Recombinant adeno-associated virus vector carrying TYR gene and application of recombinant adeno-associated virus vector in treatment of OCA1 disease

Abstract

The invention discloses a recombinant adeno-associated virus vector carrying a TYR gene and application of the recombinant adeno-associated virus vector in treatment of OCA1 diseases. The recombinant adeno-associated virus of the tissue specific expression TYR protein designed by the invention is prepared by co-transfecting HEK293 cells with REP protein of AAV, CAP protein expression plasmids of selected serotypes, helper plasmids and the vector provided by the invention, and packaging; the recombinant adeno-associated virus comprises an expression cassette of a human TYR gene, through suprachoroidal space injection, effective targeted infection of RPE can be realized, and tissue-specific efficient and continuous expression of TYR protein is realized, so that melanin deficiency caused by diseases is improved, retina electrophysiology a and b wave intensity is improved, and the purpose of treating ocular skin albinism I (OCA1) diseases is achieved. The medicine has the characteristic of simple and convenient administration, and is higher in safety and minimum in induced inflammation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant adeno-associated virus vector carrying a TYR gene and an application thereof in treating (oculocutaneous albinism type I) OCA1 disease. Background Art

[0002] Oculocutaneous albinism (OCA) is an autosomal recessive genetic disease and the most common type of albinism. It can be divided into oculocutaneous albinism type I to type VIII (OCA1 to OCA8) according to different pathogenic genes. OCA1 is caused by mutations in the tyrosinase (TYR) gene and is a more common type of oculocutaneous albinism, with a prevalence of about 1 / 40,000 of the world's population. TYR is located on chromosome 11q14.3, spans 5 exons, and encodes tyrosinase containing 529 amino acids. It has been reported that there are more than 100 mutations in this locus. The severity of the disease phenotype caused depends on the degree of damage to tyrosinase activity. Tyrosinase is the key rate-limiting enzyme in the biosynthesis of melanin. Mutations in the TYR gene lead to the production of non-functional or reduced-function tyrosinase, resulting in no melanin formation or reduced melanin formation. Due to the lack of melanin, OCA1 patients will experience symptoms such as decreased visual acuity, abnormal refractive errors, decreased or absent stereoscopic vision, nystagmus, iris transillumination, normal optic disc morphology and foveal hypoplasia, which can be severe enough to cause blindness. In addition, OCA1 patients will also have fair skin and white or yellowish hair.

[0003] For the treatment of diseases with loss of normal gene function, the most direct method is to use vectors to introduce normal genes into the patient's body to achieve therapeutic effects. In recent years, clinical studies on gene therapy have continued to emerge, and major breakthroughs have been made in the fields of some genetic diseases and malignant tumors, bringing new hope to patients with some diseases that are ineffective, prone to relapse or difficult to treat with traditional therapies. In 2009, Gargiulo et al. constructed an AAV1 viral vector that produces the human TYR gene under the transcriptional control of the cytomegalovirus (CMV) promoter, and delivered it to the eye tissue of 1-month-old Tyrc-2j albino mice by subretinal injection. After removing the eyeball, it was found that pigmentation was visible in some retinal areas, the iris had diffuse pigmentation, and melanin was present in the retinal epithelium and choroid.

[0004] To achieve efficient and specific expression of AAV in the eye, the design of AAV vectors, the method of administration and the selection of AAV serotypes are all very important. For the design of AAV vectors, screening for suitable transgenic expression frames is particularly critical for the development of AAV gene therapy products for specific genetic diseases. Promoters such as CMV / CBA / CAG can drive high levels of gene expression in the eye, but as broad-spectrum promoters, they may drive gene expression in non-target areas. In addition, non-tissue-specific expression will increase the amount of AAV vectors used and increase the risk of immunity. Moreover, studies have found that broad-spectrum promoters (such as CMV promoters, etc.) are toxic to photoreceptors and retinal pigment epithelial cells in the eye (PubMed: 30833387), affecting the treatment effect of the disease and clinical transformation applications. Summary of the invention

[0005] In order to overcome the defects in the design and application of AAV vectors in the prior art, the present invention provides a TYR protein expression frame, a recombinant adeno-associated virus vector and its application.

