Gene therapy drug aiming at RDH12 mutation

Abstract

The invention discloses a gene therapy drug aiming at RDH12 mutation, and relates to the technical field of biology, and the technical key points are as follows: an AAV-hRDH12 gene therapy drug is constructed, and a vitreous cavity injection method is adopted to introduce a retinal photoreceptor cell, so that the retinal photoreceptor cell expresses complete and active RDH12 protein and participates in a visual circulation pathway, and the RDH12 gene therapy drug is obtained. And the dysfunction caused by the RDH12 gene variation is rescued. According to the invention, codon-optimized human RDH12-cDNA is packaged by an improved AAV vector, so that the human RDH12-cDNA expresses complete and active RDH12 protein and participates in a visual circulation pathway, dysfunction caused by RDH12 gene variation is rescued, and the visual function of the RDH12 is improved or the disease progress is delayed.

Description

[0001] Priority and related applications

[0002] This application claims priority to Chinese patent application CN202410528860.6, filed on April 29, 2024, entitled “A gene therapy drug for RDH12 mutation”, and all contents of the application including the appendix are incorporated into this disclosure as a reference. Technical Field

[0003] The present invention relates to the field of biotechnology, in particular to an anti-human RDH12 gene expression frame carried by a recombinant adeno-associated virus vector and a gene medicine comprising the gene expression frame, belonging to the field of biotechnology. Background Art

[0004] Inherited retinal diseases (IRDs) are a group of congenital and inherited retinal diseases characterized by retinal lesions. These diseases have clinical and genetic heterogeneity, complex clinical manifestations, and diverse inheritance patterns. Currently, nearly 300 genes are known to cause the disease. IRD is currently the main cause of irreversible blinding eye disease.

[0005] Mutations in the retinol dehydrogenase 12 (RDH12) gene can lead to severe inherited retinal degenerative diseases, such as Leber congenital amaurosis (LCA), early-onset severe retinal dystrophy (EOSRD), autosomal dominant and recessive retinitis pigmentosa (ADRP and ARRP), and cone-rod dystrophy (CORD). Most patients have early-onset severe retinal dystrophy as the clinical phenotype, with significant visual impairment in childhood, gradual progression of the disease, and significant macular atrophy. The macular tissue on optical coherence tomography (OCT) of patients atrophies and is significantly sunken. Due to RDH12 gene mutations, patients have severe vision loss since childhood, and there is no effective treatment. Most patients have difficulty taking care of themselves, which imposes a heavy burden on their families and society.

[0006] To this end, the present invention aims to provide a gene therapy drug targeting RDH12 mutation to solve the above problems. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems and provide a gene therapy drug for RDH12 mutation. The codon-optimized human RDH12-cDNA is packaged in a modified AAV vector and injected into the vitreous cavity to express complete and active RDH12 protein, participate in the visual cycle pathway, rescue the functional abnormalities caused by RDH12 gene mutation, improve visual function or delay disease progression.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a gene therapy drug for RDH12 mutation, wherein the gene therapy drug comprises three plasmids: a phepler plasmid, a RepCap plasmid pAAV-RC2_IVT13 with a serotype of IVT13, and a pssAAV-CB7-RDH12opt-hGH pA plasmid, wherein the gene sequence of the pssAAV-CB7-RDH12 opt-hGH pA plasmid is SEQ ID NO: 3.

[0010] In some preferred technical solutions, the drug is used to treat hereditary retinopathy.

[0011] In a second aspect, the present invention provides a method for preparing a gene therapy drug for RDH12 mutation as described in the first aspect of the present invention, characterized in that the gene therapy drug is prepared by the following steps:

[0012] S1. Obtain the CDS sequence from the mRNA sequence of human RDH12 and optimize its codons to obtain RDH12 opt;

[0013] S2, seamlessly cloning the RDH12 opt sequence into the AAV vector plasmid pssAAV-CB7-Fluc-hGH pA carrying the firefly luciferase reporter gene expression frame, replacing the Fluc sequence in the pssAAV-CB7-Fluc-hGH pA to obtain pssAAV-CB7-RDH12 opt-hGH pA, the expression frame of the pssAAV-CB7-RDH12 opt-hGH pA vector plasmid is CB7-RDH12opt-hGH pA;

[0014] S3. Use IVT13 with an amino acid sequence such as SEQ ID NO: 4 as the shell, and package and recombinant the pssAAV-CB7-RDH12 opt-hGH pA through a three-plasmid packaging system to obtain the rAAV IVT13-RDH12 gene therapy drug.

