CN118460545B

CN118460545B - An expression cassette for inducible expression of Rep polypeptide

Info

Publication number: CN118460545B
Application number: CN202410910284.1A
Authority: CN
Inventors: 陈意雄; 林卿; 蒋天祺; 盛晓菁
Original assignee: Shanghai Lingyi Biotechnology Co ltd
Current assignee: Shanghai Lingyi Biotechnology Co ltd
Priority date: 2024-07-08
Filing date: 2024-07-08
Publication date: 2025-02-11
Anticipated expiration: 2044-07-08
Also published as: CN118460545A

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

Expression cassette for inducible expression of Rep polypeptide

Technical Field

The disclosure belongs to the field of genetic engineering, and in particular relates to an expression cassette for inducible expression of Rep polypeptides.

Background

AAV currently has many advantages as the most commonly used vector for gene therapy. Currently, large-scale production of AAV is typically performed using three plasmid cotransfection of suspension 293 cells. However, this approach has disadvantages of high production cost, insufficient yield, and difficult amplification, which severely limit the cost control and production reliability of AAV vectors for commercial applications. Therefore, there is an urgent need in the industry to find ways to increase packaging efficiency of transiently transfected viruses and to establish AAV-producing cell lines.

Disclosure of Invention

In one aspect, the present disclosure provides an inducible P19 promoter, wherein the inducible P19 promoter comprises an intron downstream of the TATA box, and wherein two copies of a TetO inducing element are inserted into the intron. Preferably, the two copies of the TetO inducing element are inserted after the 5 'common motif of the intron or before the 3' polypyrimidine region.

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

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

In another aspect, the present disclosure provides an expression cassette for inducible expression of a Rep polypeptide, wherein the expression cassette comprises an inducible P19 promoter of the present disclosure described above, the inducible P19 promoter being used to induce expression of a Rep52 polypeptide and a Rep40 polypeptide.

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

In some embodiments, the inducible promoter is derived from a P5, EF1A, RSV, CMV, MNDU, hCEF, or SV40 promoter, preferably from an EF1A, RSV or CMV promoter.

In another aspect, the present disclosure provides a vector comprising the inducible P19 promoter of the present disclosure or an expression cassette for inducible expression of a Rep polypeptide described above.

In another aspect, the present disclosure provides a viral packaging system comprising the vector of the present disclosure described above, and other essential element expression plasmids for packaging a virus.

In some embodiments, the virus is selected from an AAV virus, the serotype of which 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 engineered based on AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAVrh.8、AAVrh.10、AAVrh.39、AAVHSC15、AAVHSC17.

In another aspect, the present disclosure provides a cell line comprising a vector of the present disclosure described above, or a viral packaging system of the present disclosure described above.

To achieve the inducible expression of each Rep protein (including Rep78/68, rep 52/40), the P5 and P19 promoters that initiate Rep expression are typically replaced with inducible promoters. However, since the P19 promoter for expressing Rep52/40 is nested in the expression cassette of Rep78/68, direct replacement will disrupt the expression cassette, affecting Rep protein expression. In order to solve the problems, the inventor designs an induction control element in an intron and inserts the intron into a P19 promoter to achieve the effect of induction expression without affecting the expression of the whole expression frame, and further replaces the P5 promoter with the induction promoter, thereby realizing the induction expression of each protein of Rep.

By adopting the expression cassette for inducible expression of the Rep polypeptide, the expression quantity of the Rep protein after induced expression is high, the background leakage is low before induction, and the inducible plasmid containing the expression cassette can be used for realizing the packaging of the inducible AAV virus. At the same time, the structure can be stably integrated into cells in various ways to construct an induced expression AAV production cell line.

Drawings

The present disclosure may be more fully understood with reference to the following drawings.

FIG. 1 shows Western blot results of expression of each Rep protein using an inducible plasmid.

FIG. 2 shows the expression levels of the Luciferase reporter gene in Huh-7 cells infected with cell lysates of AAV packaged with each inducible plasmid.

FIG. 3 shows the expression levels of the Luciferase reporter gene in Huh-7 cells infected with cell lysates of AAV packaged with each inducible plasmid.

FIG. 4 shows Western blot results of expression of each Rep protein using an inducible plasmid.

FIG. 5 shows expression levels of the Luciferase reporter gene in HEK293T cells infected with cell lysates of AAV packaged with respective inducible plasmids.

FIG. 6 shows expression levels of the Luciferase reporter gene in HEK293T cells infected with cell lysates of AAV packaged with respective inducible plasmids.