[0006] The present invention provides a TYR protein expression cassette, which 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.

[0007] Preferably, the TYR protein expression frame can be composed of promoter-intron-target gene sequence-regulatory element-polyA signal, wherein the promoter is selected from the broad-spectrum cytomegalovirus CMV (Cytomegaloviru) promoter (sequence such as SEQ ID NO.3), the retinal pigment epithelium-specific human BEST1 (Bestrophin-1) promoter (sequence such as SEQ ID NO.4), and the human RPE65 (Retinoid isomerohydrolase RPE65) promoter (sequence such 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 SV40PolyA, bGH polyA, hGH polyA or rBG polyA sequence; the promoter, intron, target gene sequence and polyA are connected by a bond or a nucleotide linker sequence. As a preferred embodiment of the present invention, the promoter is selected from RPE65 promoter; the intron sequence is selected from SV40 intron, and the sequence is shown in SEQ ID NO.6; polyA is selected from bGHployA, and the sequence is shown in SEQ ID NO.7; the regulatory element is WPRE, and the sequence is shown in SEQ ID NO.8.

[0008] The present invention also discloses a recombinant adeno-associated virus vector carrying the TYR gene, wherein the AAV REP protein and the selected serotype CAP protein expression plasmid, the auxiliary plasmid and the cis-pathogenic plasmid vector of the present invention are co-transfected into HEK293 cells for packaging to prepare a recombinant adeno-associated virus that tissue-specifically expresses the TYR protein; the AAV8.hRPE65p.hTYRco vector specifically expressed in RPE cells carries the nucleotide sequence of the sequence-optimized human TYR protein; and the vector is injected into the suprachoroidal space to achieve the purpose of treating 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 one of the following serotypes: AAV1, AAV2, AAV7m8, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-LK03, AAVAnc80. Preferably, 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, which contains the TYR protein expression frame or the recombinant adeno-associated virus vector.

[0012] The present invention further discloses the use of the above-mentioned TYR protein expression frame, 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 preparation, which contains the above-mentioned recombinant adeno-associated virus vector and a pharmaceutically acceptable carrier or excipient.

[0014] Furthermore, the present invention provides a suprachoroidal injection preparation for treating oculocutaneous albinism type I, wherein the suprachoroidal injection preparation 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 a recombinant adeno-associated virus vector to a patient by means of 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.

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

[0017] To achieve efficient and specific expression of AAV in the eye, the design of AAV vectors, the mode of administration and the selection of AAV serotypes are all very important. For AAV vectors, designing the best transgenic expression frame is of great significance for the efficiency and cell specificity of AAV transduction of the retina. The inventors of the present application found through research that for the TYR expression frame of the present invention, the promoters designed based on RPE65, BEST1, etc. are all retinal pigment epithelial (RPE) specific promoters, which can achieve specific expression in RPE cells, and its targeting and safety are better. In addition, sequence optimization of the expressed gene can also effectively improve the gene expression level. AAV is divided into a variety of serotypes, and the in vivo targeting and infection efficiency of different serotypes are different. A large number of studies have found that after subretinal administration, AAV2 targets photoreceptor cells and RPE cells, but mainly infects photoreceptor cells, infects RPE cells to a lesser extent, and AAV2 delivery induces adaptive immune cells to infiltrate into the neural retina, inducing more inflammation. And AAV8 can effectively infect RPE, and the induced inflammation is minimal. In addition, different injection methods will also affect the transduction effect of AAV vectors. Subretinal injection is the injection of drugs into the subretinal space between photoreceptor cells and RPE, which requires the retinal neuroepithelial layer to be separated from the RPE to form a subretinal space. Subretinal injection is an effective method for delivering genes directly to retinal pigment epithelial cells or photoreceptor cells, but this method is highly invasive and can easily cause retinal detachment, which may cause gliosis, photoreceptor degeneration, and visual impairment, etc. In addition, the drug diffusion range is limited and can only achieve local transduction. Suprachoroidal injection has recently been demonstrated to be a new 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 suprachoroidal injection of AAV8 gene delivery vectors can enable widespread expression of transgenes throughout the retinal pigment epithelium and photoreceptors in rats, non-human primates, and pigs, and may become a non-invasive treatment for retinal diseases. It avoids the risk of vitrectomy and separation of photoreceptors from the retinal pigment epithelium. A single suprachoroidal injection of an expression vector produces transgene expression levels comparable to those of a subretinal injection of the same vector and dose, and transgene expression can be increased with multiple suprachoroidal vector injections, providing a noninvasive outpatient approach to ocular gene delivery for many patients.The RPE cell-specific AAV8.hRPE65p.hTYRco vector designed in this protocol can safely and efficiently express tyrosinase protein specifically in RPE cells after being injected into the suprachoroidal space, promote the biosynthesis of melanin and pigmentation of RPE, effectively restore the retinal function and vision of disease model rats, and has a wider range of pigmentation. No toxic reactions such as retinal detachment and atrophy occur, showing very good safety.

