A recombinant adeno-associated virus vector targeting RB1 and its construction method and application

Abstract

The present application relates to the technical field of gene vector construction, and specifically discloses a targeted RB1 A recombinant adeno-associated virus vector and its construction method and application. The expression framework of the vector includes: AAV2 Cap Gene, AAV2 Rep Gene, RB1 Gene. The targeting provided in this application RB1 The recombinant adeno-associated virus vector can efficiently and accurately target retinal progenitor cells, providing a basis and direction for further promoting the targeted treatment research of RB.

Description

Technical Field

[0001] The present application relates to the technical field of gene vector construction, and more specifically, to a recombinant adeno-associated virus vector targeting RB1 and a construction method and application thereof. Background Art

[0002] Retinoblastoma (RB) is an intraocular malignant tumor that originates from embryonic retinal cells. It is inherited in an autosomal dominant manner and is common in childhood, mostly before the age of 4. The clinical manifestation is the early appearance of a gray-white mass on the fundus. When the mass grows inside the eye, the external eye is normal, so it is difficult to detect. RB1 gene mutation is the most important genetic pathogenic driver aberration of RB, and functional loss caused by RB1 gene mutation is also the main cause of RB. Therefore, gene therapy targeting the RB1 gene is the fundamental way to overcome RB.

[0003] However, more than 3,000 RB-specific RB1 gene mutations have been discovered so far. The types and sites of RB1 gene mutations are variable, and there are no hotspot mutations, so it is difficult to treat by repairing specific sites. In addition, the RB1 gene fragment is large, and there is a lack of a vector system for safely and effectively delivering the RB1 gene, making RB1 gene therapy difficult to implement. The above problems have seriously hindered the research on targeted treatment of RB. Summary of the invention

[0004] The present application provides a recombinant adeno-associated virus vector targeting RB1 and its construction method and application. In order to further promote the research on targeted therapy of RB, the present application successfully constructed a recombinant adeno-associated virus (rAAV2-RB1) that efficiently and accurately targets retinal progenitor cells, which can efficiently replenish and reconstruct the function of the RB1 gene. On this basis, the present application conducts intervention experiments with the PDX animal model of RB to verify the effectiveness and safety of the recombinant adeno-associated virus provided by the present application on RB, in order to establish a reliable solution for the targeted RB1 gene therapy of RB.

[0005] In a first aspect, the present application provides a recombinant adeno-associated virus vector targeting RB1, using the following technical solution:

[0006] A recombinant adeno-associated virus vector targeting RB1, wherein the expression framework of the vector comprises: the Cap gene of AAV2, the Rep gene of AAV2, and the RB1 gene.

[0007] Optionally, the nucleotide sequence of the RB1 gene comprises the nucleotide sequence shown in SEQ ID NO 1-SEQ ID NO 3.

[0008] In a specific embodiment, the nucleotide sequence of the RB1 gene comprises the nucleotide sequence shown in SEQ ID NO 1.

[0009] In a specific embodiment, the nucleotide sequence of the RB1 gene comprises the nucleotide sequence shown in SEQ ID NO 2.

[0010] In a specific embodiment, the nucleotide sequence of the RB1 gene comprises the nucleotide sequence shown in SEQ ID NO 3.

[0011] Optionally, the nucleotide sequence of the Cap gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 4-SEQ ID NO 6.

[0012] In a specific embodiment, the nucleotide sequence of the Cap gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 4.

[0013] In a specific embodiment, the nucleotide sequence of the Cap gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 5.

[0014] In a specific embodiment, the nucleotide sequence of the Cap gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 6.

[0015] Optionally, the nucleotide sequence of the Rep gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 7 to SEQ ID NO 9.

[0016] In a specific embodiment, the nucleotide sequence of the Rep gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 7.

[0017] In a specific embodiment, the nucleotide sequence of the Rep gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 8.

[0018] In a specific embodiment, the nucleotide sequence of the Rep gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 9.