Detailed Description

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

In one aspect, the present disclosure provides an inducible P19 (also referred to as iP 19) promoter, the inducible P19 promoter comprising an intron downstream of a TATA box (TATA box) with two copies of a TetO inducing element inserted therein. Preferably, the two copies of the TetO inducing element are inserted after the 5 'common motif of the intron or before the 3' polypyrimidine region.

The term "TetO-inducing element" as used herein refers to a tetracycline regulatory gene (TetO) that is capable of modulating expression of a protein of interest by altering the conformation of the regulatory protein by inducing drugs such as tetracycline and Doxycycline (DOX), etc. In the inducible P19 promoters of the present disclosure, the proteins of interest are Rep52 and Rep40 polypeptides.

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

In some embodiments, there is at least a separation of 2-5 bp, preferably a separation of 2 bp, preferably a TC between the two copies of the TetO operator.

The term "5 'common motif" as used herein refers to the site of U1 snRNP pairing recognition, and the term "3' multimeric pyrimidine region" refers to the site of U2 snRNP recognition.

In some embodiments, the two copies of the TetO inducing 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-inducing element are inserted at positions immediately adjacent to the 5 'terminal common motif and/or the 3' terminal polypyrimidine region.

In some embodiments, the intron sequence in the inducible P19 promoter is selected from the group consisting of SEQ ID NOS.1-18. Wherein, in the introns shown in SEQ ID NO. 1-14, the TetO inducing element is inserted after the 5' terminal common motif. The 5' common motif shown in these sequences is a TetO pre-gt initiated sequence that will vary from one intron to another. In the introns shown in SEQ ID NOS.15-18, a TetO inducing element is inserted before the 3' -terminal polypyrimidine region. The 3' terminal polypyrimidine region shown in these sequences is the sequence that begins ct/tt after the TetO sequence, which also varies from one intron to another. Preferably, two copies of the TetO inducing element are inserted after the 5' common motif of the intron.

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

To achieve the induced expression of each Rep polypeptide, preferably, the P5 promoter expressing the Rep78 polypeptide and the Rep68 polypeptide is replaced with an inducible promoter.

The inducible promoter used may be "endogenous", "exogenous" or "heterologous" with respect 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 a promoter that is 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.

In some embodiments, the inducible promoter may be, for example, a promoter derived from P5, EF1A, RSV, CMV, MNDU, hCEF1, or SV 40. Wherein the inducible promoter is preferably derived from the EF1A, RSV or CMV promoter.

In the expression cassette of the present disclosure for inducible expression of the Rep polypeptide, the inducible expression of the Rep78/68 polypeptide is achieved by an inducible promoter and the expression of the Rep52/40 polypeptide is achieved by an inducible P19 promoter of the present disclosure. It will be appreciated that the inducible P19 promoter is located within and forms part of the coding sequences of Rep78 and Rep 68.

In another aspect, the present disclosure provides a vector comprising the promoter or expression cassette of the present disclosure described above.

In the present disclosure, the vector may be specifically selected according to the AAV serotype to be expressed, and for example, may be an RC plasmid for AAV8 packaging, or the like. The vector can be obtained by introducing the inducible promoter or expression cassette of the present disclosure by genetic engineering means on the basis of a commercially available or self-prepared plasmid.

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

As genes required for packaging AAV viruses, there may generally be included (a) a nucleic acid template comprising at least one AAV ITR sequence, (b) AAV sequences sufficient to replicate the nucleic acid template and to be packaged into an AAV capsid (e.g., AAV rep sequences and AAV cap sequences encoding an AAV capsid), and (c) coding sequences comprising a helper 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 5 'and 3' of the heterologous nucleic acid sequence, respectively.

In the present disclosure, as the virus packaging system, for example, one plasmid, two plasmid, three plasmid or multi-plasmid adeno-associated virus packaging system comprising one or more plasmids including AAV Rep protein coding region sequences, AAV Cap protein VP1, VP2, VP3 coding region sequences, ITR sequences, and, as required, target gene coding sequences and auxiliary gene coding sequences may be mentioned. Wherein, the Rep52 polypeptide and the Rep40 polypeptide are induced to express under the control of an inducible P19 promoter.

In some embodiments, the viral packaging system comprises a first plasmid comprising a coding region for a Rep protein, a second plasmid comprising coding regions for Cap proteins VP1, VP2, and VP3, and a third plasmid comprising a coding region for a gene of interest.

In some embodiments, the viral packaging system comprises a first plasmid comprising the Rep protein, and the coding regions for Cap proteins VP1, VP2, and VP3, and a second plasmid comprising the coding region for the gene of interest.