[0018] The beneficial effects achieved by the present invention are:

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

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

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

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

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

[0024] Figure 5 This is a graph showing the in vitro melanin production and deposition results of the vectors of the TYR protein wild-type gene sequence (hTYRwt) and the sequence-optimized TYR gene sequence (hTYRco).

[0025] Figure 6 It is a schematic diagram of the plasmid vector containing the CMV expression cassette designed by the present invention.

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

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

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

[0029] Fig.10 This is a schematic diagram of taking pictures of the eyeballs and observing the pigmentation conditions 3 months after administration of the three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p).

[0030] Fig.11 The images are of the fundus of mice taken one month after administration of three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p) to observe the production of melanin.

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

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

[0033] Fig.14 This is a schematic diagram of the three-dimensional structure of the box used to conduct a visual cliff experiment to evaluate the vision of experimental mice.

[0034] Fig.15 Three AAV gene therapy candidate vectors (CMV, RPE65p, BEST1p) were designed respectively. The vision of the treated group mice was evaluated through the visual cliff test 3 months after administration. Schematic diagram of the therapeutic effect.

[0035] Fig.16 Three AAV candidate gene therapy vectors (CMV, RPE65p, BEST1p) were designed respectively. After 12 months of administration, the mouse eyeballs were taken and photographed to record the results of melanin deposition.

[0036] Fig.17 Three AAV candidate gene therapy vectors (CMV, RPE65p, BEST1p) were designed respectively, and the fundus of mice was photographed at different time points after drug administration to observe the production of melanin.

[0037] Fig.18Three AAV candidate gene therapy vectors (CMV, RPE65p, BEST1p) were designed respectively. After 12 months of administration, the eyeballs were collected for paraffin embedding and sectioned for Masson-Fontana staining.

[0038] Fig.19 Schematic diagram of the evaluation results showing that under dark-adapted conditions, mice treated with both candidate vectors showed significant enhancement of b-wave and a-wave amplitudes at higher stimuli.

[0039] Fig. 20 Schematic diagram of the evaluation results showing that under light-adapted green light stimulation, mice treated with both candidate vectors showed significant enhancement in b-wave and a-wave amplitudes at higher stimulation.

[0040] Fig.21 Schematic diagram of the evaluation results showing that under light-adapted ultraviolet stimulation, mice treated with both candidate vectors showed significant enhancement in b-wave and a-wave amplitudes at higher stimulation.

[0041] Fig. 22 This is a schematic diagram of the evaluation results of the visual recovery of disease model mice assessed through the visual cliff experiment 12 months after drug administration.

[0042] Fig.23 It is a protein electrophoresis diagram of the expression of functional TYR protein levels by the preferred AAV gene therapy vector injected into the suprachoroidal space and subretina.

[0043] Fig.24 It is the preferred AAV gene therapy vector for suprachoroidal and subretinal injection. After 12 months of administration, the eyeballs are taken and photographed to record the results of melanin deposition.