[0019] Optionally, the order of the RB1 gene, the Cap gene of the AAV2, and the Rep gene of the AAV2 is Cap-Rep-RB1, Rep-RB1-Cap, Rep-Cap-RB1, Cap-RB1-Rep, RB1-Cap-Rep, and RB1-Rep-Cap.

[0020] In a specific embodiment, the order of the RB1 gene, the Cap gene of AAV2, and the Rep gene of AAV2 is Cap-Rep-RB1.

[0021] In a specific embodiment, the order of the RB1 gene, the Cap gene of AAV2, and the Rep gene of AAV2 is Rep-RB1-Cap.

[0022] In a specific embodiment, the order of the RB1 gene, the Cap gene of AAV2, and the Rep gene of AAV2 is Rep-Cap-RB1.

[0023] In a specific embodiment, the order of the RB1 gene, the Cap gene of AAV2, and the Rep gene of AAV2 is Cap-RB1-Rep.

[0024] In a specific embodiment, the order of the RB1 gene, the Cap gene of AAV2, and the Rep gene of AAV2 is RB1-Cap-Rep.

[0025] In a specific embodiment, the order of the RB1 gene, the Cap gene of AAV2, and the Rep gene of AAV2 is RB1-Rep-Cap.

[0026] In a second aspect, the present application provides a method for constructing a recombinant adeno-associated virus vector targeting RB1, characterized in that the construction method specifically comprises the following steps:

[0027] The vector was linearized by enzyme digestion, and primers were designed to extract the target fragment from the template, amplify and recover the target fragment by gel, and the target fragment recovered by gel was connected with the linearized vector for recombination and transformed into DH5α competent cells. Positive clones were identified by PCR, plasmids were extracted by shaking and sequenced, and finally the vector plasmid containing the target fragment was constructed.

[0028] In a third aspect, the present application provides a use of a recombinant adeno-associated virus vector targeting RB1 in the preparation of a composition for gene therapy of retinoblastoma.

[0029] In order to further promote the research on targeted therapy of RB, this application successfully constructed a recombinant adeno-associated virus (rAAV2-RB1) that efficiently and accurately targets retinal progenitor cells, which can restore the function of the RB1 gene. On this basis, this application conducted an intervention experiment with the PDX animal model of RB to verify the effectiveness and safety of the vector provided by this application for RB, in order to establish a reliable solution for targeted RB1 gene therapy of RB.

[0030] In summary, this application has the following beneficial effects:

[0031] 1. The recombinant adeno-associated virus (rAAV2-RB1) targeting RB1 provided in the present application can provide a new direction for the treatment of RB, improve the effectiveness of RB treatment, and reduce the rate of enucleation.

[0032] 2. Experimental analysis showed that the injection of rAAV2-RB1 in the RB PDX animal model can significantly inhibit the growth of retinoblastoma and has good biosafety in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the construction and packaging of the rAAV2-RB1 vector provided in this application.

[0034] Figure 2 The results of validation of rAAV2-RB1 targeting retinal cell expression in Example 1 (A-RB1 RNA expression level in mouse eyes; B-RB1 protein expression level in mouse eyes; C-immunofluorescence staining results of mouse retina).

[0035] Figure 3 This is the result of establishing the RB PDX model.

[0036] Figure 4 This is the efficacy evaluation of rAAV2-RB1 in Example 1 on the RB PDX animal model cohort (A-overall experimental design diagram; B-appearance of eye tumors in RB PDX model mice after treatment; C-gross image of RB tumors after treatment; D-RB tumor weight after treatment; E-volume growth curve of mouse eye tumors during treatment).

[0037] Figure 5 This is the safety evaluation of rAAV2-RB1 of Example 1 in the RB PDX animal model cohort (A-HE staining results of important organs of RBPDX model mice and immunohistochemical staining results of Flag protein after treatment; B-histological morphology of eyeballs of RB PDX model mice after treatment; C-electroretinogram detection results of RB PDX model mice after treatment). DETAILED DESCRIPTION

[0038] Combination Figure 1 The recombinant adeno-associated virus (rAAV2-RB1) targeting RB1 provided in this application is based on the AAV2 vector, which is responsible for encoding the target gene and two terminal inverted repeat sequences. The helper plasmid contains the Cap gene encoding the viral capsid protein required for AAV packaging and the Rep gene involved in viral replication, as well as the adenovirus Helper plasmid. After the three plasmids were co-transfected into 293T cells, the AAV virus began to replicate and package. The obtained virus particles were purified by ultracentrifugation, and the virus titer was measured.

