Capsid protein mutants for improving the retinal penetration ability of AAV virus and their applications

Abstract

The present invention belongs to the field of viral vectors, and discloses a capsid protein mutant that improves the retinal penetration ability of AAV virus and its application. The capsid protein mutant is obtained by replacing the amino acids 561 to 588 of the wild-type AAV2 virus capsid protein with the polypeptide DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSARQTSGGLSR. The capsid protein mutants of some examples of the present invention can effectively improve the retinal penetration ability of AAV virus, and fundamentally solve the problem of the efficiency of AAV vitreous cavity injection in infecting the posterior layer of the retina. The capsid protein mutants of some examples of the present invention can effectively increase the yield of recombinant AAV virus, and are expected to improve the therapeutic effect and transgenic efficiency of recombinant AAV virus. The capsid protein mutants of some examples of the present invention can be well used in the treatment of eye diseases and the construction of eye disease model animals.

Description

Technical Field

[0001] The present invention belongs to the field of virus vectors, and in particular relates to a capsid protein mutant capable of improving the retinal penetration of AAV viruses and an application thereof. Background Art

[0002] Adeno-associated virus (AAV) has the characteristics of low immunogenicity, tissue targeting, low integration, and long-term stable expression, making it the preferred vector for in vivo treatment in the field of gene therapy. It is widely used in the research of various diseases such as genetic diseases, cancer, and some chronic diseases. However, it still faces many challenges: First, the production process is complex, the cost is high, and the production capacity is limited; second, high-dose administration may show neurotoxicity and hepatotoxicity, causing safety issues. Third, the ability to infect some tissue cells is weak. For example, it is difficult to infect the posterior retinal cells by intravitreal administration, and it is difficult to infect central nervous cells by intravenous administration. Modification and screening of AAV capsid protein to improve its targeting and transfection efficiency to specific tissues, in order to achieve lower and safer administration doses, is an effective way to solve this problem.

[0003] Due to the existence of the blood-ocular barrier, the eye has a certain immune exemption, local administration is relatively safe, and fundus diseases are mostly single-gene genetic diseases, so ophthalmology has become a hot area for gene therapy. Different fundus diseases have different pathogenesis. When using AAV virus for gene therapy, the cell layers that need to be infected are different. There are two commonly used injection methods in clinical practice: intravitreal injection and subretinal injection. Subretinal injection has a better infection effect, especially for the RPE layer and outer nuclear layer, but this method is more invasive and can easily cause retinal detachment, which may lead to photoreceptor degeneration, visual function damage and glial cell proliferation. Intravitreal injection has a lower surgical risk, but conventional serotypes such as AAV2 and AAV5 can only infect the RGC layer, and have poor infection effects on cones, rods and RPE. Therefore, the development of AAV mutants that can improve the expression ability of the whole eye or can penetrate the RGC layer and inner nuclear layer to infect outer nuclear layer cells and RPE cells is of great significance to solving the problem of delivery vectors for ophthalmic gene therapy.

[0004] Previous studies have shown that mutations in the amino acid sites 561 to 588 of Cap2 can optimize its ability to infect mouse eye tissues, but how to improve the retinal penetration ability of recombinant AAV vectors remains a challenging task. Summary of the invention

[0005] The purpose of the present invention is to overcome at least one deficiency of the prior art and to provide a capsid protein mutant and its application that improves the retinal penetration ability of AAV virus.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides: a capsid protein mutant that improves the retinal penetration ability of the AAV virus, obtained by replacing amino acids 561 to 588 of the wild-type AAV2 virus capsid protein with one of the following polypeptides:

[0008] 1)DEEEIRQTNPVATEQYGSVCTNLQRPNR

[0009] 2)DESEIATVNPVATEQYGAVSTNLQRPNR

[0010] 3) DEQEIATTNPVATEQYGVVSTNLQRGNR

[0011] 4) DDEEIATTNPVATEQYGVVSTNLQRGNR

[0012] 5)DEHEIATTNPVATENYGDCSTNLQRGNR

[0013] 6) DEDEIRTTNPVCTEQYGSVATNIQRGNR

[0014] 7)DEDEIATTNPVATEQYGSVSTNLQRGNR

[0015] 8) DEEEIRTTNPVATEQYGSVSTNLQRGNLAAQLGQKTIAAR

[0016] 9) DEEEIRTTNPVATEQYGSVSTNLQRGNASAGLLGGKPAGR

[0017] 10)DEEEIRTTNPVATEQYGSVSTNLQRGNTGGQAYGPKGLSR

[0018] 11) DEEEIRTTNPVATEQYGSVSTNLQRGNTGTGAVNRGPGLSR

[0019] 12) DEEEIRTTNPVATEQYGSVSTNLQRGNTGPATGTSPRGLSR

[0020] 13) DEEEIRTTNPVATEQYGSVSTNLQRGNTGHRSVAGMGGLSR

[0021] 14) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSARQTSGGLSR

[0022] 15) DEEEIRTTNPVATEQYGSVSTNLQRGNTGGASYGLPGSRGLSR

[0023] 16) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSASLQRGLSR

[0024] 17) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSGIKESRGLSR

[0025] 18) DEEEIRTTNPVATEQYGSVSTNLQRGNTGWSEKRPQQGLSR

[0026] 19) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSYGVKVGLSR.

[0027] In some examples of capsid protein mutants, the GenBank accession number of the wild-type AAV2 virus capsid protein is YP_680426.1.

[0028] The second aspect of the present invention provides: a gene encoding the capsid protein mutant described in the first aspect of the present invention.

[0029] In some cases of genes, codon optimization was performed depending on the expression system.

[0030] The third aspect of the present invention provides: an expression system into which the gene described in the second aspect of the present invention is inserted.

[0031] In some examples of expression systems, the expression system is a recombinant AAV vector.

[0032] In some examples of expression systems, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

[0033] The fourth aspect of the present invention provides: a composition comprising the expression system described in the third aspect of the present invention.

[0034] The fifth aspect of the present invention provides: use of the composition described in the fourth aspect of the present invention in the preparation of gene therapy preparations or transgenic preparations.

[0035] In some examples of applications, the gene therapy formulation is used to treat eye diseases.

[0036] In some application examples, the transgenic preparation is used to treat eye diseases or to construct disease animal models.

[0037] In some application examples, the animal is selected from mice, rats, cynomolgus monkeys, rhesus monkeys, etc.

[0038] In some application examples, the disease is an eye disease, particularly an eye hereditary disease.

[0039] The sixth aspect of the present invention provides: a method for constructing a transgenic animal model, comprising introducing the composition described in the fourth aspect of the present invention into the animal.

[0040] In some examples of the construction methods, the animal is selected from mice, rats, cynomolgus monkeys, rhesus monkeys, etc.

[0041] In some examples of the construction method, the transgenic animal model is a transgenic animal model for an eye disease.

[0042] In some examples of the construction methods, the eye disease is an eye genetic disease.

[0043] The beneficial effects of the present invention are:

[0044] The capsid protein mutants of some examples of the present invention can effectively improve the retinal penetration ability of AAV virus, fundamentally solving the problem of the infection efficiency of the posterior layer of the retina by AAV intravitreal injection.

[0045] The capsid protein mutants of some examples of the present invention can effectively increase the yield of recombinant AAV virus, and are expected to improve the therapeutic effect and transgenic efficiency of the recombinant AAV virus.

[0046] The capsid protein mutants of some examples of the present invention can be well used for the treatment of eye diseases and the construction of eye disease model animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The agarose gel electrophoresis identification diagram of the mutant plasmid constructed in the test example of this invention.

[0048] Figure 2 This is the pAAV2-Rep2 / Cap2 packaging plasmid map in the examples of the present invention.

[0049] Figure 3 This is the pAAV2-7M8-Rep2 / Cap2-7M8 packaging plasmid map in the examples of the present invention.

[0050] Figure 4 This is a map of the pAAV2-Mut-Rep2 / Cap2-Mut packaging plasmid constructed in the examples of the present invention.

