A gene therapy drug targeting RDH12 mutations

Abstract

This invention discloses a gene therapy drug targeting RDH12 mutations, belonging to the field of biotechnology. The key technical points are: by constructing an AAV-hRDH12 gene therapy drug, it is introduced into retinal photoreceptor cells via intravitreal injection, enabling them to express complete and active RDH12 protein, participate in the visual circulation pathway, and rescue functional abnormalities caused by RDH12 gene mutations. This invention utilizes a modified AAV vector to package codon-optimized human RDH12-cDNA, enabling it to express complete and active RDH12 protein, participate in the visual circulation pathway, rescue functional abnormalities caused by RDH12 gene mutations, improve visual function, or delay disease progression.

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 targeting RDH12 mutations”, the entire contents of which, including its appendices, are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, specifically to an anti-human RDH12 gene expression cassette carried by a recombinant adeno-associated virus vector, and a gene drug containing the gene expression cassette, belonging to the field of biotechnology. Background Technology

[0004] Inherited retinal diseases (IRDs) are a group of congenital, hereditary retinal disorders characterized by retinal damage. These diseases exhibit clinical and genetic heterogeneity, with complex clinical manifestations and diverse inheritance patterns. Nearly 300 genes are currently known to cause these diseases. IRDs are the leading cause of irreversible blindness.

[0005] Mutations in the retinol dehydrogenase 12 (RDH12) gene can lead to severe hereditary retinal degenerative diseases, such as Leber congenital amaurosis (LCA), early-onset severe retinal dystrophy (EOSRD), autosomal dominant and recessive retinitis pigmentosa (ADRP & ARRP), and cone-rod dystrophy (CORD). Most patients present with early-onset severe retinal dystrophy, exhibiting significant vision impairment in childhood. The disease progresses gradually, with marked macular atrophy, evident in optical coherence tomography (OCT) scans showing macular tissue atrophy and significant concavity. Because RDH12 gene mutations cause severe vision loss from childhood and there are no effective treatments, patients often struggle with daily living, placing a heavy burden on their families and society.

[0006] Therefore, the present invention aims to provide a gene therapy drug targeting RDH12 mutations to solve the above-mentioned problems. Summary of the Invention

[0007] The purpose of this invention is to solve the above-mentioned problems and provide a gene therapy drug targeting RDH12 mutations. The drug uses a modified AAV vector to package codon-optimized human RDH12-cDNA and is injected intravitreally to express a complete and active RDH12 protein, which participates in the visual circulation pathway, rescues the functional abnormalities caused by RDH12 gene mutations, improves visual function, or delays disease progression.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

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

[0010] In some preferred embodiments, the drug is used to treat hereditary retinal diseases.

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

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

[0013] S2. The RDH12 opt sequence is seamlessly cloned into the AAV vector plasmid pssAAV-CB7-Fluc-hGH pA carrying the firefly luciferase reporter gene expression cassette, and the Fluc sequence in pssAAV-CB7-Fluc-hGH pA is replaced to obtain pssAAV-CB7-RDH12 opt-hGH pA. The expression cassette of the pssAAV-CB7-RDH12 opt-hGH pA vector plasmid is CB7-RDH12opt-hGH pA.

[0014] S3. Using IVT13, an amino acid sequence such as SEQ ID NO:4, as a shell, the recombinant pssAAV-CB7-RDH12 opt-hGH pA is packaged using 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 using a three-plasmid packaging system in step S3 is as follows:

[0016] HEK 293T cells were seeded in 100 mm culture dishes and grown to 70%–80% confluence for plasmid transfection. 5 μg of pssAAV-CB7-RDH12 opt-hGH pA plasmid, 5 μg of the IVT13-based RepCap plasmid pAAV-RC2_IVT13, and 10 μg of phepler plasmid (purchased and stored by the applicant from Cell Biolabs. This plasmid contains adenovirus-derived helper genes E2A, E4, and VA RNA required for the preparation of recombinant AAV virus from HEK293 (or HEK293T) cells) were co-transfected into HEK293T cells. Cells and supernatant were harvested 72 hours post-transfection. Cells were pelleted by low-speed centrifugation, and lysed by adding 0.5% (w / v) sodium deoxycholate. 50 U / mL Benzonase nuclease and 2 mM sodium deoxycholate were then added. The free DNA molecules were digested by incubation with MgCl2 at 37°C for 2 hours. Simultaneously, the supernatant was precipitated on ice with a 1:5 volume ratio of 40% PEG8000 and 2.5M NaCl solution. The cell lysate and supernatant were mixed and centrifuged. The supernatant was then purified by ultracentrifugation with iodixanol to obtain the rAAVIVT13-RDH12 gene therapy drug.