[0039] The expression framework of the recombinant adeno-associated virus vector targeting RB1 provided in the present application includes: the Cap gene of AAV2, the Rep gene of AAV2, and the RB1 gene.

[0040] Among them, the nucleotide sequence of the RB1 gene includes the nucleotide sequence shown in SEQ ID NO 1-SEQ ID NO 3; the nucleotide sequence of the Cap gene of AAV2 includes the nucleotide sequence shown in SEQ ID NO 4-SEQ ID NO 6; the nucleotide sequence of the Rep gene of AAV2 includes the nucleotide sequence shown in SEQ ID NO 7-SEQ ID NO 9.

[0041] After screening, when the nucleotide sequence of the RB1 gene includes the nucleotide sequence shown in SEQ ID NO 1; the nucleotide sequence of the Cap gene of AAV2 includes the nucleotide sequence shown in SEQ ID NO 4; and the nucleotide sequence of the Rep gene of AAV2 includes the nucleotide sequence shown in SEQ ID NO 7, the relative expression level of RB1 mRNA and the relative expression level of RB1 protein are both high.

[0042] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application are clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application.

[0043] The present application is further described in detail below in conjunction with embodiments, drawings, comparative examples and verification test results.

[0044] Example

[0045] Example 1

[0046] This embodiment provides a recombinant adeno-associated virus (rAAV2-RB1) targeting RB1.

[0047] In the rAAV2-RB1, the RB1 gene includes the nucleotide sequence described in SEQ ID NO 1, the Cap gene includes the nucleotide sequence described in SEQ ID NO 4, and the Rep gene includes the nucleotide sequence described in SEQ ID NO 7. The order of the three genes is Cap-Rep-RB1, as shown in Table 1.

[0048] The construction method of the above rAAV2-RB1 is as follows: the vector is linearized by restriction digestion (purchased from Shandong Weizhen Biotechnology Co., Ltd., AAV serotype is AAV2), and primers are designed to extract the target fragment from the template, amplify and gel-recover the target fragment, connect the gel-recovered target fragment with the linearized vector for recombination and transform DH5α competent cells. Positive clones are identified by PCR, plasmids are extracted by shaking and sequenced, and finally a vector plasmid containing the target fragment is constructed.

[0049] The full-length DNA sequence of rAAV2-RB1 was synthesized by a third-party company.

[0050] Example 2

[0051] This embodiment provides a recombinant adeno-associated virus vector targeting RB1. The difference between this embodiment and embodiment 1 is that: in the rAAV2-RB1, the RB1 gene includes a nucleotide sequence as described in SEQ ID NO 2, as shown in Table 1.

[0052] Example 3

[0053] This embodiment provides a recombinant adeno-associated virus vector targeting RB1. The difference between this embodiment and embodiment 1 is that: in the rAAV2-RB1, the RB1 gene includes a nucleotide sequence as described in SEQ ID NO 3, as shown in Table 1.

[0054] Example 4

[0055] This embodiment provides a recombinant adeno-associated virus vector targeting RB1. The difference between this embodiment and embodiment 1 is that: in the rAAV2-RB1, the Cap gene includes the nucleotide sequence described in SEQ ID NO 5, as shown in Table 1.

[0056] Example 5

[0057] This embodiment provides a recombinant adeno-associated virus vector targeting RB1. The difference between this embodiment and embodiment 1 is that: in the rAAV2-RB1, the Cap gene includes the nucleotide sequence described in SEQ ID NO 6, as shown in Table 1.

[0058] Example 6

[0059] This embodiment provides a recombinant adeno-associated virus vector targeting RB1. The difference between this embodiment and embodiment 1 is that: in the rAAV2-RB1, the Rep gene includes the nucleotide sequence described in SEQ ID NO 8, as shown in Table 1.