[0044] Fig.25 It is the preferred AAV gene therapy vector for suprachoroidal and subretinal injection, and the resulting image shows the visual function recovery of disease model rats.

[0045] Fig.26 It is the preferred AAV gene therapy vector for suprachoroidal and subretinal injection. Three months after administration, OCT examination was performed on disease model rats to evaluate the retinal structural status.

[0046] Fig. 27 It is the preferred AAV gene therapy vector for suprachoroidal and subretinal injection. Twelve months after administration, the eyeballs of the disease model rats were removed for paraffin embedding and sectioned for H&E staining. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

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

[0049] The wild-type human TYR gene (hTYRwt, SEQ ID NO.1) and the sequence-optimized TYR gene (hTYRco, SEQ ID NO.2) were synthesized and pAAV.CMV.hTYRwt.bGH ( Figure 1 , SEQ ID NO.9) and pAAV.CMV.hTYRco.bGH ( Figure 2 , SEQ ID NO.10) plasmid expression vector.

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

[0051] The pAAV.CMV.hTYRwt.bGH and pAAV.CMV.hTYRco.bGH in Example 1 were transfected into HEK293 cells cultured in 24-well plates with the same plasmid amount (250 ng) using PEImax transfection reagent. Proteins were extracted 72 hours later, and the expression levels of TYR proteins were compared using Western blot. The results showed that sequence optimization can significantly improve the expression level of proteins ( Figure 3 ), by comparing the grayscale values ​​of the protein bands, the expression level of TYR protein increased by 2.26 times after sequence optimization ( Figure 4 ). Cell pellets were collected 72 hours after transfection and it was found that the cells transfected with the expression vector pAAV.CMV.hTYRco.bGH had more pigmentation than the cells transfected with the expression vector pAAV.CMV.hTYRwt.bGH ( Figure 5 ), indicating that sequence optimization promoted the biosynthesis and pigmentation of melanin.

[0052] Example 3: Design and construction of candidate gene therapy vectors

[0053] In order to further evaluate the expression of hTYRco in vivo, the efficacy and safety of gene therapy. Through sequence analysis, the retinal pigment epithelial cell-specific promoters RPE65 promoter (RPE65p, SEQ ID NO.5) and BEST1 promoter (BEST1p, SEQ ID NO.4) were designed and synthesized, and the viral packaging cis plasmid vectors 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) were constructed by T4 ligation subcloning. Figure 8 , SEQ ID NO.13) a total of 3 vectors.

[0054] Example 4: AAV virus preparation and purification

[0055] Referring to the method for packaging and purifying recombinant AAV virus reported by Martin Lock et al. [Lock M, Alvira M, Vadeberghe LH, Samanta A, Toelen J, Debyser Z, Wilson JM: Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Human gene therapy 2010, 21(10): 12591271.], PEI was used to express the Rep and Cap protein expression plasmids (pAAV2 / 8), the auxiliary plasmid (pAdΔF6) and the 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.13) were co-transfected into HEK293 cells to package and prepare gene therapy vector AAV8.CMV.hTYRco( Fig. 9 a, SEQ ID NO.14), AAV8.hBEST1p.TYRco( Fig. 9 b, SEQ IDNO.15) and AAV8.hRPE65p.hTY-Rco ( Fig. 9c, SEQ ID NO.16), 48 h after transfection, cells and culture supernatant were harvested, AAV virus was purified by iodixanol ultracentrifugation, and virus titer was determined by digital quantitative PCR (ddPCR). The virus titer of AAV8.CMV.hTYRco was 2.81×10 13 GC / mL, AAV8.hBEST1p.TYRco virus titer is 4.85×10 13 GC / mL, the titer of AAV8.hRPE65p.hTYRco virus was 4.52×10 13 GC / mL.