[0015] In some preferred technical solutions, the operation of packaging and recombining the pssAAV-CB7-RDH12 opt-hGH pA by a three-plasmid packaging system in step S3 is:

[0016] HEK 293T cells were inoculated in a 100 mm culture dish and grown to a confluence of 70% to 80% for plasmid transfection; 5 μg of pssAAV-CB7-RDH12 opt-hGH pA plasmid, 5 μg of RepCap plasmid pAAV-RC2_IVT13 with serotype IVT13 and 10 μg of phepler plasmid (the plasmid was purchased and stored by the applicant from Cell Biolabs. The plasmid contains the adenovirus-derived auxiliary function genes E2A, E4 and VA RNA required for the co-transfection of three plasmids into HEK293 (or HEK293T) cells to prepare recombinant AAV viruses.) were co-transfected into HEK293T cells using polyethyleneimine (PEI), and cells and supernatant were harvested 72 hours after transfection; cells were precipitated by low-speed centrifugation, and cells were lysed by adding 0.5% (w / v) sodium deoxycholate, 50 U / mL Benzonase nuclease and 2 mM MgCl2, incubate at 37°C for 2 hours to digest free DNA molecules, and at the same time, precipitate the supernatant on ice with a 1:5 volume of 40% PEG8000, 2.5M NaCl solution, mix the cell lysate and the supernatant and centrifuge, take the supernatant, and purify it by iodixanol ultracentrifugation purification method to obtain rAAVIVT13-RDH12 gene therapy drug.

[0017] In a third aspect, the present invention provides use of the gene therapy drug for RDH12 mutation as described in the first aspect of the present invention in the preparation of a pharmaceutical composition for treating hereditary retinopathy.

[0018] In this scheme, the RDH12 gene is located on human autosome 14q23. The protein it encodes contains 316 amino acids and belongs to retinol dehydrogenases (RDHs). It is a member of the short-chain dehydrogenase / reductase family. Its main function in the visual cycle is to catalyze all-trans retinal to produce all-trans retinol. The structure of the RDH12 protein is highly conserved among different species, and the similarity between humans, mice and rats is as high as 85%. Currently, several studies have constructed mouse models with Rdh12 gene mutations or knockouts, but no retinal degeneration has been found. In vitro experiments have found that the function of RDH12 to convert all-trans retinal to all-trans retinol is severely reduced, and its function can be restored by subretinal injection of AAV-hRDH12. The AAV-hRDH12 gene therapy drug is constructed and introduced into retinal photoreceptor cells by intravitreal injection, so that it expresses complete and active RDH12 protein, participates in the visual cycle pathway, and rescues the functional abnormalities caused by RDH12 gene mutations. Compared with subretinal injection, intravitreal injection reduces direct contact and trauma to the retina. This helps reduce the risk of damage to the retina during treatment and reduces possible complications.

[0019] Compared with the prior art, this solution has the following beneficial effects:

[0020] 1. The gene expression cassette provided by the present invention can achieve high-level expression of RDH12;

[0021] 2. The gene delivery vector provided by the present invention can effectively deliver RDH12 into cells;

[0022] 3. The gene therapy drug provided by the present invention can achieve sustained expression of RDH12 in vivo;

[0023] 4. The gene therapy drug provided can rescue the enzymatic function of RDH12 in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of the pssAAV-CB7-Fluc-hGH pA vector in an embodiment of the present invention;

[0025] Figure 2 Schematic diagram of the structure of the pssAAV-CB7-RDH12 opt-hGH pA vector in an embodiment of the present invention;

[0026] Figure 3 Schematic diagram of the expression of PSSAAV-CB7-RDH12 OPT-HGH PA plasmid in 293T cells in an embodiment of the present invention;

[0027] Figure 4 1 is a schematic diagram of the experimental results of Rdh12 gene knockout rats in the embodiments of the present invention, wherein A is a schematic diagram of exons 2-3 of Rdh12 in knockout rats; B is a schematic diagram of DNA analysis of the expression of Rdh12 in wild-type, heterozygous and homozygous rats; C is a bar graph of the mRNA level detection of Rdh12 in knockout rats and wild-type rats;

[0028] Figure 5 It is a line graph group of ERG and OCT results of Rdh12 knockout rats and wild-type rats at 12 weeks in the embodiment of the present invention, wherein A and B are the amplitudes of a wave and b wave of dark-adapted ERG under stimulation of different light intensities, the horizontal axis represents different stimulation light intensities, and the vertical axis represents amplitude; C and D are the peak times of a wave and b wave under stimulation of different light intensities, the horizontal axis represents different stimulation light intensities, and the vertical axis represents peak time; E is fundus photography and OCT of model mice and wild-type mice; F is a statistical graph of the outer nuclear layer thickness of Rdh12 knockout mice and wild-type mice;

