Recombinant adeno-associated virus vector carrying TYR gene, and use thereof in treatment of OCA1 disease

Abstract

Provided are a recombinant adeno-associated virus vector carrying the TYR gene, and the use thereof in the treatment of OCA1 disease. A recombinant adeno-associated virus capable of tissue-specific expression of the TYR protein is prepared by co-transfecting HEK293 cells with the REP protein of AAV, a CAP protein expression plasmid and a helper plasmid of a selected serotype, and the vector and then packaging same. The recombinant adeno-associated virus comprises an expression cassette of the human TYR gene, so that by means of suprachoroidal injection, the recombinant adeno-associated virus can effectively and targetedly infect the RPE and realize efficient and continuous tissue-specific expression of the TYR protein, thereby relieving disease-caused melanin deficiency, increasing the intensities of retinal electrophysiological a and b waves and achieving the purpose of treating oculocutaneous albinism type I (OCA1). In addition, the recombinant adeno-associated virus vector has the characteristics of simple and convenient administration, high safety and minimal induced inflammation.

Description

Recombinant adeno-associated virus vector carrying the TYR gene and its application in the treatment of OCA1 disease Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a recombinant adeno-associated virus vector carrying the TYR gene and its application in the treatment of OCA1 disease (oculocutaneous albinism type I). Background Technology

[0002] Oculocutaneous albinism (OCA) is an autosomal recessive genetic disorder and the most common type of albinism. Based on the causative gene, it can be classified into OCA1 to OCA8 (types I to VIII). OCA1 is caused by mutations in the tyrosinase (TYR) gene and is the most common type of oculocutaneous albinism, with a prevalence of approximately 1 in 40,000 people worldwide. TYR is located on chromosome 11q14.3, spanning five exons, and encodes a tyrosinase containing 529 amino acids. More than 100 mutations have been reported at this locus. The severity of the resulting disease phenotype depends on the degree of impairment of tyrosinase activity. Tyrosinase is a key rate-limiting enzyme in melanin biosynthesis; mutations in the TYR gene lead to the production of non-functional or reduced-functioning tyrosinase, resulting in no melanin formation or reduced melanin production. Due to the lack of melanin, OCA1 patients may experience symptoms such as decreased vision, abnormal refractive errors, decreased or absent stereoscopic vision, nystagmus, iris transillumination, normal optic disc morphology, and foveal hypoplasia. The severity of these symptoms can be enough to cause blindness. In addition, OCA1 patients may also have fair skin and white or pale yellow hair.

[0003] For the treatment of diseases involving the loss of normal gene function, the most direct method is to use a vector to introduce the normal gene into the patient's body to achieve a therapeutic effect. In recent years, clinical research on gene therapy has emerged continuously, and significant breakthroughs have been achieved in some genetic diseases and malignant tumors, bringing new hope to patients with diseases that are ineffective with traditional therapies, prone to relapse, or difficult to treat. In 2009, Gargiulo et al. constructed an AAV1 viral vector that uses cytomegalovirus (CMV) promoter transcription to control the production of the human TYR gene, and delivered it to the eye tissue of 1-month-old Tyrc-2j albino mice via subretinal injection. After enucleation, pigmentation was found in some retinal areas, diffuse pigmentation in the iris, and melanin in the retinal epithelium and choroid.

[0004] To achieve efficient and specific AAV expression in the eye, the design of the AAV vector, the administration route, and the selection of the AAV serotype are all crucial. For AAV vector design, screening for suitable transgenic expression cassettes is particularly critical for developing AAV gene therapy products targeting specific genetic diseases. Promoters such as CMV / CBA / CAG can drive high-level gene expression in the eye, but as broad-spectrum promoters, they may drive gene expression in non-target regions. Furthermore, non-tissue-specific expression increases the dosage of the AAV vector, raising the risk of immunization. Moreover, studies have found that broad-spectrum promoters (such as the CMV promoter) are toxic to photoreceptors and retinal pigment epithelial cells in the eye (PubMed: 30833387), affecting therapeutic efficacy and clinical translation. Summary of the Invention

[0005] To overcome the shortcomings in the design and application of AAV vectors in the prior art, this invention provides a TYR protein expression cassette, a recombinant adeno-associated virus vector, and its application.