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

[0057] The gene therapy candidate vectors AAV8.CMV.hTYRco, AAV8.hBEST1p.TYRco, and AAV8.hRPE65p.hTYRco prepared in Example 4 were injected into OCA1 disease model mice (B6(Cg)-Tyr c-2J / J , 2 weeks old, n = 10). According to the injection dose of candidate gene therapy vector, they were divided into low-dose treatment group (3×10 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 starting one month after administration ( Fig.11 ), and 3 months after administration, the eyeballs were photographed to observe the pigmentation ( Fig.10 ), and extracted the protein in the eyeballs of mice in the high-dose group, and further analyzed the expression of TYR protein by WB. The results showed that compared with the untreated group, the eye tissues of the treated mice all expressed functional TYR protein, among which the eyeballs of mice injected with the candidate vector AAV8.CMV.hTYRco had the highest expression of TYR protein, followed by mice injected with the candidate vector AAV8.hRPE65p.hTYRco, and the eyeballs of mice injected with the candidate vector AAV8.hBEST1p.TYRco had the lowest expression of TYR protein ( Fig.12 , Fig.13 The eyeballs of mice in the treatment group showed obvious pigmentation, and the three candidate vectors were all in the medium-dose treatment group (1×10 9The melanin content of mice injected with the candidate vector AAV8.hRPE65p.hTYRco was the highest at the same dose, followed by mice injected with the candidate vector AAV8.CMV.hTYRco, and the least pigmented was mice injected with the candidate vector AAV8.hBEST1p.TYRco. The visual acuity of the mice in the treatment group was evaluated by the visual cliff test 3 months after administration ( Fig.14 , Fig.15 ), the results showed that compared with untreated mice, the vision of the treated mice was significantly improved and comparable to that of wild-type mice. The mice injected with the candidate vector AAV8.CMV.hTYRco and the candidate vector AAV8.hRPE65p.hTYRco chose the safe zone at a higher rate than the mice injected with the candidate vector AAV8.hBEST1p.TYRco, showing better vision. In summary, the therapeutic effects of the AAV gene therapy candidate vectors AAV8.CMV.hTYRco and AAV8.hRPE65p.hTYRco on disease model mice are better than those of the candidate vector AAV8.hBEST1p.TYRco.

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

[0059] The long-term efficacy was evaluated using the superior vector of Example 5. Fundus photography was performed monthly to observe the production of melanin starting from one month after injection and continued for 12 months ( Fig.17 ); The results showed that the melanin production in the fundus of the mice was basically stable 2 months after the injection, and there was no significant change in pigmentation as the mice aged. At 12 months, the eyeballs of the mice were taken and photographed through a stereoscope to more directly observe the production of melanin in the retinal pigment epithelium of the mice ( Fig.16 ). The results showed that the retinal pigment epithelium of the mice in the treatment group produced melanin. At the same dose, the melanin produced in the retinal pigment epithelium of mice treated with the candidate vector AAV8.CMV.hTYRco was less than that produced after treatment with the candidate vector AAV8.hRPE65p.hTYRco. It was also found that mice injected with the candidate vector AAV8.CMV.hTYRco not only produced melanin in the retinal pigment epithelium, but also had obvious melanin production on the iris. In addition, the retinal pigment epithelial cells of mice treated with medium doses of the two candidate therapeutic vectors produced more melanin than those in other dose groups. After 12 months of administration, the eyeballs were removed for paraffin embedding and sectioned for Masson-Fontana staining ( Fig.18 ), the same trend was observed.