[0029] Figure 6 : is a schematic diagram group of enzyme function detection results of RDH12 knockout rats and wild-type rats in the embodiment of the present invention, wherein A is a waveform diagram of all-trans retinal and all-trans retinol in mass spectrometry analysis; B is a catalytic efficiency diagram within 0-45min; C is a bar graph of the content of all-trans retinal converted to all-trans retinol by RDH12 catalysis at 45min;

[0030] Figure 7 is a bar graph of mRNA expression after gene therapy with different doses of drugs in the embodiments of the present invention;

[0031] Figure 8 1 is a schematic diagram of the results of enzyme function detection after different doses of drug gene therapy in the embodiment of the present invention, wherein A is a waveform diagram of all-trans retinal and all-trans retinol in mass spectrometry analysis; B is a catalytic efficiency diagram within 0-45 minutes; C is a bar graph of the content of all-trans retinal converted into all-trans retinol by RDH12 catalysis at 45 minutes. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0034] Embodiment 1:

[0035] The materials used in the present invention are as follows:

[0036] Plasmid: phelper plasmid, purchased and stored from Cell Biolabs. This plasmid contains the adenovirus-derived auxiliary function genes E2A, E4 and VARNA required for the co-transfection of three plasmids into HEK293 (or HEK293T) cells to prepare recombinant AAV viruses;

[0037] The RC plasmid pAAV2 / 8 used for packaging AAV8 was purchased from Addgene (Cat. No. 112864). The plasmid originated from Professor James M. Wilson of the University of Pennsylvania.

[0038] HEK293T cell line (ATCC, CRL-3216);

[0039] Lipo3000 (Thermo, L3000015);

[0040] 1M Tris-HCl (Protesci, PBS011S-BR100);

[0041] 0.05M NaOH (Protesci, PBS019S-BR500);

[0042] PCR Mix (TOROIVD, KAO-201);

[0043] Sucrose (Macklin, S818045);

[0044] Trisaminomethane acetate (MCE, HY-D0227B);

[0045] Dithiothreitol (MCE, HY-15917);

[0046] All-trans retinal (Sigma-Aldrich, R2500);

[0047] All-trans retinol (Sigma-Aldrich, 95144);

[0048] NAPDH; (Thermo, N1630);

[0049] Methanol (Sigma-Aldrich, 322415);

[0050] Acetonitrile (Merck, 40064184);

[0051] Methyl tert-butyl ether (Sigma-Aldrich, 650560);

[0052] Tropicamide compound (Santen, J20180051);

[0053] Carbomer eye drops (Bausch and Lomb, J20150018)

[0054] RDH12 antibody (Aviva Systems Biology, ARP52972_P050);

[0055] α-Tubulin antibody (Proteintech, 66031);

[0056] Secondary antibody-rabbit (Proteintech, PR30011);

[0057] Secondary antibody-mouse (Proteintech, PR30012);

[0058] Trizol(Invitrogen,15596018CN);

[0059] Reverse transcription kit (Takara, RR036A);

[0060] qPCR kit (Bio-Rad, 1725121);

[0061] BCA kit (Thermo, 23227);

[0062] The experimental groups included Rdh12- / -SD rats and wild-type SD rats (purchased from Saiye Experimental Animal Technology Co., Ltd.).

[0063] Embodiment 2:

[0064] Codon-optimized plasmid vector construction:

[0065] In order to construct an AAV vector plasmid carrying the human RDH12 expression frame, this example first codon-optimized the open reading frame of RDH12, and then constructed an expression vector plasmid.

[0066] The specific process is as follows:

[0067] The CDS sequence (SEQ ID NO: 1) of human RDH12 was obtained from the mRNA sequence (NM_152443.3) registered in NCBI. Codon optimization was performed to obtain RDH12 opt (SEQ ID NO: 2). The RDH12 opt sequence was sent to General Bio (Anhui) Co., Ltd. for synthesis and seamlessly cloned into the AAV vector plasmid pssAAV-CB7-Fluc-hGH pA ( Figure 1 ), and the Fluc sequence was replaced to obtain pssAAV-CB7-RDH12 opt-hGH pA( Figure 2 ). The expression cassette of the vector plasmid is CB7-RDH12 opt-hGH pA (SEQ ID NO: 3).

