein the inducible promoter is preferably derived from EF1A, RSV or CMV promoter.

[0036] In the expression frame of the inducible expression Rep polypeptide disclosed herein, the induced expression of Rep78 / 68 polypeptide is achieved by an inducible promoter, and the expression of Rep52 / 40 polypeptide is achieved by the inducible P19 promoter disclosed herein. It is understood that the inducible P19 promoter is located within the coding sequence of Rep78 and Rep68 and forms a part of the coding sequence.

[0037] In another aspect, the present disclosure provides a vector comprising the promoter or expression cassette of the present disclosure.

[0038] In the present disclosure, the vector can be specifically selected according to the AAV serotype to be expressed, for example, it can be an RC plasmid for AAV8 packaging, etc. The vector can be obtained by introducing the inducible promoter or expression frame of the present disclosure through genetic engineering means on the basis of a commercially available or self-prepared plasmid.

[0039] In another aspect, the present disclosure provides a virus packaging system, which comprises the above-mentioned vector of the present disclosure and an expression plasmid of other necessary elements for packaging viruses.

[0040] In some embodiments, the virus is selected from AAV virus, and 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 their variants.

[0041] 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.

[0042] In the present disclosure, the virus packaging system may be, for example, a one-plasmid, two-plasmid, three-plasmid or multi-plasmid adeno-associated virus packaging system composed of one or more 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. Wherein, the Rep52 polypeptide and the Rep40 polypeptide are induced to express under the regulation of the inducible P19 promoter.

[0043] In some specific embodiments, the virus packaging system comprises a first plasmid, a second plasmid and a third plasmid, wherein the first plasmid comprises the coding region of the Rep protein, the second plasmid comprises the coding region of the Cap proteins VP1, VP2 and VP3, and the third plasmid comprises the coding region of the target gene.

[0044] In some specific embodiments, the virus packaging system comprises a first plasmid and a second plasmid, wherein the first plasmid comprises the coding region of the Rep protein and the Cap proteins VP1, VP2 and VP3, and the second plasmid comprises the coding region of the target gene.

[0045] In another aspect, the present disclosure provides a cell line comprising the inducible P19 promoter or expression cassette of the present disclosure, or comprising the vector of the present disclosure or the virus packaging system of the present disclosure.

[0046] In the present disclosure, the inducible P19 promoter is stably integrated into the host cell genome or contained in a vector, thereby being stably integrated into the cell.

[0047] 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.

[0048] The method for producing the cell line of the present invention, i.e., the method for introducing the inducible P19 promoter, 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.

[0049] 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.

[0050] The term "promoter" as used herein means a control sequence, which is a region of a polynucleotide sequence that controls the transcription initiation and rate of a coding sequence such as a gene or a transgenic gene. The promoter can be constitutive, inducible, repressible, or tissue-specific. In some embodiments, the promoter is used together with the polynucleotides described herein to improve transcription efficiency. In some embodiments, the promoter described herein is an inducible P19 promoter.

[0051] 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.

[0052] 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).

[0053] 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.

[0054] 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.

[0055] 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, the 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.

[0056] Example

[0057] The following examples are illustrative only and are not intended to limit the scope or content of the present disclosure in any way.

[0058] Western blot detection of Cap protein

[0059] FectoVIR-AAV transfection reagent was used to transfect the Rep protein expression plasmid or co-transfect multiple plasmids in suspended HEK293 cells. 72 h after transfection, a total of about 3 M cells were collected and washed by centrifugation with dPBS, and repeated 3 times. After washing, the cells were resuspended in RIPA lysis buffer and ultrasonically lysed. After lysis, the supernatant was taken as the sample to be tested by Western Blot, 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 each Rep protein was detected using anti-AAV2 Replicase mouse monoclonal, 303.9 (Progen 61069).

[0060] AAV virus packaging

[0061] FectoVIR-AAV transfection reagent was used to perform multi-plasmid co-transfection virus packaging 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°C for 1 h. After digestion, centrifuge at 10,000 g for 10 minutes to remove cell debris, and the supernatant was the crude AAV virus extract.

[0062] Titer detection of AAV virus crude extract

[0063] The crude virus extract was diluted and treated with DNase, and then the sample was heated at 95°C to release the AAV viral genome. After further serial dilution of the lysate, the genome titer of the crude AAV viral extract was detected using the BioRad QX200 ddPCR system.