[0006] The present invention provides a TYR protein expression cassette, which includes a human RPE65 promoter with a nucleotide sequence as shown in SEQ ID NO.5 and a TYR gene with a nucleotide sequence as shown in SEQ ID NO.2.

[0007] Preferably, the TYR protein expression cassette can be composed of a promoter, an intron, a target gene sequence, a regulatory element, and a polyA signal, wherein the promoter is selected from the broad-spectrum cytomegalovirus (CMV) promoter (sequence as SEQ ID NO.3), the retinal pigment epithelium-specific human BEST1 (Bestrophin-1) promoter (sequence as SEQ ID NO.4), and the human RPE65 (Retinoid isomerohydrolase RPE65) promoter (sequence as SEQ ID NO.5); the intron is selected from the SV40 intron; the regulatory element is selected from WPRE; the target gene is selected from the human TYR coding gene; the polyA signal sequence is selected from SV40 PolyA, bGH polyA, hGH polyA, or rBG polyA sequences; the promoter, intron, target gene sequence, and polyA are connected by a bond or nucleotide linkage sequence. As a preferred embodiment of the present invention, the promoter is selected from the RPE65 promoter; the intron sequence is selected from the SV40 intron, as shown in SEQ ID NO.6; polyA is selected from bGH polyA, as shown in SEQ ID NO.7; and the regulating element is WPRE, as shown in SEQ ID NO.8.

[0008] This invention also discloses a recombinant adeno-associated virus vector carrying the TYR gene. A recombinant adeno-associated virus expressing the TYR protein in tissue-specific form is prepared by co-transfecting HEK293 cells with the AAV REP protein, the selected serotype's CAP protein expression plasmid, helper plasmid, and the cis-terminated plasmid vector described in this invention. The AAV8.hRPE65p.hTYRco vector, specifically expressed in RPE cells, carries the sequence-optimized nucleotide sequence of the human TYR protein. This is then administered via suprachoroidal injection to treat oculocutaneous albinism type I.

[0009] The vector virus of the recombinant adeno-associated virus vector of the present invention is an adeno-associated virus selected from any of the following serotypes: AAV1, AAV2, AAV7m8, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-LK03, AAVAnc80. Preferably, it is AAV2, AAV5, AAV8, or AAV9.

[0010] Preferably, the full sequence of the above-mentioned recombinant adeno-associated virus vector is shown in SEQ ID NO.16.

[0011] The present invention also discloses a host cell containing the above-mentioned TYR protein expression cassette or the above-mentioned recombinant adeno-associated virus vector.

[0012] The present invention further discloses the use of the above-mentioned TYR protein expression cassette, the above-mentioned recombinant adeno-associated virus vector, or the above-mentioned host cell in the preparation of a drug for treating oculocutaneous albinism type I.

[0013] Furthermore, the present invention provides a pharmaceutical formulation comprising the above-described recombinant adeno-associated virus vector, and a pharmaceutically acceptable carrier or excipient.

[0014] Furthermore, the present invention provides a suprachoroidal injection formulation for treating oculocutaneous albinism type I, wherein the suprachoroidal injection formulation comprises the above-mentioned recombinant adeno-associated virus vector.

[0015] The present invention also discloses a method for treating oculocutaneous albinism type I, comprising administering a sufficient dose of recombinant adeno-associated virus vector to the patient via suprachoroidal injection; wherein the recombinant adeno-associated virus vector comprises a human RPE65 promoter with a nucleotide sequence as shown in SEQ ID NO.5 and a TYR gene with a nucleotide sequence as shown in SEQ ID NO.2.