[0068] SEQ ID NO:1 (human RDH12 CDS nucleotide sequence)

[0069] 5’atgctggtcaccttgggactgctcacctccttcttctcgttcctgtatatggtagctccatccatcaggaagttctttgctggtggagtgtgtagaacaaatgtgcagcttcctggcaaggtagtggtgatcactggcgccaacacgggcattggcaaggagacggccagagagctcgctagccgaggagcccgagtctatattgcctgcagagatgtactgaagggggagtctgctgccagtgaaatccgagtggatacaaagaactcccaggtgctggtgcggaaattggacctatccgacaccaaatctatccgagcctttgctgagggctttctggcagaggaaaagcagctccatattctgatcaacaatgcgggagtaatgatgtgtccatattccaagacagctgatggctttgaaacccacctgggagtcaaccacctgggccacttcctcctcacctacctgctcctggagcggctaaaggtgtctgcccctgcacgggtggttaatgtgtcctcggtggctcaccacattggcaagattcccttccacgacctccagagcgagaagcgctacagcaggggttttgcctattgccacagcaagctggccaatgtgctttttactcgtgagctggccaagaggctccaaggcaccggggtcaccacctacgcagtgcacccaggcgtcgtccgctctgagctggtccggcactcctccctgctctgcctgctctggcggctcttctccccctttgtcaagacggcacgggagggggcgcagaccagcctgcactgcgccctggctgagggcctggagcccctgagtggcaagtacttcagtgactgcaagaggacctgggtgtctccaagggcccgaaataacaaaacagctgagcgcctatggaatgtcagctgtgagcttctaggaatccggtgggagtag3’;

[0070] SEQ ID NO:2 (RDH12 opt nucleotide sequence)

[0071] 5’atgctggtgacactgggcctgctgacaagcttctttagctttctttatatggtggctcctagcatcaggaagttctttgcaggaggagtgtgcaggaccaatgtgcagctgcctggcaaggtggtggtcatcacaggagccaatacaggaataggcaaggaaacagccagagaactggccagcagaggagccagggtgtacatagcctgtagagatgtgctgaaaggggagagtgcagcttctgagatcagggtggacacaaagaacagccaggtgctggtcagaaagctggacctgtcagatacaaaaagcatcagagcctttgcagagggcttcctggcagaggaaaagcagctgcacatcctgatcaacaatgcaggagttatgatgtgcccctacagcaagacagctgatggctttgagacacacctgggagttaaccacctgggacacttcctgctgacctacctgctgctggaaaggctgaaggtgtcagcccctgccagagtggtgaatgtgtctagtgtggcccaccatattggcaagatccccttccatgacctgcaaagtgagaagagatactccagaggctttgcctactgccacagcaagctggctaatgtgctgttcaccagagagctggccaagaggctgcagggcacaggagtgaccacatatgcagtgcacccaggagtggtgagatctgaactggtgaggcactccagcctgctgtgcctgctgtggaggctgttcagcccttttgtgaagacagccagagagggagctcagaccagcctgcactgtgccctggcagaaggtcttgagcctctgagtggcaaatacttcagtgactgcaagaggacctgggtctctccaagagccagaaacaacaaaacagctgagagactgtggaatgtgtcttgtgaactgctgggcatcagatgggagtaa3’;

[0072] SEQ ID NO:3 (CB7-RDH12 opt-hGH pA expression cassette nucleotide sequence)

[0073]

[0074] Embodiment 3:

[0075] Virus packaging and testing:

[0076] In this example, the pssAAV-CB7-RDH12 opt-hGH pA vector plasmid constructed in Example 1 was used to further package the virus. With reference to the literature Xiao X, et al. J Virol. 1998; 72(3): 2224-2232.), certain adjustments were made on this basis. The three-plasmid packaging system was used to package the recombinant AAV, and the outer shell used IVT13 with an amino acid sequence such as SEQ ID NO: 4 as the outer shell.

[0077] Wherein, the amino acid sequence of SEQ ID NO:4 is:

[0078] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN AAARGSLAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL.