In another aspect, the present disclosure provides a cell line comprising the inducible P19 promoter or expression cassette of the present disclosure described above, or comprising the vector of the present disclosure described above or the viral packaging system of the present disclosure described above.

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

Host cells suitable for use in the cell lines of the present disclosure are not particularly limited and are known in the art. Preferably, the host cell displays constitutive E1A expression. In preferred embodiments, the cell line is selected from HEK293 cells (including cells obtained by genetic editing, engineering, acclimatization, and/or selection of HEK293 cells, such as HEK293T, HEK293F, or HEK293FT cells), CAP cells, per.c6 cells, and the like.

Methods for generating the cell lines of the present disclosure, i.e., methods for introducing the inducible P19 promoter, vector or viral packaging system of the present disclosure into a suitable host cell, are not particularly limited and are known in the art. For example, cells can be transfected with the vectors or the virus packaging system.

The terms "nucleotide" and "polynucleotide" are used interchangeably herein to 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 derivatized nucleotide bases.

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 may be constitutive, inducible, repressible, or tissue specific. In some embodiments, promoters are used with the polynucleotides described herein to increase transcription efficiency. In some embodiments, the promoters described herein are inducible P19 promoters.

The term "intron" as used herein means a spacer sequence within a gene that is not present in a mature RNA molecule and is excised by processing after transcription.

The term "UTR" or "untranslated region" as used herein means any fragment located on either side of an mRNA strand or the coding sequence of a corresponding DNA strand. If it is located at the 5 'end, it is referred to as the 5' untranslated region (5 'UTR), whereas if it is located at the 3' end, it is referred to as the 3 'untranslated region (3' UTR).

The term "expression cassette" as used herein refers to the complete elements required for expression of a gene, including operably linked promoters and gene coding sequences.

The term "vector" as used herein refers to a nucleic acid comprising, consisting essentially of, or consisting of an intact replicon such that the vector may be replicated when placed into a cell by, for example, transfection, infection, or transformation procedures. It will be appreciated in the art that once inside the cell, the vector may replicate as an extrachromosomal (episomal) element, or may be integrated into the host cell chromosome. The vector may comprise nucleic acid derived from a retrovirus, adenovirus, herpes virus, baculovirus, modified baculovirus, papilloma virus, AAV viral vector, lentiviral vector, adenovirus vector, alphaviral vector, etc., preferably the vector described herein is selected from AAV vector, adenovirus vector, or lentiviral vector.

The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with that name and belonging to the genus parvoviridae-dependent parvovirus. Adeno-associated virus is a single stranded DNA virus that grows only in cells, with some functions provided by co-infected helper viruses. All AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes and all bear three associated capsid proteins (caps). At least 13 sequentially numbered naturally occurring AAV serotypes are known in the art. Non-limiting exemplary serotypes for use in the methods disclosed herein include any of these 13 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes such as AAV-DJ and AAV php.b. AAV particles comprise, consist essentially of, or consist of three major viral proteins VP1, VP2, and VP 3. In embodiments, the AAV comprises an AAV capsid protein selected from the group consisting of AAVPHP.B、AAVrh74、AAV 110、AAV 204、AAV 214、AAV 214A、AAV 214e、AAV 214e8、AAV 214e9、AAV 214el 0、AAV ITB102_45 and AAV 214 AB. In embodiments, the AAV refers to serotype AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAVrh.8、AAVrh.10、AAVrh.39、AAVHSC15、AAVHSC17, as well as novel AAV serotypes engineered 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 of the AAV serotypes isolated from human and non-human mammals, or variants thereof.

Examples

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

Western blot detection of Cap protein

Transfection of Rep protein expression plasmids or cotransfection of multiple plasmids was performed in suspension HEK293 cells using FectoVIR-AAV transfection reagents. After transfection, 72 h, the total amount of cells was collected about 3M, washed by centrifugation with pbs, and repeated 3 times. After washing was completed, resuspended with RIPA lysate and sonicated. Taking the supernatant after the completion of the cleavage to obtain a Western Blot sample to be detected, and quantifying BCA protein of the sample. In Western Blot, proteins of the same amount were loaded on SDS-PAGE according to the quantitative result of BCA proteins, and subjected to electrophoresis, followed by transfer, and expression of each protein of Rep was detected using anti-AAV2 REPLICASE MOUSE MONOCLONAL, 303.9 (Progen 61069).