[0051] Figure 5This is a map of the pAAV-CAG-EGFP-HGH expression plasmid in the virus packaging three-plasmid system in the embodiment of the present invention.

[0052] Figure 6 This is a map of the pHelper auxiliary plasmid in the three-plasmid system for virus packaging in an embodiment of the present invention.

[0053] Figure 7 The yield results of AAV2 wild type, 7M8 and AAV2 mutant prepared by the scheme in the test example of the present invention.

[0054] Figure 8 These are in vivo fluorescent fundus photographs of mice infected with AAV2 wild type, 7M8 and some AAV2 mutants prepared according to the scheme in the test example of the present invention.

[0055] Fig. 9 The retinal tissue sections of mice infected with AAV2 wild type, 7M8 and some AAV2 mutants prepared according to the protocol in the test example of the present invention.

[0056] Fig.10 It is an amino acid sequence alignment diagram of AAV2 wild type, 7M8 and AAV2 mutants (AAV2-ROD-01 to ROD-19) according to the embodiments of the present invention. DETAILED DESCRIPTION

[0057] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels. Example

[0058] This embodiment provides a variety of adeno-associated virus variants (AAV2-ROD-01 to ROD-19, AAV2-Mut214 to AAV2-Mut227) based on AI design and previous studies. The preparation of the adeno-associated virus variants includes the following steps:

[0059] Table 1 Amino acid sequence information of AAV2 mutants with substitutions at positions D561 to R588 in Cap2

[0060]

[0061] 1. Plasmid construction

[0062] The nucleic acid sequences corresponding to the amino acid sequences of AAV2-ROD-01 to ROD-19 and AAV2-Mut214 to AAV2-Mut227 (as shown in Table 1) were synthesized (GenScript Biotechnology) and cloned into the pAAV2-Rep2 / Cap2 plasmid by a seamless method. The sequences of Cap2 561 to 588 of the wild-type AAV2 were replaced with the mutant sequences to construct the pAAV-mutant plasmid vector. The ligation products were transformed into competent Escherichia coli Stbl3, and a single colony was picked for expansion and plasmid extraction for sequencing verification and restriction digestion verification. The sequencing verification was consistent with the theoretical sequence, and the results were identified by agarose gel electrophoresis. Figure 1 As shown, the mutant plasmid was successfully constructed.

[0063] 2. AAV2-WT, 7M8 and AAV mutant virus packaging

[0064] AAV2-WT, 7M8 or AAV2-mutant packaging plasmids were co-transfected with pAAV-CAG-EGFP-hGH expression plasmid and pHelper helper plasmid into suspension HEK293T cells.

[0065] Virus packaging includes the following steps:

[0066] A. Cell culture

[0067] (1) Take out a tube of suspended 293T cells from the liquid nitrogen tank, put it in a 37℃ water bath, shake it quickly to thaw the cells, add it to a centrifuge tube containing 5ml Wayne 293 serum-free medium, centrifuge it at 200×g for 5min, discard the supernatant, resuspend it in 10ml Wayne 293 serum-free medium, place it in a 125ml triangular shake flask, add Wayne 293 serum-free medium to 20ml, and culture it in a shaking incubator at 120rpm, 37℃, and 5% CO2;

[0068] (2) After 48-72 hours of culture, the cell density reaches 2-3E+6 cells / ml, and the cells can be subcultured;

[0069] (3) Take out the seed cells to the clean bench, shake well and then pipette about 500 μl of sample, take 20 μl of sample and add 20 μl of trypan blue to mix, pipette 20 μl of the mixture into the cell counting plate, read the data three times on the automatic cell counter, and take the average value as the cell density;

[0070] (4) Take an appropriate amount of cells, dilute them to 0.6-0.65E+6 cells / ml with fresh Wayne 293 serum-free medium, and culture them in a 500ml triangular shake flask with a culture volume of 25ml per flask. Culture them in a shaking CO2 cell culture incubator at 120rpm, 37°C, and 5% CO2.