[0079] The specific construction method of the RepCap plasmid pAAV-RC2_IVT13 with serotype IVT13 is as follows:

[0080] Step 1) Construction of intermediate plasmid RC2_IVB-NotI plasmid

[0081] A reverse P5 promoter sequence was added upstream of the Rep sequence on pAAV-RC2 (purchased from CellBiolabs, catalog number: VPK-410-SER2), and a NotI restriction endonuclease site was inserted at 1752 bp of the Cap2 sequence. The intermediate plasmid RC2_IVB-NotI plasmid was commissioned to Anhui General Biotechnology to complete the construction. The specific sequence is as follows:

[0082] RC2_IVB-NotI plasmid vector sequence (SEQ ID NO: 5)

[0083]

[0084] Step 2) Construction of AAV capsid protein expression plasmid encoding IVT13

[0085] The AAV capsid protein expression plasmid encoding IVT13 was constructed using the Gibson assembly method (for specific steps, see Gibson Chemical Transformation Protocol (E2611), Gibson assembly of the PCR fragment and the fragment of the intermediate plasmid RC2_IVB-NotI plasmid linearized by NotI enzyme as constructed in step 1) above, and then obtain the AAV capsid plasmid encoding IVT13. In the process of obtaining the PCR fragment, PCR has no template, and the primers can be paired by themselves for PCR amplification. The primer sequences are as follows:

[0086] Forward primer (SEQ ID NO:6):

[0087] TGCCGCAAGACTGCCCCTAGCAGCGGCGTTGCCTCTCTGGAGGTTGGTAGATACAGAACCCATACTG

[0088] Reverse primer (SEQ ID NO:7):

[0089] GCCGCTGCTAGGGGCAGTCTTGCGGCAAGACAAGCAGCTACCGC AGATGTCAACACACAAGGCG

[0090] Experimental results: The constructed plasmid DNA was identified by restriction digestion and Sanger sequencing, which proved that the expression plasmid of IVT13 capsid protein was successfully constructed. The amino acid sequence of the AAV capsid protein expressed in the obtained AAV capsid protein expression plasmid is shown in SEQ ID NO:4.

[0091] HEK 293T cells were inoculated in a 100 mm culture dish and grown to a confluence of 70% to 80% for plasmid transfection. 5 μg of pssAAV-CB7-RDH12 opt-hGH pA plasmid, 5 μg of RepCap plasmid pAAV-RC2_IVT13 with serotype IVT13, and 10 μg of phepler plasmid (purchased and preserved by the applicant from Cell Biolabs. The plasmid contains the adenovirus-derived auxiliary function genes E2A, E4, and VA RNA required for the co-transfection of three plasmids into HEK293 (or HEK293T) cells to prepare recombinant AAV viruses.) were co-transfected into HEK293T cells using polyethyleneimine (PEI), and cells and supernatant were harvested 72 hours after transfection. The cells were precipitated by low-speed centrifugation, and lysed by adding 0.5% (w / v) sodium deoxycholate, adding 50U / mL Benzonase nuclease and 2mM MgCl2, incubating at 37°C for 2 hours to digest free DNA molecules, and precipitating the supernatant on ice with a 1:5 volume of 40% PEG8000, 2.5M NaCl solution, mixing the cell lysate and supernatant, centrifuging, taking the supernatant, and purifying it by iodixanol ultracentrifugation purification method to finally obtain rAAVIVT13-RDH12. The purified AAV titer was then determined by qPCR and stored in a -80°C refrigerator.

[0092] The specific determination method is as follows:

[0093] The physical titer of the prepared AAV viral genome was determined by qPCR. The specific process is as follows:

[0094] Design primers and probes for qPCR detection based on the exact same sequence of RDH12 opt:

[0095] RDH12-QF: 5'-CTGCTGGAAAGGCTGAAGGT-3' (SEQ ID NO: 8);

[0096] RDH12-QR: 5'-TCTCTTCTCACTTTGCAGGTCATG-3' (SEQ ID NO: 9);

[0097] RDH12-QP: 5'-TGGCCCACCATATTGGCAAGATCC-3' (SEQ ID NO: 10);

[0098] RDH12-QF and RDH12-QR were used as primers, and RDH12-QP was used as a probe. The 5' end of the probe was labeled with FAM fluorescent protein, and the 3' end was connected to BlackBerry quencher. The primers and probe were synthesized by ThermoFisher Scientific. RDH12-QF and RDH12-QR were used as primers to specifically amplify a 108 bp fragment in the RDH12 opt sequence. The TaqMan probe binding method was used with 1.0×10 8 pssAAV-CB7-RDH12 opt-hGH pA plasmid with 10 copies / mL and its 10-fold gradient dilution samples were used as standards, and the physical titer of the viral genome was detected using Premix Ex Taq (Probe qPCR) reagent (TaKaRa, Beijing, China) and a fluorescent quantitative PCR instrument (model: Q5, ThermoFisher). The operation process is described in the instructions of the reagents used. The virus processing method is described in the literature (Aurnhammer C, et al. Hum Gene Ther Methods. 2012; 23(1): 18-28.).