[0016] In the above method, the injection dose of the recombinant adeno-associated virus vector is 1×10⁻⁶. 10 GC / eye up to 1×10 13 GC / eye, one injection only.

[0017] To achieve efficient and specific AAV expression in the eye, the design of the AAV vector, the administration method, and the selection of the AAV serotype are all crucial. For AAV vectors, designing the optimal transgenic expression cassette is of great significance for the efficiency and cell specificity of AAV transduction in the retina. The inventors of this application, through research, discovered that the TYR expression cassette designed for this invention, based on promoters such as RPE65 and BEST1, are retinal pigment epithelial (RPE) specific promoters, enabling specific expression within RPE cells with better targeting and safety. In addition, sequence optimization of the expressed gene can also effectively improve gene expression levels. AAV exists in various serotypes, each with different in vivo targeting and infection efficiencies. Numerous studies have found that after subretinal administration, AAV2 targets photoreceptor cells and RPE cells, but primarily infects photoreceptor cells, with a smaller degree of infection in RPE cells. Furthermore, AAV2 delivery induces adaptive immune cell infiltration into the neuroretina, inducing significant inflammation. In contrast, AAV8 can effectively infect RPE with minimal induced inflammation. Moreover, different injection methods also affect the transduction efficacy of the AAV vector. Subretinal injection involves injecting drugs into the subretinal space between photoreceptor cells and the retinal pigment epithelium (RPE). This requires separating the retinal neuroepithelium from the RPE to create the subretinal space. Subretinal injection is an effective method for directly delivering genes to retinal pigment epithelial cells or photoreceptor cells. However, this method is highly invasive, prone to retinal detachment, and may cause glial cell proliferation, photoreceptor degeneration, and visual impairment. Furthermore, drug diffusion is limited, achieving only local transduction. Suprachoroidal injection has recently been demonstrated as a novel route for ocular drug delivery. The suprachoroidal space is a potential space along the inner surface of the sclera. Fluorescently labeled particles injected near the limbus flow circumferentially around the eye, allowing for large-area exposure. Studies have shown that choroidal injection of the AAV8 gene delivery vector enables widespread expression of the transgene throughout the retinal pigment epithelium and photoreceptors in rats, non-human primates, and pigs, potentially serving as a non-invasive treatment for retinal diseases. It avoids the risks associated with vitrectomy and the separation of photoreceptor cells from the retinal pigment epithelium. A single choroidal injection of the expression vector yields transgene expression levels comparable to subretinal injections with the same vector and dosage, and multiple choroidal injections can increase transgene expression. For many patients, this offers a non-invasive ocular gene delivery treatment that can be performed on an outpatient basis.The AAV8.hRPE65p.hTYRco vector designed in this protocol, which specifically expresses RPE cells, can safely and efficiently express tyrosinase protein in RPE cells after intrachoroidal injection, promoting melanin biosynthesis and pigmentation in RPE, effectively restoring retinal function and vision in disease model rats, with a wider range of pigmentation, and without toxic reactions such as retinal detachment or atrophy, demonstrating very good safety.

[0018] The beneficial effects achieved by this invention are as follows:

[0019] The recombinant adeno-associated virus vector carrying the TYR gene provided by this invention can specifically and safely and efficiently express tyrosinase protein in RPE cells through non-invasive injection, promote melanin biosynthesis and pigmentation in RPE, effectively restore retinal function and vision in disease model rats, and achieve a wider range of pigmentation without toxic reactions such as retinal detachment or atrophy, and with minimal induced inflammatory response, showing very good targeting and safety, and has potential clinical application prospects. Attached Figure Description

[0020] Figure 1 is a schematic diagram of the plasmid expression vector constructed from the wild-type TYR gene sequence of the TYR protein.

[0021] Figure 2 is a schematic diagram of the plasmid expression vector constructed from the sequence-optimized TYR gene sequence.