[0071] B. Cell transfection

[0072] (1) Cultivate the above cells for 48 hours, and the cell density reaches 3-3.8E+6 cells / ml, and the cells can be transfected;

[0073] (2) For each bottle of cells, add packaging plasmid, pHelper, and pAAV-CAG-EGFP--hGH (100 μg in total) to 4 mL of DMEM medium and mix thoroughly to prepare the "plasmid dilution solution".

[0074] (3) Take another 1 mL of DMEM medium and add 300 μg of PEI to mix well, which is the "PEI dilution solution";

[0075] (4) Pour the "PEI diluent" into the "plasmid diluent", mix quickly, and let stand at room temperature for 25 minutes to form a transfection complex; add the transfection complex dropwise into the cell solution while gently shaking;

[0076] (5) Place the culture flask in a shaking CO2 cell culture incubator and culture under the following conditions: 120 rpm, 37°C, and 5% CO2.

[0077] C. Virus Harvest

[0078] (1) After transfection, cells were cultured for 72 hours and virus harvesting was started.

[0079] (2) Collect the cell suspension in each bottle into a centrifuge bottle, centrifuge at 1000g for 5 min, collect the supernatant into a new centrifuge bottle, add 0.245mL of 50% PEG8000 solution (containing 0.5mol / L NaCl) per ml of supernatant, mix thoroughly, and let stand at 2-8℃ overnight;

[0080] (3) Add an appropriate amount of 0.5% TritonX-100 cell lysis buffer (containing universal nuclease) to the cell pellet, treat at 37°C for 1-2h, then add 1 / 10 volume of 5 mol / L NaCl, mix thoroughly and centrifuge at 4°C and 3000g for 15min, collect the supernatant, which is the "viral crude extract" and temporarily store at 2-8°C;

[0081] (4) Centrifuge the supernatant after PEG8000 precipitation at 4°C and 3000g for 15 min, discard the supernatant, and resuspend it in the "viral crude extract" in step (3) for later use.

[0082] D. Virus purification

[0083] 1) Take an Ultra-Clear centrifuge tube, add 0.5mL 60% Iodixanol, 2mL 40% Iodixanol, 1.5mL 25% Iodixanol, 1.5mL 15% Iodixanol, the collected virus suspension, and finally the cell lysate to balance at the bottom;

[0084] 2) 10°C, 230,000 g, 8°C, 9°C ultracentrifugation for 18 h;

[0085] 3) Take an ultrafiltration tube and soak the filter membrane with 1 mL of PBS buffer;

[0086] 4) Use a pipette to carefully extract the middle layer of 40% Iodixanol-60% Iodixanol in the ultracentrifuge tube, avoiding protein, and transfer it to the ultrafiltration tube;

[0087] 5) Add an appropriate amount of PBS buffer and blow evenly, centrifuge at 4500g for 3-5 min, and repeat this step 5-7 times to remove Iodixanol;

[0088] 6) Add 1 mL of PBS buffer and pipette for 40-50 times to form a virus suspension, then transfer to an EP tube;

[0089] 7) Use a 5 mL syringe to draw the virus suspension in the EP tube and filter it through a 0.22 μm filter. Collect 20 μL of the virus liquid as a test sample and dispense it into 100 μL tubes to obtain adeno-associated virus variants.

[0090] Comparative Example 1

[0091] This comparative example provides a wild-type adeno-associated virus (AAV2-WT), which is prepared according to the method of Example 1 with respect to the AAV2-mutant, the only difference being that the sequence of the adeno-associated virus capsid protein adopts the wild-type AAV2 capsid protein sequence (SEQ ID NO.1).

[0092] Comparative Example 2

[0093] This comparative example provides an AAV2 adeno-associated virus mutant (AAV2-7M8) with strong whole-eye expression ability that has been used in ophthalmic clinical trials. It is prepared according to the method of Example 1 with the AAV2- mutant, and the only difference is that the adeno-associated virus capsid protein sequence adopts the mutant AAV2-7M8 capsid protein sequence (SEQ ID NO.2).