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

[0061] ERG testing was performed on the mice 12 months after administration. Dark-adapted ERG showed that mice treated with both candidate vectors exhibited significant enhancement of b-wave and a-wave amplitudes at higher stimuli ( Fig.19 ). Light-adapted ERG showed that under green light stimulation, both candidate vector-treated mice showed significant enhancement of b-wave and a-wave amplitudes at higher stimulation ( Fig. 20 ), under ultraviolet stimulation, the b wave of mice treated with the candidate vector AAV8.hRPE65p.hTYRco was significantly enhanced under higher stimulation, but the a wave was not significantly enhanced. The b wave and a wave of mice treated with the candidate vector AAV8.CMV.hTYRco were not significantly enhanced ( Fig.21 The medium-dose treatment group (1×10 9 The stimulation response of mice in the GC / eye group was stronger than that of the other groups. Under the strongest stimulation of dark adaptation, the b wave increased by 76.34% and the a wave increased by 53.15%; under the strongest stimulation of light adaptation green light, the b wave increased by 57.87% and the a wave increased by 11.84%; under the strongest stimulation of light adaptation ultraviolet light, the b wave increased by 49.23% and the a wave increased by 4.2%. After 12 months of administration, the visual cliff test was used to evaluate the visual recovery of the disease model mice ( Fig. 22 ). The results showed that the vision of the treated mice was significantly improved compared with that of the untreated mice. At the same dose, the vision recovery of the mice treated with the candidate vector AAV8.CMV.hTYRco was comparable to that of the mice treated with the candidate vector AAV8.hRPE65p.hTYRco. The medium-dose treatment group (1×10 9 The visual recovery of mice treated with GC / eye was more significant than that of the other dose treatment groups and was comparable to that of wild-type mice.

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

[0063] According to 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 injections. A low-dose treatment group (6×10 9 GC / eye), medium-dose treatment group (2×10 10 GC / eye) and high-dose treatment group (6×10 10GC / eye). After 2 months of administration, the eye proteins of rats were extracted, and the expression of TYR protein in the eyes of rats under different injection methods was evaluated by WB. The results showed that at the same dose, the expression level of TYR protein in the eyes of rats injected with suprachoroidal space was lower than that in the eyes of rats injected with subretinal space, but compared with the untreated eye tissue, TYR protein still increased significantly ( Fig.23 ). After 12 months of administration, the eyeballs were taken and photographed to record the melanin deposition. The results showed that at the same dose, the melanin deposition in the eyes of rats injected with the suprachoroidal space was more dispersed and the pigmentation covered a wider area, while the melanin deposition in the eyes of rats injected with the subretinal space was more concentrated and the pigment was darker ( Fig.24 ).

[0064] Example 9: The visual function of the disease model rats injected with suprachoroidal space was better restored

[0065] After 12 months of drug administration, the rats were tested for ERG dark adaptation. The ERG dark adaptation results showed that compared with the untreated group, the a-wave and b-wave amplitudes of the rats in the suprachoroidal space injection (SCS) and subretinal (SP) low, medium, and high dose treatment groups were significantly increased. Under different stimulation intensities, the a-wave and b-wave amplitudes of the rats injected into the suprachoroidal space were more significantly increased than those of the rats injected into the subretinal space at the same dose. 10 The amplitudes of a and b waves of GC / eye rats increased the most. Under the strongest stimulation of dark adaptation, the b wave increased by 120.1% and the a wave increased by 123.0% compared with the untreated group ( Fig.25 ).

[0066] Example 10: Suprachoroidal space injection has a higher success rate and safety

[0067] Three months after administration, the rats underwent OCT examination. The OCT results showed that the retina of some rats injected with subretinal space had a small-scale atrophy near the injection site, while the retina of rats injected with suprachoroidal space did not have this phenomenon, and the retinal structure was intact and normal ( Fig.26 ), and the results were independent of the injection dose and candidate vector. 12 months after the injection, the rat eyeballs were taken for pathological tissue sectioning and staining, and the same phenomenon was found ( Fig. 27 ), indicating that the success rate and safety of suprachoroidal injection are higher than those of subretinal injection.

Claims

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

2.

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

6.

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

8.

4. A recombinant adeno-associated virus vector comprising the TYR protein expression cassette according to any one of claims 1 to 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 one of the following serotypes: AAV1, AAV2, AAV7m8, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-LK03, and AAVAnc80.

6. The recombinant adeno-associated virus vector according to claim 4, characterized in that: The complete 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 to 3, or the recombinant adeno-associated virus vector according to any one of claims 4 to 6.

8. Use of the TYR protein expression cassette according to any one of claims 1 to 3, or the recombinant adeno-associated virus vector according to any one of claims 4 to 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 according to any one of claims 4 to 6, and a pharmaceutically acceptable carrier or excipient.

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

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