[0022] Figure 3 is a protein electrophoresis diagram showing the in vitro expression levels of the wild-type TYR protein gene sequence (hTYRwt) and the sequence-optimized TYR gene sequence (hTYRco) in a vector.

[0023] Figure 4 is a schematic diagram of the in vitro protein expression levels of the vectors containing the wild-type TYR protein gene sequence (hTYRwt) and the sequence-optimized TYR gene sequence (hTYRco).

[0024] Figure 5 shows the results of in vitro melanin production and deposition using vectors containing the wild-type TYR protein gene sequence (hTYRwt) and the sequence-optimized TYR gene sequence (hTYRco).

[0025] Figure 6 is a schematic diagram of the plasmid vector containing the CMV expression frame designed in this invention.

[0026] Figure 7 is a schematic diagram of the plasmid vector containing the BEST1p expression cassette designed in this invention.

[0027] Figure 8 is a schematic diagram of the plasmid vector containing the RPE65p expression cassette designed in this invention.

[0028] Figure 9 is a schematic diagram of AAV gene therapy candidate vectors with three expression cassettes (CMV, RPE65p, BEST1p) designed respectively.

[0029] Figure 10 is a schematic diagram showing the pigmentation observation of the eyeballs 3 months after administration of three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p).

[0030] Figure 11 shows the results of melanin production observed in mouse fundus images one month after administration of three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p).

[0031] Figure 12 is a protein electrophoresis diagram showing the expression levels of functional TYR protein in three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p).

[0032] Figure 13 is a schematic diagram of the functional TYR protein expression levels of three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p).

[0033] Figure 14 is a schematic diagram of the three-dimensional structure of the box used to assess the vision of experimental mice in the visual cliff test.

[0034] Figure 15 shows a schematic diagram of the treatment effect of three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p) designed respectively, and the visual acuity of the mice in the treatment group was evaluated by the visual cliff test 3 months after administration.

[0035] Figure 16 shows the results of photographing mouse eyeballs to record melanin deposition 12 months after the design of three AAV candidate gene therapy vectors (CMV, RPE65p, BEST1p).

[0036] Figure 17 shows the results of melanin production in mice at different time points after administration of three AAV candidate gene therapy vectors (CMV, RPE65p, BEST1p).

[0037] Figure 18 shows the results of eyeballs being harvested 12 months after administration of three AAV candidate gene therapy vectors (CMV, RPE65p, BEST1p) for paraffin embedding and sectioning for Masson-Fontana staining.

[0038] Figure 19 is a schematic diagram of the evaluation results of mice treated with the two candidate vectors under dark adaptation conditions, showing significant enhancement of the amplitude of the b wave and a wave under higher stimulation.

[0039] Figure 20 is a schematic diagram illustrating the evaluation of the significant increase in the amplitude of the b-wave and a-wave in mice treated with two candidate carriers under high stimulation under light-adapted green light.

[0040] Figure 21 is a schematic diagram illustrating the evaluation of the significant increase in the amplitude of the b-wave and a-wave in mice treated with both candidate carriers under high-level UV stimulation.

[0041] Figure 22 is a schematic diagram of the results of the visual cliff test to assess the visual recovery of the disease model mice 12 months after drug administration.

[0042] Figure 23 is a protein electrophoresis diagram showing the expression level of functional TYR protein by the preferred AAV gene therapy vector injected into the suprachoroidal space and subretinal space.

[0043] Figure 24 shows the results of melanin deposition after 12 months of intrachoroidal and subretinal injection of the preferred AAV gene therapy vector.

[0044] Figure 25 shows the results of visual function recovery in rat disease models after injection of the preferred AAV gene therapy vector into the suprachoroidal space and subretinal space.

[0045] Figure 26 shows the results of OCT assessment of retinal structural status in disease model rats three months after injection of the preferred AAV gene therapy vector into the suprachoroidal space and subretinal space.