[0099] Experimental results: After testing, the physical titer of the packaged rAAVIVT13-RDH12 viral genome was 1.0E+13vg / mL.

[0100] Embodiment 4:

[0101] In vitro expression of pssAAV-CB7-RDH12 opt-hGH pA plasmid:

[0102] To determine the expression of the rAAV vector plasmid carrying the RDH12 expression frame. In this example, lipo3000 (ThermoFisher) was used to transfect the pssAAV-CB7-RDH12 opt-hGH pA plasmid constructed in Example 2 into HEK293T cells in vitro. After 48 hours of transfection, the cell protein was extracted. Western blotting was used to detect intracellular RDH12 protein. Protein was extracted using RIPA+PMSF and the concentration was quantified using a BCA kit. Gel preparation: 12% separation gel + 5% concentrated gel, protein sample 98°C metal bath for 10 minutes, 3000rpm, 2min centrifugation, sample electrophoresis, 300mA constant current for 1h transfer, 5% skim milk blocking for 1h, RDH12 primary antibody incubated overnight at 4°C, the primary antibody was recovered the next day, washed 3 times with TBST, 8min each time, and then incubated with secondary antibody at room temperature on a shaker for 1h, the secondary antibody was recovered, and the membrane was washed 3 times with TBST, 8min each time. Using Bio-Rad ChemiDoc TMThe MP Imager imaged the membrane under chemiluminescent blotting, and the results showed that it can be expressed in cells ( Figure 3 ).

[0103] Embodiment 5:

[0104] Construction and identification of Rdh12 KO rat model:

[0105] The rat strain used in this study was SD rats, and exons 2-3 of the rat Rdh12 gene were knocked out ( Figure 4 A). CRISPR / Cas9 technology was used to design sgRNA, and RDH12 gene knockout rats were obtained by high-throughput electroporation of fertilized eggs. After sexual maturity, sperm was cryopreserved to verify the F0 with reproductive transmission ability, and then subsequent breeding was continued. The rats used in the experiment were purchased from Saiye Biotechnology Co., Ltd. The rats were raised in the Animal Center of Peking Union Medical College Hospital, with room temperature controlled at 20-25℃, free access to water and food. The breeding room uses a timed fluorescent lighting device, and rat feed and bedding are regularly replaced every week. After adulthood, the rats are mated in cages at an appropriate time according to the needs of the experiment. The breeding, experimental operations and sacrifice of all experimental rats were approved by the Animal Ethics Committee of the Animal Experiment Center of Peking Union Medical College Hospital. The genotype of rats aged 3-4 weeks (w) was identified. 3mm of the rat tail was taken and the DNA was extracted by the alkaline lysis and heating rapid method. The specific method is as follows: 180μL of 50mM NaOH solution was added to the EP tube containing the rat tail, vortexed and mixed, and placed in a 98℃ metal bath for 10min. 20μL of 1M Tris-HCL (PH=8) was added to the centrifuge tube, vortexed and mixed thoroughly, centrifuged at 12000rpm for 5min, and the supernatant was used as a DNA template for PCR. PCR reaction was performed using 2% agarose gel, 5μL of PCR product was loaded into each well, and electrophoresis was performed at 120V for 25min for development. The amplified product size of wild-type rats is 1607 and 672bp, the amplified product of heterozygotes includes 1607, 672 and 459bp, and the amplified product size of homozygotes is only 459bp ( Figure 4B). To detect the knockout effect of Rdh12 gene, the level of Rdh12 mRNA was detected by qPCR. The rat retina was collected, 1 mL Trizol (Sigma) was added and mixed, fully lysed on ice, 200 μL chloroform was added and mixed, and centrifuged at 4°C, 12000 rpm for 15 min; 500 μL supernatant was taken and 500 μL isopropanol (equal volume) was added, mixed and allowed to stand for 10 min; centrifuged at 4°C, 12000 rpm for 10 min, the supernatant was discarded, and the RNA was precipitated at the bottom of the tube; 1 ml 75% ethanol was added for washing, and the RNA was gently mixed to suspend the RNA, and centrifuged at 4°C, 7500 rpm for 5 min; the supernatant was discarded (carefully blotted dry), dried in a fume hood for 10-20 min, and 20-50 μL sterilized DEPC water was added to dissolve the RNA sample; the OD value was measured to quantify the RNA concentration. Then reverse transcription (Takara) was performed, and cDNA was used for qPCR (BioRad) reaction, GAPDH was used as the internal reference gene, and each sample was repeated 3 times to reduce errors; the detection was performed on the ABI7500 quantitative instrument, using 2 -ΔΔCT The results showed that compared with the normal control group, the mRNA content of Rdh12 in the knockout rats was significantly reduced ( Figure 4 C).