[0060] Example 7

[0061] This embodiment provides a recombinant adeno-associated virus vector targeting RB1. The difference between this embodiment and embodiment 1 is that: in the rAAV2-RB1, the Rep gene includes the nucleotide sequence described in SEQ ID NO 9, as shown in Table 1.

[0062] Example 8

[0063] This example provides a recombinant adeno-associated virus vector targeting RB1. The difference between this example and Example 1 is that the order of the three genes is Cap-RB1-Rep, as shown in Table 1.

[0064] Example 9

[0065] This example provides a recombinant adeno-associated virus vector targeting RB1. The difference between this example and Example 1 is that the order of the three genes is RB1-Cap-Rep, as shown in Table 1.

[0066] Example 10

[0067] This example provides a recombinant adeno-associated virus vector targeting RB1. The difference between this example and Example 1 is that the order of the three genes is RB1-Rep-Cap, as shown in Table 1.

[0068] Embodiment 11

[0069] This example provides a recombinant adeno-associated virus vector targeting RB1. The difference between this example and Example 1 is that the order of the three genes is Rep-RB1-Cap, as shown in Table 1.

[0070] Example 12

[0071] This example provides a recombinant adeno-associated virus vector targeting RB1. The difference between this example and Example 1 is that the order of the three genes is Rep-Cap-RB1, as shown in Table 1.

[0072] Comparative Example

[0073] Comparative Example 1

[0074] Comparative Example 1 provides a recombinant adeno-associated virus vector targeting RB1. The difference between this comparative example and Example 1 is that in the rAAV2-RB1, the RB1 gene is the RB1 exon sequence, and the Accession in NCBI is NM_000321.3.

[0075] Comparative Example 2

[0076] Comparative Example 2 provides a viral vector targeting RB1. The difference between this comparative example and Example 1 is that the viral vector is a lentivirus (Shanghai GeneCare Gene Medical Technology Co., Ltd.), and the RB1 gene has a nucleotide sequence as described in SEQ ID NO 1.

[0077] Verification test

[0078] 1. Verification of rAAV2-RB1 targeted expression

[0079] The rAAV2-RB1 prepared in the above example was verified for targeted expression.

[0080] (1) Verification method

[0081] The constructed rAAV2-RB1 was injected into the vitreous cavity (a cavity located in the posterior 4 / 5 of the eyeball, behind the lens and in front of the retina, filled with colorless and transparent colloid, i.e., vitreous body) of nude mice (mutant mice with congenital thymus defects) through a microsyringe, and then the wound was covered with erythromycin ointment.

[0082] After 3 weeks, RNA and protein were extracted from the mouse retina, and the mouse eyeballs were separated to prepare pathological sections. The tissue sections were placed in a humidified box, and diluted RB1 antibody (Mouse anti-RB1 purchased from CST, USA) was added to the tissue sections to completely cover the tissue section samples, and incubated at room temperature for 1 hour; after rinsing with PBS for 3 times, fluorescent secondary antibody (purchased from Sigma-Aldrich, USA) was added to the sections to cover the samples, and the humidified box was covered and incubated at room temperature for 1 hour; rinsed with PBS for 3 times; a coverslip was taken, and 1 drop of Gelvatol (purchased from MERCK, USA, polyvinyl alcohol mounting medium containing DABCO, anti-fading) was added to the center, and a reverse transfer slide was pressed on the coverslip, and placed at room temperature in the dark for 20-30 minutes. After it solidified, it was observed under a fluorescence microscope (purchased from OLYMPUS, Japan) and the results were recorded.

[0083] (2) Verification results

[0084] The verification results are shown in Table 1. Figure 2 These are the verification results of rAAV2-RB1 targeting retinal progenitor cell expression in Example 1, wherein Figure A shows the RB1 RNA expression level in mouse eyes; Figure B shows the RB1 protein expression level in mouse eyes; and Figure C shows the immunofluorescence staining results of mouse retinal progenitor cells.