[0046] Figure 27 shows the results of H&E staining of eyeballs from disease model rats 12 months after administration of the preferred AAV gene therapy vector injected into the suprachoroidal space and subretinal space. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0048] Example 1: Optimization of TYR protein target gene sequence and construction of expression vector

[0049] The whole-genome wild-type human TYR gene (hTYRwt, SEQ ID NO.1) and the sequence-optimized TYR gene (hTYRco, SEQ ID NO.2) were synthesized and then expressed as plasmids pAAV.CMV.hTYRwt.bGH (Figure 1, SEQ ID NO.9) and pAAV.CMV.hTYRco.bGH (Figure 2, SEQ ID NO.10) respectively by enzyme digestion and ligation.

[0050] Example 2: Validation of TYR protein expression level in vitro

[0051] HEK293 cells cultured in 24-well plates were transfected with the same plasmid amount (250 ng) of pAAV.CMV.hTYRwt.bGH and pAAV.CMV.hTYRco.bGH from Example 1 using PEImax transfection reagent. Protein was extracted after 72 h, and the expression level of TYR protein was compared using Western blotting. The results showed that sequence optimization significantly improved protein expression (Figure 3). By comparing the gray values ​​of the protein bands, the expression level of TYR protein increased by 2.26 times after sequence optimization (Figure 4). Cell pellets collected 72 h after transfection showed that cells transfected with expression vector pAAV.CMV.hTYRco.bGH exhibited more pigmentation than those transfected with expression vector pAAV.CMV.hTYRwt.bGH (Figure 5), indicating that sequence optimization promoted melanin biosynthesis and pigmentation.

[0052] Example 3: Design and Construction of Gene Therapy Candidate Vectors

[0053] To further evaluate the expression of hTYRco in vivo, the efficacy of gene therapy, and its safety, the retinal pigment epithelial cell-specific promoters RPE65 (RPE65p, SEQ ID NO.5) and BEST1 (BEST1p, SEQ ID NO.4) were designed and synthesized through sequence analysis. Three viral packaging cis-plasmid vectors were constructed via T4 ligation subcloning: pAAV.CMV.hTYRco.WPRE.bGH (Figure 6, SEQ ID NO.11), pAAV.hBEST1p.hTYRco.WPRE.bGH (Figure 7, SEQ ID NO.12), and pAAV.hRPE65p.hTYRco.WPRE.bGH (Figure 8, SEQ ID NO.13).

[0054] Example 4: Preparation and purification of AAV virus

[0055] Referring to the method for packaging and purifying recombinant AAV viruses reported by Martin Lock et al. [Lock M, Alvira M, Vadeberghe LH, Samanta A, Toelen J, Debyser Z, Wilson JM: Rapid, simple, and versatile manfacturing of recombinant adeno-associated viral vectors at scale. Human gene therapy 2010, 21(10):12591271.], PEI was used to package and purify AAV Rep and Cap protein expression plasmids (pAAV2 / 8), helper plasmids (pAdΔF6), and AAV packaging cis plasmids (pAAV.CMV.hTYRco.WPRE.bGH (SEQ ID NO.11), pAAV.hBE-ST1p.hTYRco.WPRE.bGH (SEQ ID NO.12) and pAAV.hRPE65p.hTYRco.WPRE.bGH (SEQ ID NO.12)). NO.13) Gene therapy vectors AAV8.CMV.hTYRco (Figure 9a, SEQ ID NO.14), AAV8.hBEST1p.TYRco (Figure 9b, SEQ ID NO.15), and AAV8.hRPE65p.hTY-Rco (Figure 9c, SEQ ID NO.16) were prepared by co-transfecting HEK293 cells. After 48 h of transfection, cells and culture supernatant were harvested. AAV virus was purified by ultracentrifugation with iodixanol, and viral titer was determined by digital quantitative PCR (ddPCR). The viral titer of AAV8.CMV.hTYRco was 2.81 × 10⁻⁶. 13 GC / mL, the viral titer of AAV8.hBEST1p.TYRco was 4.85 × 10⁻⁶. 13 GC / mL, the viral titer of AAV8.hRPE65p.hTYRco was 4.52 × 10⁻⁶. 13 GC / mL.