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

[0018] In this protocol, the RDH12 gene is located on human chromosome 14q23. Its encoded protein contains 316 amino acids and belongs to the retinol dehydrogenases (RDHs), a member of the short-chain dehydrogenase / reductase family. Its main function in the visual cycle is to catalyze the conversion of all-trans-retinal to all-trans-retinol. The RDH12 protein structure is highly conserved across different species, showing over 85% similarity between human, mouse, and rat proteins. Several studies have constructed mouse models with RDH12 gene mutations or knockouts, but no retinal degeneration has been observed. However, in vitro experiments have shown that the RDH12 function in converting all-trans-retinal to all-trans-retinol is severely reduced, and subretinal injection of AAV-hRDH12 can restore its function. AAV-hRDH12 gene therapy drug was constructed and introduced into retinal photoreceptor cells via intravitreal injection, enabling them to express complete and active RDH12 protein, which participates in the visual circulation pathway and rescues functional abnormalities caused by RDH12 gene mutations. Compared to subretinal injection, intravitreal injection reduces direct contact and trauma to the retina. This helps reduce the risk of retinal damage during treatment and minimizes potential complications.

[0019] Compared with existing technologies, the beneficial effects of this solution are:

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

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

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

[0023] 4. The gene therapy drugs provided can salvage the enzymatic function of RDH12 in vitro. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the pssAAV-CB7-Fluc-hGH pA carrier structure in an embodiment of the present invention;

[0025] Figure 2 This is a schematic diagram of the pssAAV-CB7-RDH12 opt-hGH pA carrier structure in an embodiment of the present invention;

[0026] Figure 3 This is a 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 This 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 Rdh12 expression in wild-type, heterozygous and homozygous rats; C is a bar chart of Rdh12 mRNA level detection in knockout rats and wild-type rats;

[0028] Figure 5 This is a set of line graphs showing the ERG and OCT results of Rdh12 knockout rats and wild-type rats at 12W in this embodiment of the invention. A and B are the amplitudes of the a and b waves of dark-adapted ERG under different light intensities, with the horizontal axis representing different light intensities and the vertical axis representing the amplitude; C and D are the peak times of the a and b waves under different light intensities, with the horizontal axis representing different light intensities and the vertical axis representing the peak time; E is the fundus photography and OCT of the model rats and wild-type rats; F is a statistical graph of the outer nuclear layer thickness of Rdh12 knockout rats and wild-type rats.

[0029] Figure 6 This is a schematic diagram of the enzyme function detection results of RDH12 knockout rats and wild-type rats in the embodiments of the present invention. A is the waveform diagram of all-trans retinaldehyde and all-trans retinol in mass spectrometry analysis; B is the catalytic efficiency diagram within 0-45 min; C is the bar chart of the content of all-trans retinaldehyde catalyzed by RDH12 to all-trans retinol at 45 min.

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

[0031] Figure 8 This is a set of schematic diagrams showing the results of enzyme function detection after gene therapy with different doses of drugs in the embodiments of the present invention. In this diagram, A is a waveform diagram of all-trans retinaldehyde and all-trans retinol in mass spectrometry analysis; B is a catalytic efficiency diagram within 0-45 min; and C is a bar chart of the content of all-trans retinaldehyde converted to all-trans retinol by RDH12 catalysis at 45 min. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0034] Example 1:

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

[0036] Plasmid: phelper plasmid, purchased from Cell Biolabs and stored here. This plasmid contains adenovirus-derived helper genes E2A, E4, and VARNA, which are required for the preparation of recombinant AAV virus by co-transfection of HEK293 (or HEK293T) cells with three plasmids;

[0037] The RC plasmid pAAV2 / 8 used for packaging AAV8 was purchased from Addgene (catalog number: 112864), and the plasmid was derived 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] Triaminomethane acetate (MCE, HY-D0227B);

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

[0046] All-trans retinaldehyde (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] Compound tropicamide (Santen, J20180051);

[0053] Carbomer Eye Drops (Bausch & 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 group consisted of Rdh12- / - SD rats and wild-type SD rats (purchased from Cyagen Laboratory Animal Technology Co., Ltd.).

[0063] Example 2:

[0064] Construction of codon-optimized plasmid vectors:

[0065] To construct an AAV vector plasmid carrying the human RDH12 expression frame, this embodiment first optimizes the codons of the open reading frame of RDH12, and then constructs the expression vector plasmid.