AAV viral packaging

Cotransfection virus packaging of multiple plasmids was performed in suspension HEK293 cells using FectoVIR-AAV transfection reagents. After transfection, 72 h, cells were collected by centrifugation, resuspended in lysis buffer and lysed by 3 freeze thawing cycles in liquid nitrogen. After lysis, 80 IU/mL nuclease 37℃was added to the lysate to digest 1 h. After digestion, the mixture is centrifuged for 10 minutes by using a centrifuge 10000 g to remove cell debris, and the supernatant is taken as AAV virus crude extract.

Titer detection of AAV crude extracts

After dilution of the crude virus extract, treatment with DNase was performed, and the samples were then subjected to thermal cleavage at 95 ℃ to release AAV viral genomes. After further gradient dilution of the lysate, the genomic titer of the AAV virus crude extract was detected using the BioRad QX200 ddPCR system.

AAV virus viability assay

The day before infection, 96-well plates were plated with Huh-7 cells at a density of 20000 cells/well. On the day of infection, 5. Mu.L of AAV crude extract was directly added to the wells for infection. After 72 h infection incubations, 96-well plates were removed and 100 μl of Luciferase detection reagent (bi cloud sky RG 056M) was directly added to each well. After incubation at room temperature for more than 5 minutes, signal readings from each well were measured using a chemiluminescent format of the microplate reader to represent AAV viral viability.

In the following examples, the intron sequences of the inducible P19 promoter are shown in Table A and the expression cassette sequences are shown in Table B.

Table A

In the above table, capital letters indicate TetO sequences.

Table B

In the above table, capital letters represent intron sequences, bold indicates TATA boxes, and underlined portions indicate promoter sequences.

EXAMPLE 1 construction and screening of inducible P19 promoter

(1) Construction of inducible P19 promoter

The P19 promoter initiates expression of Rep52/40, but because it is nested in the Rep78/68 expression cassette, the addition of an induction element directly into the P19 promoter would disrupt the Rep78 expression cassette. Thus, in this example, two copies of the TetO inducible element (TCCCTATCAGTGATAGAGA, SEQ ID NO: 24) were designed in different introns, and these introns containing the TetO inducible element were inserted 1 bp into the P19 TATA box, thus constituting an inducible P19 promoter for inducible expression of Rep 52/40. These expression cassettes that induce Rep52/40 expression were constructed on plasmid RC2/8 (Addgene 112864) for AAV8 packaging, replacing the original Rep expression cassette. The constructed plasmids are shown in Table 1 below.

TABLE 1

(2) Screening inducible P19 promoter

Each of the inducible P19 plasmids in Table 1 was co-transfected with Helper plasmid (Cell Biolabs 340202) and TetR-krab control element plasmid （Szulc, J.et al.A versatile tool for conditional gene expression and knockdown.Nat Methods,3, 109-116 (2006)）, respectively, in suspension HEK293 cells, and expression of Rep52/40 protein was induced by addition of DOX after transfection. The co-transfected cells were harvested three days after culture, lysed and assayed for expression of each protein of Rep using Western Blot (WB). WB results are shown in fig. 1. As can be seen from FIG. 1, some plasmids showed better Rep52/40 protein induction, while the insertion of introns did not affect the expression of Rep 78/68.

The images were subjected to gray scale analysis using ImageJ software to quantify the expression amounts of Rep78 and Rep52 proteins expressed by each plasmid in fig. 1, and the results are shown in table 2 below.

TABLE 2

In the above table, "4.0E+04" means 4.0X10 ⁴, and so on.

(3) Testing the expression of RC plasmid containing inducible P19 promoter on AAV8 Virus packaging

The selected RC plasmid (LYR 1P023, 026, 027, 028, 029, 036) containing the inducible P19 promoter and AAV010 (ITR plasmid) and Helper plasmid (Cell Biolabs 340202) were subjected to three-plasmid transient virus packaging in suspension HEK293 cells. As a control, both the inducible construct reported in U.S. Pat. No. 3, 20200199627A1 (LYR 1P033 from LONZA, comprising SEQ ID NO: 9) and the conventionally used non-inducible RC2/8 control plasmid were transfected. Rep52/40 protein is induced to express by adding DOX after transfection so as to achieve the effect of inducing AAV8 virus packaging. Cell lysates were collected 3 days after transfection, huh-7 cells in 96-well plates were infected, and the expression level of the Luciferase reporter gene in the infected cells was detected 3 days after infection, and the yield and activity of each group of virus packages were judged.