[0056] Example 5: In vivo screening and validation of AAV8 gene therapy vector

[0057] Using the gene therapy candidate vectors AAV8.CMV.hTYRco, AAV8.hBEST1p.TYRco, and AAV8.hRPE65p.hTYRco prepared in Example 4, OCA1 disease model mice were injected subretinally with B6(Cg)-Tyr. c-2J / J (2 weeks old, n=10). Patients were divided into a low-dose treatment group (3×10⁻⁶) based on the injection dose of the gene therapy candidate vector.8 GC / eye), medium-dose treatment group (1×10 9 GC / eye) and high-dose treatment group (3×10) 9 GC / eye). Fundus photography was performed one month after drug administration (Figure 11), and eyeballs were photographed three months after drug administration to observe pigmentation (Figure 10). Proteins were extracted from the eyes of mice in the high-dose group, and the expression of TYR protein was further analyzed by Western blotting. The results showed that, compared with the untreated group, functional TYR protein was expressed in the ocular tissues of mice in the treatment group. The mice injected with candidate vector AAV8.CMV.hTYRco had the highest TYR protein expression in their eyes, followed by mice injected with candidate vector AAV8.hRPE65p.hTYRco, and the mice injected with candidate vector AAV8.hBEST1p.TYRco had the lowest TYR protein expression in their eyes (Figures 12 and 13). The eyes of mice in the treatment group all showed obvious pigmentation, and the three candidate vectors were all in the medium-dose treatment group (1×10⁻⁶). 9 The mice injected with GC (in the eye) showed the highest melanin content. At the same dose, mice injected with candidate vector AAV8.hRPE65p.hTYRco showed the most melanin deposition, followed by mice injected with candidate vector AAV8.CMV.hTYRco, while mice injected with candidate vector AAV8.hBEST1p.TYRco showed the least pigmentation. Three months after administration, the visual acuity of the treated mice was assessed using the visual cliff test (Figures 14 and 15). The results showed that the visual acuity of the treated mice was significantly improved compared to untreated mice and comparable to that of wild-type mice. Mice injected with candidate vectors AAV8.CMV.hTYRco and AAV8.hRPE65p.hTYRco showed a higher proportion of choosing the safe zone than mice injected with candidate vector AAV8.hBEST1p.TYRco, indicating better visual acuity. In summary, the AAV gene therapy candidate vectors AAV8.CMV.hTYRco and AAV8.hRPE65p.hTYRco showed better therapeutic effects on disease model mice than the candidate vector AAV8.hBEST1p.TYRco.

[0058] Example 6: Evaluation of the long-term efficacy of the preferred AAV gene therapy vector

[0059] The long-term efficacy was evaluated using the superior carrier from Example 5. Fundus photography was performed monthly starting one month after injection to observe melanin production for 12 months (Figure 17). Results showed that melanin production in the mouse fundus stabilized approximately two months after injection, and pigmentation did not significantly change with age. At 12 months, eyeballs were photographed using a stereoscope to more directly observe melanin production in the retinal pigment epithelium (Figure 16). Results showed that melanin production was observed in the retinal pigment epithelium of all mice in the treatment group. At the same dose, mice treated with candidate carrier AAV8.CMV.hTYRco produced less melanin in the retinal pigment epithelium than those treated with candidate carrier AAV8.hRPE65p.hTYRco. Furthermore, it was found that mice injected with candidate carrier AAV8.CMV.hTYRco not only produced melanin in the retinal pigment epithelium but also showed significant melanin production in the iris. Furthermore, the medium-dose treatment groups of mice using both candidate therapeutic vectors produced more melanin in their retinal pigment epithelial cells than the other dose groups. Twelve months after administration, eyeballs were harvested for paraffin embedding and sectioning for Masson-Fontana staining (Figure 18), and the same trend was observed.