[0064] AAV virus activity detection

[0065] 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.

[0066] In the following examples, the intron sequence of the inducible P19 promoter is shown in Table A, and the expression cassette sequence is shown in Table B.

[0067] Table A

[0068]

[0069] In the above table, capital letters represent TetO sequences.

[0070] Table B

[0071]

[0072]

[0073]

[0074] In the above table, capital letters represent intron sequences, bold represents TATA box, and underlined parts represent promoter sequences.

[0075] Example 1 Construction and screening of inducible P19 promoter

[0076] (1) Construction of inducible P19 promoter

[0077] The P19 promoter initiates the expression of Rep52 / 40, but since it is nested in the Rep78 / 68 expression frame, adding an inducible element directly to the P19 promoter will destroy the Rep78 expression frame. Therefore, in this embodiment, two copies of the TetO inducible element (TCCCTATCAGTGATAGAGA, SEQ ID NO: 24) are designed in different introns, and these introns containing the TetO inducible element are inserted into the P19 TATA frame 1 bp later to form an inducible P19 promoter for inducible initiation of Rep52 / 40 expression. These expression frames that can induce Rep52 / 40 expression are constructed into the plasmid RC2 / 8 (Addgene 112864) for AAV8 packaging, replacing the original Rep expression frame. The constructed plasmids are shown in Table 1 below.

[0078] Table 1

[0079]

[0080] (2) Screening of inducible P19 promoter

[0081] Each inducible P19 plasmid in Table 1 was combined with the Helper plasmid (Cell Biolabs 340202) and the TetR-krab control element plasmid (Szulc, J. et al. A versatile tool for conditional gene expression and knockdown. Nat Methods,3, 109-116 (2006)) were co-transfected in suspended HEK293 cells, and the expression of Rep52 / 40 protein was induced by adding DOX after transfection. The co-transfected cells were collected and lysed after three days of culture, and the expression of each Rep protein was detected by Western Blot (WB). The WB results are shown in Figure 1 As shown. Figure 1 It can be seen that some plasmids showed good induction effect of Rep52 / 40 protein, and the insertion of intron did not affect the expression of Rep78 / 68.

[0082] The images were analyzed using ImageJ software for quantification. Figure 1 The expression levels of Rep78 and Rep52 proteins expressed by each plasmid are shown in Table 2 below.

[0083] Table 2

[0084]

[0085] In the above table, “4.0E+04” means 4.0×10 4 , and so on.

[0086] (3) Testing the performance of RC plasmid containing inducible P19 promoter in AAV8 virus packaging

[0087] The selected RC plasmids (LYR1P023, 026, 027, 028, 029, 036) containing the inducible P19 promoter were transiently transfected with AAV010 (ITR plasmid) and Helper plasmid (Cell Biolabs 340202) in suspended HEK293 cells for three-plasmid viral packaging. As a control, the inducible structure reported by US20200199627A1 (LYR1P033 from LONZA, containing SEQ ID NO: 9) and the conventional non-inducible RC2 / 8 control plasmid were transfected at the same time. After transfection, the expression of Rep52 / 40 protein was induced by adding DOX to achieve the effect of inducing AAV8 virus packaging. Cell lysate was collected 3 days after transfection and infected Huh-7 cells in 96-well plates. The expression level of the Luciferase reporter gene in the infected cells was detected 3 days after infection, and the yield and activity of virus packaging in each group were determined.

[0088] The results after normalization of Luciferase reporter gene expression levels are as follows Figure 2 As shown. Figure 2It can be seen that RC plasmids LYR1P027, 028, and 029 have better induced virus packaging effects while maintaining good virus packaging yield and activity after induction. Compared with the prior art LYR1P033, LYR1P027, 028, and 029 have lower background when not induced, indicating less leakage of Rep52 / 40 protein expression.

[0089] Example 2 Construction and screening of inducible Rep expression plasmids

[0090] (1) Construction of a novel inducible Rep expression cassette

[0091] The expression of inducible Rep78 / 68 proteins can be achieved by replacing the P5 promoter with an exogenous inducible promoter. Specifically, the P5 promoter was replaced with the inducible iEF1A, iRSV and iCMV promoters on the plasmids LYR1P027 and 028 selected in Example 1, so that the expression of each Rep78 / 68 / 52 / 40 protein can be induced. The constructed plasmids are shown in Table 3.