[0106] Table 1 PCR and qPCR primer sequences

[0107]

[0108] Table 2 PCR system: system 25 μL, template 1.5 μL

[0109]

[0110] Table 3 PCR program

[0111]

[0112] Embodiment 6:

[0113] Phenotypic detection of Rdh12- / - rats:

[0114] To understand the effects of Rdh12 knockout on the function and structure of the rat retina, we performed electroretinography (ERG) and optical coherence tomography (OCT) tests on RDH12 knockout rats and wild-type rats. We performed ERG and OCT tests on the two groups of rats at 8, 12, and 16 weeks after birth.

[0115] ERG detection was performed using the Celeris system (Diagnosys LLC). Rats were dark-adapted overnight for dark-view ERG recording. 10 minutes before recording, pupils were dilated with tropicamide (1%). Rats were anesthetized using a gas anesthesia machine. During the entire ERG procedure, the animals were placed on a heating plate to maintain the body temperature at 37°C. Two electrodes were placed in the direction of the eye axis, the resistance was measured, and the resistance was adjusted to below 5Ω to start the detection. The dark-adapted ERG protocol included 5 steps, with the stimulation intensity ranging from 0.003, 0.01, 0.1, 1 to 10 cd.s / m 2 The light intensity of the rat retina was stimulated to induce discharge. All flashes were presented without background illumination, with a constant interstimulus interval of 5 seconds for dim flashes and 30 seconds for bright flashes to maintain dark adaptation. After 5 minutes of light adaptation, the rat retina was exposed to 1, 3.16, 10, and 31.6 cd.s / m 2 The light intensity stimulated the rat retina and caused it to discharge. After the test, Bausch and Lomb eye gel was applied to the rat's eyes.

[0116] Fundus examination was performed using Micron IV Retinal Imaging Microscope (Phoenix Research Labs). Before the examination, one drop of compound tropicamide eye drops was dripped into each eye of the rat to dilate the pupil. Then gas anesthesia was performed and the rat was placed on a bed. The eye was placed close to the lens, and the eye was always placed in the middle of the lens. The distance was adjusted. After the fundus image was clear, the brightness and focus were adjusted to make the fundus image clearer. The OCT image was adjusted so that the optic disc was in the middle, and the contrast, brightness and compensation were adjusted to make the image clearer. After the adjustment was clear, the picture was taken using the picture overlay mode. After the shooting was completed, Bausch and Lomb gel was dripped on the rat, and the lens was wiped with a cotton swab dipped in distilled water.

[0117] The results are as follows Figure 5 After sufficient dark adaptation, the results of different light intensity stimulation were analyzed and there was no statistical difference in the amplitude and peak time of ERG a wave and b wave between the two groups ( Figure 5 AD), no obvious changes in the fundus ( Figure 5 E), the thickness of the retinal outer nuclear layer had no statistical difference ( Figure 5 F).

[0118] RDH12 is a retinol dehydrogenase. In the visual cycle, RDH12 promotes the conversion of all-trans retinal to all-trans retinol. To explore the changes in the enzymatic function of RDH12, we extracted rat retinas for in vitro enzymatic function detection. 200 μL of a solution consisting of sucrose (0.25 M), trisaminomethane acetate (25 mM, PH=7), and dithiothreitol (1 mM) was added to the retinal homogenate of one mouse (two eyes); the retinal homogenate was sonicated on ice (speed: 30 times / second, ultrasound for 10 s and stop for 20 s, and cycled 3 times); after the disruption was completed, it was centrifuged at 1000×g for 5 min at 4°C; the unbroken cells (precipitate) were removed and the supernatant was aspirated; the protein concentration was measured using a BCA kit, the OD value was detected using an enzyme reader, a standard curve was drawn based on the standard sample, and a formula was obtained; the concentration of the sample was calculated according to the formula; the retinal homogenates of the two eyes were mixed, 40 ug of protein was taken, and a solution containing 200 μm NAPDH and 200μM all-trans-retinal in hepes buffer (pH=7). Incubate at 37℃ for 0min and 45min. Prepare mass spectrometry standard solution: concentrations of 1000ng / mL, 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2ng / mL, 1ng / mL; the solvent is acetonitrile, and the solution is a mixture of retinal and retinol. Add 200μL of the incubated solution 1:1 to 200μL acetonitrile, vortex for 1min, then add 1.2mL methyl tert-butyl ether, vortex for 1min, add internal standard, centrifuge at 14000g for 10min, take the upper solvent and spin dry; re-dissolve with 1:3 = water: methanol, and detect on a mass spectrometer. It was found that the enzyme activity of the knockout mouse was much lower than that of the wild-type rat ( Figure 6 ).