[0060] Example 7: Preferred AAV gene therapy candidate vector restores visual function and vision in disease model mice

[0061] ERG analysis was performed on mice 12 months after drug administration. Dark-adapted ERG showed that mice treated with both candidate vectors exhibited significant increases in the amplitude of b-waves and a-waves under higher stimulation (Figure 19). Light-adapted ERG showed that under green light stimulation, mice treated with both candidate vectors exhibited significant increases in the amplitude of b-waves and a-waves under higher stimulation (Figure 20). Under ultraviolet stimulation, mice treated with candidate vector AAV8.hRPE65p.hTYRco showed significant increases in b-waves but no significant increases in a-waves under higher stimulation, while mice treated with candidate vector AAV8.CMV.hTYRco showed no significant increases in either b-waves or a-waves (Figure 21). The medium-dose treatment group of candidate vector AAV8.hRPE65p.hTYRco (1×10⁻⁶) showed the greatest increase in b-waves. 9The GC / eye mice showed a stronger stimulus response compared to other groups. Under the strongest dark-adapted stimulus, the b-wave increased by 76.34% and the a-wave by 53.15%; under the strongest light-adapted green light stimulus, the b-wave increased by 57.87% and the a-wave by 11.84%; and under the strongest light-adapted ultraviolet light stimulus, the b-wave increased by 49.23% and the a-wave by 4.2%. Twelve months after administration, the visual recovery of the disease model mice was assessed using the visual cliff test (Figure 22). The results showed that the visual acuity of the treated mice was significantly improved compared to the untreated mice. At the same dose, the visual recovery of mice treated with the candidate vector AAV8.CMV.hTYRco was comparable to that of mice treated with the candidate vector AAV8.hRPE65p.hTYRco. The medium-dose treatment groups of the two candidate vectors (1×10⁻⁶) showed... 9 The visual recovery in GC / eye mice was significantly greater than in other treatment groups and comparable to that in wild-type mice.

[0062] Example 8: Evaluation of the efficacy of AAV gene therapy in two injection methods in a disease model rat.

[0063] Based on Examples 5-7, the optimal AAV gene therapy vector was determined to be AAV8.hRPE65p.hTYRco. This virus was delivered to the retina of OCA1 disease model rats via suprachoroidal and subretinal injection. A total of 6 × 10⁶ low-dose treatment groups were established. 9 GC / eye), medium-dose treatment group (2×10 10 GC / eye) and high-dose treatment group (6×10) 10 Three dosage groups (GC / eye) were used. Two months after administration, ocular proteins were extracted from rats, and the expression of TYR protein in the eyes of rats under different injection methods was assessed by Western blotting. The results showed that, at the same dose, the expression level of TYR protein in the eyes of rats injected through the suprachoroidal cavity was lower than that in rats injected through the subretinal cavity, but TYR protein was still significantly increased compared with untreated eye tissue (Figure 23). Twelve months after administration, eyeballs were photographed to record the melanin deposition. The results showed that, at the same dose, the melanin deposition in the eyes of rats injected through the suprachoroidal cavity was more dispersed and covered a wider area, while the melanin deposition in rats injected through the subretinal cavity was more concentrated and the pigment was darker (Figure 24).

[0064] Example 9: Rats with a disease model treated with suprachoroidal injection showed better recovery of visual function.

[0065] Twelve months after drug administration, rats underwent ERG dark adaptation testing. ERG dark adaptation results showed that, compared to the untreated group, rats in the low, medium, and high-dose treatment groups (supracrustal injection (SCS) and subretinal injection (SP)) showed significant increases in both a-wave and b-wave amplitudes. Under different stimulation intensities, the a-wave and b-wave amplitudes in rats treated with the supracrustal injection were significantly higher than those in rats treated with the same dose of subretinal injection. The high-dose supracrustal injection group (6×10⁻⁶) showed the most significant increase. 10 In GC / eye rats, the amplitudes of waves a and b were the most significantly increased. Under the strongest dark-adapted stimulus, wave b was 120.1% stronger than the untreated group, and wave a was 123.0% stronger than the untreated group (Figure 25).