The results of the expression level homogenization of the Luciferase reporter gene are shown in FIG. 2. As shown in FIG. 2, the RC plasmids LYR1P027, 028 and 029 have good effect of inducing virus packaging while maintaining good yield and activity of virus packaging after induction. Compared with LYR1P033 in the prior art, LYR1P027, 028, 029 had lower background in the non-induction, suggesting less leakage of Rep52/40 protein expression.

EXAMPLE 2 construction and selection of inducible Rep expression plasmids

(1) Construction of novel inducible Rep expression cassette

Expression of the inducible Rep78/68 protein can be achieved by replacing the P5 promoter with an exogenous inducible promoter. Specifically, inducible iEF, A, iRSV and iCMV promoters were used in place of the P5 promoter on plasmids LYR1P027 and 028 selected in example 1, thereby allowing each protein of Rep78/68/52/40 to induce expression. The plasmids constructed are shown in Table 3 below.

TABLE 3 Table 3

(2) Testing the expression of inducible Rep expression plasmids in AAV8 Virus packaging

Three plasmid transient virus packaging was performed on suspension HEK293 with the inducible Rep plasmid and AAV010 (ITR plasmid) and Helper plasmid (Cell Biolabs 340202). As a control, the RC2/8 control plasmid (Addgene 112864) was transfected simultaneously. Rep78/68/52/40 protein is induced to express by adding DOX after transfection so as to achieve the effect of inducing AAV8 virus packaging.

Cell lysates were collected 3 days after transfection, huh-7 cells in 96-well plates were infected, and the expression level of the Luciferase reporter gene in the infected cells was detected 3 days after infection, and the yield and activity of each group of virus packages were judged. The results of the expression level homogenization of the Luciferase reporter gene are shown in FIG. 3. Lysed cells were also harvested and Rep protein expression was detected using Western Blot (WB), the WB results are shown in FIG. 4.

The above virus packaging results and WB results both indicate that by adopting the Rep expression frames, the Rep proteins obtain good induction expression effects and have lower background before induction. At the same time, the Rep expression frames can also support the induction packaging of AAV viruses.

Example 3 expansion of serotypes

(1) Construction of different AAV serotypes of plasmids containing an inducible Rep expression cassette

Based on the same engineering pattern of plasmid LYR1P545, RC2/2 (Addgene 104963) and RC2/5 (Addgene 104964) control plasmids were engineered, respectively, to construct different AAV serotypes of plasmids containing the novel inducible Rep expression cassette, as shown in Table 4 below.

TABLE 4 Table 4

(2) Packaging of individual serotypes of virus using plasmids containing novel inducible Rep expression cassettes

AAV2 and AAV5 were packaged separately using the plasmids in Table 4. Viral packaging was performed by co-transfection with Helper plasmid (Cell Biolabs 340202) and AAV010 (ITR plasmid) in suspension HEK293 cells. Cell lysates were collected after 72 h transfection to infect HEK293T cells, and Luciferase expression was examined after 72: 72 h infection to represent virus packaging yield and viability, and compared to conventional Rep-Cap plasmid-transfected packaged virus. The results of the homogenized luciferases are shown in FIGS. 5 and 6.

The above virus packaging results suggest that induction packaging of various serotypes of AAV virus can be supported by the use of these Rep expression cassettes.

Incorporated by reference

The entire contents of each patent and scientific document referred to herein is incorporated by reference for all purposes.

Equivalency of

The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The above embodiments should therefore be regarded as illustrative in all respects, rather than limiting on the invention described herein. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An inducible P19 promoter, wherein said inducible P19 promoter comprises an intron downstream of the TATA box, and wherein two copies of a TetO inducing element are inserted into said intron, wherein the nucleotide sequence of said intron is selected from any 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 TetO inducing element are inserted after the 5 'common motif of the intron or before the 3' polypyrimidine region.

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

5. The expression cassette of claim 4, wherein the P5 promoter expressing the Rep78 polypeptide and the Rep68 polypeptide is replaced with an inducible promoter.

6. The expression cassette of claim 5, wherein the inducible promoter is derived from a P5, EF1A, RSV, CMV, MNDU, hCEF1 or SV40 promoter.

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

8. A viral packaging system comprising the vector of claim 7, and an expression plasmid for other essential elements for packaging a virus.

9. The viral packaging system according to claim 8, wherein the virus is selected from the group consisting of AAV viruses, serotypes of which are 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 engineered based on AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAVrh.8、AAVrh.10、AAVrh.39、AAVHSC15、AAVHSC17.

10. A cell line comprising the vector of claim 7, or comprising the viral packaging system of claim 8 or 9.