[0094] Test Case

[0095] 1. Virus titer detection

[0096] (1) Virus lysis

[0097] 1) Take 20 μL of the virus samples of the embodiment and the comparative example respectively;

[0098] 2) Add 1 μL each of 10% SDS, 0.5 mol / L EDTA, and proteinase K and mix well;

[0099] 3) Incubate at 56°C for 1 hour in a thermomixer, then at 90°C for 10 minutes;

[0100] 4) Take 10ul of virus lysate and dilute it 10-fold to 10,000-fold to obtain virus dilution solution for later use.

[0101] (2) Preparation of standard samples for standard curve

[0102] The concentration is 2×10 12 The plasmid standard of 10 copies / mL was diluted 10-fold with ddH2O into 6 gradients as the standard curve template 2×10 11 copies / mL, 2×10 10 copies / mL, 2×10 9 copies / mL, 2×10 8 copies / mL, 2×10 7 copies / mL, 2×10 6 copies / mL.

[0103] (3) Quantitative qPCR

[0104] 1) Take a 0.2mL PCR tube and prepare the following reaction system, with 3 replicates for each virus dilution. 2× qPCR Mix 10μL; 0.2μL each of forward and reverse primers; 5μL virus dilution; 4.2μL ddH2O;

[0105] Amplification primers:

[0106] Forward primer 5′- GGAACCCCTAGTGATGGAGTT-3′ (SEQ ID NO. 38);

[0107] Reverse primer 5'- CGGCCTCAGTGAGCGA-3' (SEQ ID NO. 39).

[0108] 2) PCR amplification

[0109] Pre-denaturation: 95°C, 2 min;

[0110] 40× cycles: 95°C, 15 s; 60°C, 60 s.

[0111] 3) qPCR data processing: Virus titer = dilution factor × viral gene array copy number.

[0112] The present invention scheme transfects AAV2-WT virus packaging three-plasmid system (including pAAV2, pAAV-CAG-EGFP, pHelper) and AAV2-mutant virus packaging three-plasmid system (including pAAV2-mutant, pAAV-CAG-EGFP, pHelper) into HEK293T cells in equal amounts, and harvests the virus solution by cell lysis, purifies it by iodixanol ultracentrifugation, and evaluates its yield by QPCR detection of virus titer. Figure 2 This is the pAAV2-Rep2 / Cap2 packaging plasmid map in the embodiment of the present invention; Figure 3 This is the pAAV2-7M8-Rep2 / Cap2-7M8 packaging plasmid map in the embodiment of the present invention; Figure 4 This is the pAAV2-Mut-Rep2 / Cap2-Mut packaging plasmid map constructed in the examples of the present invention; Figure 5 This is the pAAV-CAG-EGFP-HGH expression plasmid map of the virus packaging three-plasmid system in the embodiment of the present invention; Figure 6 This is a map of the pHelper auxiliary plasmid in the three-plasmid system for virus packaging in an embodiment of the present invention.

[0113] The experimental results are as follows Figure 7 As shown in the figure, it can be seen that 8 mutants have a yield lower than 0.5 times that of AAV2-WT, and are identified as low-yield variants and are not verified in vitro; the remaining 25 variants have a yield of 0.54 to 2.63 times that of AAV2-WT, among which the AAV2-ROD-15 variant has the highest yield. These 25 mutants were selected for mouse retinal infection effect testing. The virus yield of Comparative Example 2 (AAV2-7M8) is 0.89 times that of AAV2-WT.

[0114] , Mouse retinal infection effect test

[0115] (1) The samples of the embodiment and comparative example were diluted to 2E+9 vg / µl, and injected into the vitreous cavity of C57BL-6J mice, with 1.5 µl injected into each eye (total virus amount was 3E+9 vg), and 3 mice were injected with each virus;

[0116] (2) 3 weeks after virus injection, the mice were anesthetized and fluorescent fundus imaging (OCT) was performed using the Micron IV small animal retinal imaging system. The imaging parameters were: Gain (dB)-10dB, Frame Rate (fps)-2fps, Exposure-Auto;

[0117] (3) The mouse eyeballs were removed, fixed, embedded in paraffin, sectioned, stained with DAPI, and observed and photographed under a fluorescence microscope.