[0066] Example 10: Higher success rate and safety of suprachoroidal injection

[0067] Three months after administration, rats underwent OCT examination. OCT results showed that some rats injected subretinally experienced small-scale retinal atrophy near the injection site, while rats injected suprachoroidally did not exhibit this phenomenon, and their retinal structures remained intact and normal (Figure 26). Furthermore, the results were independent of the injection dose and candidate carrier. Twelve months after injection, histopathological sections and staining of rat eyeballs revealed the same phenomenon (Figure 27), indicating that suprachoroidal injection has a higher success rate and safety profile than subretinal injection.

Claims

1. A TYR protein expression cassette, characterized in that, The TYR protein expression cassette includes the human RPE65 promoter with the nucleotide sequence shown in SEQ ID NO.5 and the TYR gene with the nucleotide sequence shown in SEQ ID NO.

2.

2. The TYR protein expression cassette according to claim 1, characterized in that, It also contains intron SV40, the nucleotide sequence of which is shown in SEQ ID NO.

6.

3. The TYR protein expression cassette according to claim 1, characterized in that, It also includes a regulatory element WPRE, the nucleotide sequence of which is shown in SEQ ID NO.

8.

4. A recombinant adeno-associated virus vector comprising the TYR protein expression cassette of any one of claims 1-3.

5. The recombinant adeno-associated virus vector according to claim 4, characterized in that, The vector virus of the recombinant adeno-associated virus vector is an adeno-associated virus selected from any of the following serotypes: AAV1, AAV2, AAV7m8, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-LK03, AAVAnc80.

6. The recombinant adeno-associated virus vector according to claim 4, characterized in that, The full sequence of the recombinant adeno-associated virus vector is shown in SEQ ID NO.

16.

7. A host cell, characterized in that, The host cell contains the TYR protein expression cassette according to any one of claims 1-3, or the recombinant adeno-associated virus vector according to any one of claims 4-6.

8. The use of the TYR protein expression cassette according to any one of claims 1-3, or the recombinant adeno-associated virus vector according to any one of claims 4-6, or the host cell according to claim 7 in the preparation of a medicament for treating oculocutaneous albinism type I.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains the recombinant adeno-associated virus vector as described in any one of claims 4-6, and a pharmaceutically acceptable vector or excipient.

10. A suprachoroidal intravascular injection preparation for treating oculocutaneous albinism type I, wherein, The suprachoroidal injection formulation comprises the recombinant adeno-associated virus vector according to any one of claims 4-6.

11. A method for treating oculocutaneous albinism type I, characterized in that, The invention comprises a recombinant adeno-associated virus vector administered to a patient in adequate dose via suprachoroidal injection; the recombinant adeno-associated virus vector comprises a human RPE65 promoter with a nucleotide sequence as shown in SEQ ID NO.5 and a TYR gene with a nucleotide sequence as shown in SEQ ID NO.

2.

12. The method for treating oculocutaneous albinism type I according to claim 11, characterized in that, The recombinant adeno-associated virus vector further comprises intron SV40, the nucleotide sequence of which is shown in SEQ ID NO.

6.

13. The method for treating oculocutaneous albinism type I according to claim 11, characterized in that, The recombinant adeno-associated virus vector also includes a regulatory element WPRE, the nucleotide sequence of which is shown in SEQ ID NO.

8.

14. The method for treating oculocutaneous albinism type I according to claim 11, characterized in that, The full sequence of the recombinant adeno-associated virus vector is shown in SEQ ID NO.

16.

15. The method for treating oculocutaneous albinism type I according to claim 11, characterized in that, The injection dose of the recombinant adeno-associated virus vector is 1×10⁻⁶. 10 GC / eye up to 1×10 13 GC / eye, one injection only.

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