[0066] The specific process is as follows:

[0067] The CDS sequence (SEQ ID NO:1) of human RDH12 mRNA sequence (NM_152443.3) registered in NCBI was obtained. Codon optimization yielded RDH12 opt (SEQ ID NO:2). The RDH12 opt sequence was sent to General Biotechnology (Anhui) Co., Ltd. for synthesis and seamless cloning into the AAV vector plasmid pssAAV-CB7-Fluc-hGH pA (carrying the firefly luciferase (Fluc) reporter gene expression cassette) stored by the applicant. Figure 1 In ), replacing the Fluc sequence yields pssAAV-CB7-RDH12 opt-hGH pA( Figure 2 The expression cassette for this 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] Example 3:

[0075] Virus packaging and detection:

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

[0077] The amino acid sequence of SEQ ID NO:4 is as follows:

[0078] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN AAARGSLAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL.

[0079] The specific construction method of RepCap plasmid pAAV-RC2_IVT13 for 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 was constructed by Anhui General Biotechnology, and the specific sequence is shown below:

[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 (see Gibson for detailed steps). Chemical Transformation Protocol (E2611) is used to assemble the PCR fragment and the NotI-digested linearized intermediate plasmid RC2_IVB-NotI plasmid constructed in step 1) above using Gibson assembly, thereby obtaining the AAV capsid plasmid encoding IVT13. During the PCR fragment acquisition process, no template is required for PCR; primers pair naturally for PCR amplification. The primer sequences are shown below:

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

[0087] TGCCGCAAGACTGCCCCTAGCAGCGGCGTTGCCTCTCTGGAGGTTGGTAGATACAGAACCATACTG

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

[0089] GCCGCTGCTAGGGGCAGTCTTGCGGCAAGACAAGCAGCTACCGC AGATGTCAACACACAAGGCG

[0090] Experimental results: The constructed plasmid DNA was identified by enzyme digestion and Sanger sequencing, confirming the successful construction of the IVT13 capsid protein expression plasmid. 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 seeded in 100 mm culture dishes and grown to 70%–80% confluence for plasmid transfection. 5 μg of pssAAV-CB7-RDH12 opt-hGH pA plasmid, 5 μg of the IVT13-based RepCap plasmid pAAV-RC2_IVT13, and 10 μg of phepler plasmid (purchased and stored by the applicant from Cell Biolabs. This plasmid contains adenovirus-derived helper genes E2A, E4, and VA RNA required for the preparation of recombinant AAV virus from HEK293 (or HEK293T) cells) were co-transfected into HEK293T cells using polyethyleneimine (PEI). Cells and supernatant were harvested 72 hours after transfection. Cells were pelleted by low-speed centrifugation, and then lysed by adding 0.5% (w / v) sodium deoxycholate. Free DNA molecules were digested by adding 50 U / mL Benzonase nuclease and 2 mM MgCl2, and incubating at 37°C for 2 hours. Simultaneously, the supernatant was precipitated on ice with a 1:5 volume solution of 40% PEG8000 and 2.5 M NaCl. The cell lysate and supernatant were mixed and centrifuged. The supernatant was then purified using iodixanol ultracentrifugation to obtain rAAVIVT13-RDH12. The purified AAV titer was then determined by qPCR and stored at -80°C.

[0092] The specific measurement method is as follows:

[0093] The physical titer of the prepared AAV virus genome was determined using qPCR. The specific procedure is as follows:

[0094] Design primers and probes for qPCR detection based on the exact same sequence as 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 ligated with BlackBerry quencher. Primers and probes were synthesized by ThermoFisher Scientific. A 108 bp fragment in the RDH12 opt sequence was specifically amplified using RDH12-QF and RDH12-QR primers. The TaqMan probe binding method was employed, with a 1.0 × 10⁻⁶ primer. 8 The pssAAV-CB7-RDH12 opt-hGH pA plasmid (copies / mL) and its 10-fold serial dilutions were used as standards. The physical titer of the viral genome was detected using Premix Ex Taq (Probe qPCR) reagent (TaKaRa, Beijing, China) and a real-time PCR instrument (model: Q5, ThermoFisher). The procedure was described in the instructions for the reagents used. Virus treatment methods were described in the literature (Aurnhammer C, et al. Hum Gene Ther Methods. 2012; 23(1):18-28.).