[0092] Table 3

[0093]

[0094] (2) Testing the performance of the inducible Rep expression plasmid during AAV8 virus packaging

[0095] The inducible Rep plasmid, AAV010 (ITR plasmid) and Helper plasmid (Cell Biolabs 340202) were used for three-plasmid transient virus packaging on suspension HEK293. As a control, the RC2 / 8 control plasmid (Addgene112864) was transfected at the same time. After transfection, DOX was added to induce the expression of Rep78 / 68 / 52 / 40 proteins to achieve the effect of inducing AAV8 virus packaging.

[0096] Three days after transfection, the cell lysate was collected and used to infect Huh-7 cells in a 96-well plate. Three days after infection, the expression level of the Luciferase reporter gene in the infected cells was detected, and the yield and activity of the virus packaging in each group were determined. The results after normalization of the Luciferase reporter gene expression level are as follows: Figure 3 At the same time, the cells were harvested and lysed, and the expression of Rep protein was detected by Western Blot (WB). The WB results are shown in Figure 4 shown.

[0097] The above virus packaging results and WB results indicate that by using these Rep expression cassettes, all Rep proteins have achieved good induced expression effects, and the background before induction is low. At the same time, these Rep expression cassettes can also support the inducible packaging of AAV viruses.

[0098] Example 3 Expansion of serotypes

[0099] (1) Construction of different AAV serotype plasmids containing inducible Rep expression cassettes

[0100] Based on the same modification method of plasmid LYR1P545, the RC2 / 2 (Addgene 104963) and RC2 / 5 (Addgene 104964) control plasmids were modified to construct different AAV serotype plasmids containing the novel inducible Rep expression cassette, as shown in Table 4.

[0101] Table 4

[0102]

[0103] (2) Using plasmids containing a novel inducible Rep expression cassette to package viruses of various serotypes

[0104] AAV2 and AAV5 were packaged using the plasmids in Table 4. Virus packaging was performed by co-transfection with Helper plasmid (Cell Biolabs340202) and AAV010 (ITR plasmid) in suspension HEK293 cells. Cell lysate was collected 72 h after transfection to infect HEK293T cells. Luciferase expression was detected 72 h after infection to represent the virus packaging yield and activity, and compared with the virus packaged by traditional Rep-Cap plasmid transfection. The Luciferase results after normalization are shown in Figure 5 , Figure 6 shown.

[0105] The above virus packaging results suggest that the use of these Rep expression cassettes can support the inducible packaging of various serotypes of AAV viruses.

[0106] Incorporated by Reference

[0107] Each patent and scientific article mentioned herein is incorporated by reference in its entirety for all purposes.

[0108] Equivalence

[0109] 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 inducible P19 promoter, characterized in that The inducible P19 promoter comprises an intron located downstream of the TATA box, and two copies of TetO inducible elements are inserted into the intron, wherein the nucleotide sequence of the intron is selected from any one of SEQ ID NOs: 1, 2, 4, 6-11 and 14.

2. The inducible P19 promoter according to claim 1, wherein The intron is located 1-10 bp downstream of the TATA box.

3. The inducible P19 promoter according to claim 1 or 2, wherein The two copies of the TetO inducible element are inserted after the common motif at the 5' end or before the polypyrimidine region at the 3' end of the intron.

4. An expression cassette for inducible expression of a Rep polypeptide, wherein: The expression cassette comprises the inducible P19 promoter according to any one of claims 1 to 3, and the inducible P19 promoter is used to induce the expression of Rep52 polypeptide and Rep40 polypeptide.

5. The expression frame according to claim 4, wherein: The P5 promoter for expressing the Rep78 polypeptide and the Rep68 polypeptide was replaced with an inducible promoter.

6. The expression cassette according to claim 5, wherein: The inducible promoter is derived from P5, EF1A, RSV, CMV, MNDU3, hCEF1 or SV40 promoter.

7. A vector comprising the inducible P19 promoter according to any one of claims 1 to 3 or the inducible expression cassette for expressing a Rep polypeptide according to any one of claims 4 to 6.

8. A virus packaging system, comprising the vector according to claim 7, and an expression plasmid of other necessary elements for packaging viruses.

9. The viral packaging system according to claim 8, wherein the virus is selected from AAV virus, and the serotype of the 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 7, or comprising the viral packaging system of claim 8 or 9.

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