[0085] Depend on Figure 2As known, 3 weeks after the rAAV2-RB1 of Example 1 was injected into the vitreous cavity of nude mice, Figure A shows that the RB1 RNA level in the mouse eye is upregulated, while Figure B shows that the expression levels of RB1 protein and Flag protein (tag protein) in the mouse eye are upregulated, and the expression of GAPDH is not affected. According to the results of Figure C, the mouse retinal layer cells can stably express RB1 protein.

[0086] Table 1 Targeting verification results of the embodiment

[0087]

[0088] As can be seen from Table 1, the relative expression levels of RB1 mRNA and RB1 protein in Example 1 are both high, so the present application selects the nucleotide sequence of SEQ ID NO 1 for the RB1 gene, the nucleotide sequence of SEQ ID NO 4 for the Cap gene, and the nucleotide sequence of SEQ ID NO 7 for the Rep gene.

[0089] 2. Establishment of RB PDX Model

[0090] Four-week-old female nude mice (provided by the SPF animal room of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine) were used as experimental materials. Using clinical tissue specimens and primary tumor cell dissociation technology, the primary tumor cell (RB) suspension was dissociated and immediately injected into the subretinal space or vitreous cavity of nude mice. With the help of fundus photography and OCT (optical coherence tomography) and other detection technologies, the tumor formation of PDX mice was confirmed, and the PDX model of RB was constructed through in vivo passage.

[0091] The build result is as follows Figure 3 shown.

[0092] Depend on Figure 3 It can be seen that compared with the control group, the PDX model of the experimental group had gray-white masses in the fundus, indicating that the RB PDX model was successfully constructed. Figure 3 It can also be seen from the test results of the anterior chamber and retina of the eye that compared with the control group, the PDX models of the experimental group showed abnormalities, further indicating that the RB PDX model was successfully constructed.

[0093] 3. Evaluation of the efficacy of rAAV2-RB1 in the RB PDX animal model cohort

[0094] The rAAV2-RB1 of Example 1 was used to evaluate the efficacy in the RB PDX animal model cohort.

[0095] The RB second-generation PDX model (P2) after serial passage and histological stability assessment was used as the experimental mouse for this part of the experiment. After the PDX P2 transplanted tumor grew for 1 week and white pupils could be seen with the naked eye, the rAAV2-RB1 of Example 1 was injected into the vitreous cavity for treatment, and the eye morphology before treatment was recorded by photographing. After administration, the morphology of the mouse eye tumor was measured and recorded every week, and relevant safety assessments were performed. The experiment was terminated after 3 weeks of treatment, the nude mice were treated by cervical dislocation, the tumor was dissected out, the tumor size was measured, and photos were taken. The tumor obtained by dissection was retained, and the relevant index detection was compared with the control group (Empty Vector) RB PDX model mice that were not treated with rAAV2-RB1.

[0096] The results are as follows Figure 4 Figure A is the overall experimental design diagram, Figure B is the appearance of the eye tumor of the RB PDX model mouse after treatment, Figure C is the general picture of the RB tumor after treatment; Figure D is the weight of the RB tumor after treatment; Figure E is the volume growth curve of the mouse eye tumor during the treatment process.

[0097] Depend on Figure 4 It can be seen from Figure B that the eye tumors of the RB PDX model mice improved after treatment. Figure C shows that the volume of the RB tumor was significantly reduced after treatment. Figures D and E show that the RB tumor weight and volume proliferation of the RB PDX model mice were reduced after treatment. This shows that the rAAV2-RB1 of the present application can efficiently and accurately inactivate retinoblastoma and significantly inhibit the proliferation of retinoblastoma.

[0098] 4. Safety evaluation of rAAV2-RB1 in the RB PDX animal model cohort

[0099] The rAAV2-RB1 of Example 1 was used for safety evaluation in the RB PDX animal model cohort.