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

[0100] Example 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 cassette, HEK293T cells were transfected in vitro with the pssAAV-CB7-RDH12 opt-hGH pA plasmid constructed in Example 2 using a Lipo3000 (ThermoFisher). Cellular proteins were extracted 48 h after transfection. Intracellular RDH12 protein was detected by Western blotting. Protein was extracted using RIPA+PMSF and its concentration was quantified using a BCA kit. Gel preparation: 12% separating gel + 5% stacking gel. Protein samples were centrifuged at 98°C for 10 min, centrifuged at 3000 rpm for 2 min, and electrophoresed. Transfer was performed using a constant current of 300 mA for 1 h, followed by blocking with 5% skim milk for 1 h. The membrane was incubated with RDH12 primary antibody overnight at 4°C. The primary antibody was recovered the next day, and the membrane was washed three times with TBST for 8 min each time. Secondary antibody was then incubated on a shaker at room temperature for 1 h, and the membrane was recovered. The membrane was washed three times with TBST for 8 min each time. Using Bio-Rad ChemiDoc TMMP imaging was used to image the membrane under chemiluminescent blotting, and the results showed that it can be expressed in cells. Figure 3 ).

[0103] Example 5:

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

[0105] The rat strain used in this study was the SD rat, and exons 2-3 of the Rdh12 gene were knocked out. Figure 4 A) Using CRISPR / Cas9 technology, sgRNA was designed. RDH12 gene knockout rats were obtained through high-throughput electroporation of fertilized eggs. After sexual maturity, sperm was collected and cryopreserved to verify the reproductive transmission capacity of F0 rats, which were then used for further breeding. All rats used in the experiment were purchased from Cyagen Biosciences Co., Ltd. The rats were housed at the Animal Center of Peking Union Medical College Hospital, with the room temperature controlled at 20-25℃, and free access to water and food. The housing was equipped with timed fluorescent lighting, and the rats' food and bedding were changed weekly. Adult rats were mated together as needed for the experiment. All animal husbandry, experimental procedures, and euthanasia procedures were approved by the Animal Ethics Committee of the Animal Experiment Center of Peking Union Medical College Hospital. Genotyping was performed on rats aged 3-4 weeks (w). A 3mm sample of rat tail was taken, and DNA was extracted using a rapid alkaline lysis and heating method. The specific method was as follows: 180μL of 50mM NaOH solution was added to an EP tube containing the rat tail, vortexed, and incubated at 98℃ for 10 min. 20μL of 1M Tris-HCl (pH=8) was added to a centrifuge tube, vortexed thoroughly, and centrifuged at 12000rpm for 5 min. The supernatant was used as the DNA template for PCR. PCR was performed, and a 2% agarose gel was prepared. 5μL of the PCR product was loaded into each well, and electrophoresis was performed at 120V for 25 min. The amplification products from wild-type rats were 1607 and 672 bp in size. The amplification products from heterozygous rats included 1607, 672, and 459 bp, while the amplification product from homozygous rats was only 459 bp in size. Figure 4B). To detect the Rdh12 gene knockout effect, Rdh12 mRNA levels were detected by qPCR. Rat retinas were collected, 1 mL of Trizol (Sigma) was added and mixed, and the mixture was thoroughly lysed on ice. 200 μL of chloroform was added and mixed, and the mixture was centrifuged at 12,000 rpm for 15 min at 4 °C. 500 μL of the supernatant was collected and 500 μL of isopropanol (equal volume) was added, mixed, and allowed to stand for 10 min. The mixture was centrifuged at 12,000 rpm for 10 min at 4 °C, the supernatant was discarded, and the RNA settled at the bottom of the tube. 1 mL of 75% ethanol was added for washing, and the RNA was gently mixed to suspend it. The mixture was centrifuged at 7,500 rpm for 5 min at 4 °C. The supernatant was discarded (and the sample was carefully aspirated), and the sample was air-dried in a fume hood for 10–20 min. 20–50 μL of sterile DEPC water was added to dissolve the RNA sample. The RNA concentration was quantified by measuring the OD value. Then reverse transcription (Takara) was performed, followed by qPCR (BioRad) using cDNA. GAPDH was used as an internal control gene. Each sample was repeated in triplicate to reduce error. Detection was performed on an ABI 7500 quantitative instrument using 2... -ΔΔCT The results were analyzed using the method described above. The results showed that, compared with the normal control group, the Rdh12 mRNA content in gene knockout rats was significantly reduced ( Figure 4 C).