[0118] In the solution of the present invention, AAV2-WT, AAV2-7M8 and variant viruses were injected into the vitreous cavity of mice. After 3 weeks of virus injection, live fluorescence photography was performed. Among the selected mutants, 19 variants had stronger GFP expression intensity than AAV2-WT. Some of the embodiments and comparative examples with better effects were selected. The results are shown in FIG. Figure 8 As shown in the figure, it can be seen that the expression of AAV2-ROD-04, AAV2-ROD-05 and AAV2-ROD-14 viruses in the whole eye is significantly stronger than that of AAV2-WT virus, and the EGFP expression intensity of comparative example 2 (AAV2-7M8) is similar to that of AAV2-WT virus.

[0119] In order to explore the infection effect on various layers of the retina, the paraffin sections of the eyeball were stained with DAPI, and the results of fluorescence microscopy showed that the variant virus could infect cells of various layers of the retina from RGC to PRC after intravitreal injection, while AAV2-WT only infected RGC layer cells. The variant virus had a stronger infection area and ability to infect the posterior layer of the retina than AAV2-WT. Fig. 9 As shown, the infection range of AAV2-ROD-04, AAV2-ROD-05 and AAV2-ROD-14 mutants is greater than that of AAV2-WT and 7M8, among which AAV2-ROD-04 and AAV2-ROD-05 can almost infect the entire retinal area. From the perspective of infection level, AAV2-WT only infects RGC layer cells, and 7M8 has a certain penetration ability, which can penetrate into the posterior layer of the retina to infect a small number of optic nerve cells. The penetration ability of AAV2-ROD-04, AAV2-ROD-05 and AAV2-ROD-14 variants is stronger than that of AAV2-WT and 7M8, and can infect cells of each layer of the retina from RGC to PRC. Among them, AAV2-ROD-05 has the best infection rate and expression effect on optic nerve cells, proving that the adeno-associated virus variants prepared by the technology of the present invention have significantly improved the infection ability of the posterior layer of the retina.

[0120] The sequence alignment results of adeno-associated virus variant capsid proteins are shown in the figure Fig.10The difference between the sequence of the capsid protein of the adeno-associated virus variant ROD-01 to ROD-19 and the wild-type AAV2 capsid protein is that the sequence of the wild-type capsid protein D561 to R588 (shown in SEQ ID NO.3) is replaced with a polypeptide sequence of 28 to 42 amino acids. Among them, ROD-01 to ROD-07 are partial base replacements, and ROD-08 to ROD-19 are multiple base insertions.

[0121] The amino acid sequence of the wild-type adeno-associated virus capsid protein Cap2 is shown in SEQ ID NO.1, and the sequence of the adeno-associated virus variant 7M8 is shown in SEQ ID NO.2.

[0122] In summary, the adeno-associated virus variant prepared by the scheme of the present invention can significantly enhance the infection range of the AAV2 virus to the retina and the infection ability to the posterior layer of the retina compared with the wild type.

[0123] The amino acid sequence of wild-type adeno-associated virus capsid protein Cap2 is SEQ ID NO.1: (protein ID: YP_680426.1).

[0124] The amino acid sequence of AAV2 adeno-associated virus mutant capsid protein Cap2-7M8 is SEQ ID NO.2:

[0125] .

[0126] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A capsid protein mutant that improves the retinal penetration ability of AAV virus, characterized in that: The polypeptide DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSARQTSGGLSR was used to replace amino acids 561 to 588 of the wild-type AAV2 virus capsid protein, and the GenBank accession number of the wild-type AAV2 virus capsid protein is YP_680426.

1.

2. A gene encoding the capsid protein mutant according to claim 1.

3. The gene according to claim 2, characterized in that Codon optimization was performed according to the expression system.

4. An expression system, into which the gene according to claim 2 or 3 is inserted, the expression system is a recombinant AAV vector, and the AAV is AAV2.

5. A composition, characterized in that Comprising the expression system described in claim 4.

6. Use of the composition according to claim 5 in the preparation of gene therapy preparations or transgenic preparations.

7. A method for constructing a transgenic animal model, comprising introducing the composition of claim 5 into the animal.

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