[0100] After 3 weeks of treatment of the RB PDX model mice in the control group and the RB PDX model mice in the test group treated with rAAV2-RB1 in Example 1, the nude mice were treated by cervical dislocation, and the heart, liver, spleen, lung, kidney and other important organs of the mice were dissected out, fixed with 4% paraformaldehyde and embedded in paraffin, and HE staining was used to detect the histological morphology of important organs. Immunohistochemistry was used to detect the expression of rAAV2-RB1 in important organs of mice (Flag protein staining), and the relevant indexes were compared with those of the RB PDX model mice in the control group. The images were captured and saved using a Leica DM6000 optical microscope at 40 times magnification.

[0101] In addition, the ocular histological morphology and visual function of the treated RB PDX model mice were detected and compared with those of the control group RB PDX model mice and the RB PDX model mice injected with normal saline. The visual function was detected by electroretinography (ERG).

[0102] The ERG detection method is as follows:

[0103] (1) Place the mice in a dark room in the animal room the night before and allow them to adapt to the dark overnight.

[0104] (2) The next morning, place the mouse cage in a dark box and move it out of the animal room to the ERG room.

[0105] (3) Take the experimental mice out of the mouse cage, and add 1-2 drops of 2.5% phenylephrine eye drops and 0.5% tropicamide eye drops to both eyes to dilate the pupils, and then add 0.5% proparacaine to numb the cornea.

[0106] (4) After the mouse is anesthetized, 1-2 drops of 0.3% hydroxypropyl methylcellulose are placed in both eyes to buffer the contact between the cornea and the electrode / stimulator.

[0107] (5) Record the mouse at 0.01cd.s / m 2 and 10cd.s / m 2 The scotopic response to white light stimulation of varying intensity.

[0108] (6) During the test, the body temperature was maintained at 37°C using the system's built-in heating element.

[0109] (7) After the experiment, tobramycin-dexamethasone eye ointment was applied to the mouse cornea, and the mouse was placed on a heating pad with a warm water bath until it recovered and then returned to the animal room.

[0110] (8) Espion V6 software was used to measure the amplitude of the ERG a-wave and b-wave after white light stimulation of different intensities. The a-wave amplitude started from the baseline and ended at the negative trough; the b-wave amplitude started from the negative trough and ended at the positive peak. The test results were recorded and analyzed.

[0111] The above results are Figure 5 Figure A shows the results of HE staining of important organs and immunohistochemical staining of Flag protein in RB PDX model mice after treatment; Figure B shows the histological morphology of eyeballs in RB PDX model mice after treatment; Figure C shows the results of electroretinogram detection in RB PDX model mice after treatment.

[0112] Depend on Figure 5It can be seen that the RB PDX model mice in the experimental group and the RBPDX model mice in the control group treated with rAAV2-RB1 in Example 1 in Figure A all showed similar histological morphology in the heart, liver, spleen, lung, and kidney of the mice, indicating that rAAV2-RB1 has good safety in the RB PDX animal model cohort and has no effect on the histological morphology of important organs of mice. At the same time, the immunohistochemical staining results of Flag protein showed that rAAV2-RB1 had no specific expression in important organs such as the heart, liver, spleen, lung, and kidney of mice, indicating that rAAV2-RB1 has good eye targeting and has not spilled over to organs other than the eye for expression.

[0113] In Figures B and C, the ocular histological morphology and electroretinogram of RB PDX model mice injected with normal saline, RB PDX model mice in the control group, and RB PDX model mice after treatment were compared. It can be seen that the ocular histological morphology and electroretinogram of RB PDX model mice after treatment did not change, indicating that the rAAV2-RB1 of the present application has good safety for ocular histological morphology and visual function.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A recombinant adeno-associated virus vector targeting RB1, characterized in that: The expression framework of the vector is composed of the Cap gene of AAV2, the Rep gene of AAV2, and the RB1 gene; the connection mode is Cap-Rep-RB1 or Rep-RB1-Cap; The nucleotide sequence of the RB1 gene comprises the nucleotide sequence shown in SEQ ID NO 1; The nucleotide sequence of the Cap gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 4; The nucleotide sequence of the Rep gene of AAV2 comprises the nucleotide sequence shown in SEQ ID NO 7.

2. Use of the recombinant adeno-associated virus vector targeting RB1 according to claim 1 in preparing a composition for gene therapy of retinoblastoma.

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