[0106] Table 1. Primer sequences for PCR and qPCR

[0107]

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

[0109]

[0110] Table 3 PCR Procedure

[0111]

[0112] Example 6:

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

[0114] To understand the effects of Rdh12 knockout on retinal function and structure in rats, we performed electroretinography (ERG) and optical coherence tomography (OCT) on both RDH12 knockout and wild-type rats. ERG and OCT were performed on both groups of rats at 8, 12, and 16 weeks of age.

[0115] ERG was performed using the Celeris system (Diagnosys LLC). Overnight dark-acclimated rats underwent dark-vision ERG recording. Ten minutes prior to recording, pupils were dilated with tropicamide (1%). Rats were anesthetized using a gas anesthesia machine. Throughout the ERG procedure, animals were placed on a heating plate to maintain body temperature at 37°C. Two electrodes were placed along the axial direction of the eye to measure resistance; the resistance was adjusted to below 5 Ω before starting the measurement. The dark-acclimated ERG protocol consisted of five steps, with stimulation intensities sequentially increasing from 0.003, 0.01, 0.1, 1 to 10 cd.s / m. 2 Light intensity stimulation was applied to the rat retina to induce firing. All flashes were presented in the absence of background illumination. The constant stimulus interval for dim flashes was 5 seconds, while the constant stimulus interval for bright flashes was as long as 30 seconds to maintain dark adaptation. After 5 minutes of light adaptation, light intensities of 1, 3.16, 10, and 31.6 cd.s / m were applied. 2 Intense light was used to stimulate the rat's retina, causing it to discharge. After the test, Bausch & Lomb ophthalmic gel was applied to the rat's eyes.

[0116] Fundus examination was performed using the Micron IV Retinal Imaging Microscope (Phoenix Research Labs). Before the examination, one drop of compound tropicamide eye drops was instilled into each eye of the rat to dilate the pupils. The rats were then anesthetized with gas and placed on a resting bed. The eyes were positioned close to the lens, always centered within it. The distance was adjusted until the fundus image was clear. The brightness and focus were then adjusted to further refine the image. The OCT image was then adjusted to center the optic disc, and further adjustments were made to contrast, brightness, and compensation to enhance clarity. Once the image was clear, it was photographed using image overlay mode. After the examination, Bausch & Lomb gel was instilled into the rats, and the lens was wiped with a cotton swab dipped in distilled water.

[0117] The results are as follows Figure 5 As shown. After sufficient dark adaptation, analysis of the results of different light intensities showed no statistically significant differences in the amplitude and peak duration of the ERG a and b waves between the two groups. Figure 5 AD), with no obvious changes in the fundus (AD). Figure 5 E), there was no statistically significant difference in the thickness of the outer nuclear layer of the retina. Figure 5 F).

[0118] RDH12 is a retinol dehydrogenase that promotes the conversion of all-trans retinaldehyde to all-trans retinol in the visual cycle. To investigate changes in the enzymatic function of RDH12, we extracted rat retina for in vitro enzymatic function testing. 200 μL of a solution composed of sucrose (0.25 M), triaminomethane acetate (25 mM, pH 7), and dithiothreitol (1 mM) was added to the retinal homogenate from one rat (both eyes). The retinal homogenate was sonicated on ice (30 times / second, 10 s sonication followed by 20 s rest, repeated 3 times). After homogenization, the homogenate was centrifuged at 1000 × g for 5 min at 4 °C. Unbroken cells (precipitate) were removed, and the supernatant was collected. Protein concentration was measured using a BCA kit, and OD values ​​were detected using a microplate reader. A standard curve was plotted based on standard samples, and the formula was derived. The concentration of the sample was calculated using the formula. Retinal homogenates from two eyes were mixed, and 40 μg of protein was added to a solution containing 200 μL of sucrose (0.25 M), triaminomethane acetate (25 mM, pH 7), and dithiothreitol (1 mM). NAPDH and 200 μm all-trans retinaldehyde were added to a Hepes buffer (pH=7). Incubation was performed at 37°C for 0 min and 45 min. Mass spectrometry standard solutions were prepared at concentrations of 1000 ng / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, and 1 ng / mL; the solvent was acetonitrile, and the solution was a mixture of retinaldehyde and retinol. 200 μL of the incubated solution was added 1:1 to 200 μL of acetonitrile, vortexed for 1 min, then 1.2 mL of methyl tert-butyl ether was added, vortexed for 1 min, an internal standard was added, and the mixture was centrifuged at 14000 g for 10 min. The supernatant was collected and evaporated to dryness; the solution was reconstituted with a 1:3 ratio of water to methanol, and the results were analyzed using a mass spectrometer. It was found that the enzyme activity in knockout rats was significantly lower than that in wild-type rats. Figure 6 ).