[0119] Embodiment 7:

[0120] Gene therapy:

[0121] To detect the therapeutic effect of rAAVIVT13-RDH12 on Rdh12- / - rats, 12 Rdh12- / - SD rats aged 8-10 weeks were randomly divided into two groups, six rats in each group, and injected intravitreally. The high-dose group received 8×10 10 vg / eye, and 4.5×10 9 vg / eye. Four weeks after injection, all rat retinas were collected, RNA was extracted from three rats, and protein was extracted from three rats. qPCR was used to detect the mRNA expression of RDH12, and it was found that the mRNA level in both the low-dose group and the high-dose group increased, and the mRNA level in the high-dose group increased more significantly ( Figure 7The enzymatic function of RDH12 was detected by mass spectrometry, using the same method as in Example 6. The results showed that the RDH12 reduction function in both the low-dose and high-dose groups was significantly improved ( Figure 8 ).

[0122] Table 4 qPCR primer sequences

[0123]

[0124] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A gene therapy drug for RDH12 mutation, characterized by: The gene therapy drug comprises three plasmids: phepler plasmid, RepCap plasmid pAAV-RC2_IVT13 with serotype IVT13, and pssAAV-CB7-RDH12 opt-hGH pA plasmid, wherein the gene sequence of pssAAV-CB7-RDH12 opt-hGH pA plasmid is SEQ ID NO:

3.

2. The method for preparing a gene therapy drug for RDH12 mutation according to claim 1, characterized in that: The gene therapy drug is prepared by the following steps: S1. Obtain the CDS sequence from the mRNA sequence of human RDH12 and optimize its codons to obtain RDH12opt; S2, seamlessly cloning the RDH12 opt sequence into the AAV vector plasmid pssAAV-CB7-Fluc-hGH pA carrying the firefly luciferase reporter gene expression frame, replacing the Fluc sequence in the pssAAV-CB7-Fluc-hGH pA to obtain pssAAV-CB7-RDH12 opt-hGH pA, the expression frame of the pssAAV-CB7-RDH12 opt-hGH pA vector plasmid is CB7-RDH12opt-hGH pA; S3. Use IVT13 with an amino acid sequence such as SEQ ID NO: 4 as the shell, and package and recombinant the pssAAV-CB7-RDH12 opt-hGH pA through a three-plasmid packaging system to obtain the rAAVIVT13-RDH12 gene therapy drug.

3. The method for preparing a gene therapy drug for RDH12 mutation according to claim 2, characterized in that: The operation of recombining the pssAAV-CB7-RDH12 opt-hGH pA by the three-plasmid packaging system in step S3 is: HEK 293T cells were inoculated in a 100 mm culture dish and grown to a confluence of 70% to 80%. 5 μg of pssAAV-CB7-RDH12 opt-hGH pA plasmid, 5 μg of RepCap plasmid pAAV-RC2_IVT13 with serotype IVT13, and 10 μg of phepler plasmid were co-transfected into HEK293T cells using polyethyleneimine, and cells and supernatant were harvested 72 hours after transfection. The cells were precipitated by low-speed centrifugation, and the cells were lysed by adding 0.5% (w / v) sodium deoxycholate, 50 U / mL Benzonase nuclease and 2 mM MgCl2 were added, and the free DNA molecules were digested at 37°C for 2 hours, and 1:5 volume of 40% PEG8000, 2.5 M The supernatant was precipitated with NaCl solution on ice, the cell lysate and the supernatant were mixed and centrifuged, the supernatant was taken, and purified by iodixanol ultracentrifugation purification method to finally obtain the rAAVIVT13-RDH12 gene therapy drug.

4. A gene therapy drug for RDH12 mutation according to claim 1, characterized in that: The medicament is used to treat hereditary retinal diseases.

5. Use of a gene therapy drug for RDH12 mutation as claimed in claim 1 in the preparation of a pharmaceutical composition for treating hereditary retinopathy.

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