[0119] Example 7:

[0120] Gene therapy:

[0121] To investigate the therapeutic effect of rAAVIVT13-RDH12 on Rdh12- / - rats, 12 8-10 week old Rdh12- / - SD rats were randomly divided into two groups of six each. The rats were administered an intravitreal injection; the high-dose group received 8 × 10⁻⁶ doses. 10 vg / eye, low-dose group: 4.5 × 10 9 Vg / eye, 4 weeks after injection, retinas of all rats were harvested; RNA was extracted from three rats and protein from the other three. qPCR was used to detect RDH12 mRNA expression levels. Elevated mRNA levels were observed in both the low-dose and high-dose groups, with a more significant increase in the high-dose group. Figure 7The enzymatic function of RDH12 was detected using mass spectrometry, following the same method as in Example 6. The results showed that the reduction function of RDH12 was significantly improved in both the low-dose and high-dose groups. Figure 8 ).

[0122] Table 4 qPCR primer sequences

[0123]

[0124] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

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

3. The pAAV-RC2_IVT13 plasmid contains a nucleic acid sequence encoding IVT13, such as SEQ ID NO:

4. The viral genome physical titer of the gene therapy drug targeting the RDH12 mutation is 1.0E+13vg / mL.

2. The method for preparing a gene therapy drug targeting RDH12 mutation as described in 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, wherein the nucleic acid sequence of the CDS is SEQ ID NO:1 and the nucleic acid sequence of RDH12opt is SEQ ID NO:

2. S2. The RDH12 opt sequence is seamlessly cloned into the AAV vector plasmid pssAAV-CB7-Fluc-hGH pA carrying the firefly luciferase reporter gene expression cassette, and the Fluc sequence in pssAAV-CB7-Fluc-hGH pA is replaced to obtain pssAAV-CB7-RDH12 opt-hGH pA. The expression cassette of the pssAAV-CB7-RDH12 opt-hGH pA vector plasmid is CB7-RDH12opt-hGH pA, and the nucleic acid sequence of CB7-RDH12 opt-hGH pA is SEQ ID NO:

3. S3. Using IVT13, an amino acid sequence such as SEQ ID NO:4, as a shell, the recombinant pssAAV-CB7-RDH12 opt-hGH pA is packaged using a three-plasmid packaging system to obtain the rAAVIVT13-RDH12 gene therapy drug. Specifically, the operation of recombinating the pssAAV-CB7-RDH12opt-hGH pA using a three-plasmid packaging system in step S3 is as follows: HEK 293T cells were seeded in 100 mm culture dishes and grown to 70%–80% confluence. 5 μg of pssAAV-CB7-RDH12 opt-hGH pA plasmid, 5 μg of the IVT13-type RepCap plasmid pAAV-RC2_IVT13, and 10 μg of phepler plasmid were co-transfected into HEK293T cells with polyethyleneimine. Cells and supernatant were harvested 72 hours post-transfection. Cells were pelleted by low-speed centrifugation, and lysed by adding 0.5% (w / v) sodium deoxycholate. Free DNA molecules were digested by adding 50 U / mL Benzonase nuclease and 2 mM MgCl2, and incubated at 37°C for 2 hours. Simultaneously, 2.5 M PEG8000 solution (1:5 volume) was used. The supernatant was precipitated with NaCl solution on ice. The cell lysate and supernatant were mixed and centrifuged. The supernatant was then purified by ultracentrifugation with iodixanol to finally obtain the rAAVIVT13-RDH12 gene therapy drug. The method for constructing the RepCap plasmid pAAV-RC2_IVT13 with serotype IVT13 is as follows: 1) Construct the RC2_IVB-NotI plasmid with the sequence SEQ ID NO:5; 2) Using the forward primer shown in SEQ ID NO:6 and the reverse primer shown in SEQ ID NO:7, a PCR fragment is obtained by PCR amplification; the PCR fragment and the fragment of the RC2_IVB-NotI plasmid described in 1) which is linearized by Not I restriction enzyme are assembled by Gibson to obtain the AAV capsid plasmid encoding IVT13.

3. The use of the gene therapy drug targeting RDH12 mutation as described in claim 1 in the preparation of a pharmaceutical composition for treating hereditary retinal degeneration caused by RDH12 gene mutation.

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