Gene therapy for retinitis pigmentosa caused by PRPF31 mutation

Abstract

The present application relates to an adeno-associated virus (AAV) vector, a nucleotide sequence thereof encoding a PRPF31 protein and a promoter operably linked thereto. The present application also relates to the vector, cells comprising the vector, a pharmaceutical composition, a kit, and a use thereof in treating retinitis pigmentosa caused by PRPF31 gene mutation.

Description

Gene therapy for retinitis pigmentosa caused by PRPF31 mutation Technical Field

[0001] This invention relates to the field of biomedicine. More specifically, this invention relates to a gene therapy drug for autosomal dominant retinitis pigmentosa caused by PRPF31 mutations. Background Technology

[0002] Retinitis pigmentosa (RP) encompasses a large class of inherited vision disorders that cause progressive degeneration of the retina (the light-sensitive membrane covering the inside of the eye). RP typically begins with visual disturbances in low light or dim light (i.e., difficulty seeing in dim environments or at dusk, or the ability to adapt or regain function in dim light after a period of exposure to bright light). Affected individuals usually become aware of the loss of peripheral vision gradually. Symptoms are common between the ages of 10 and 40, but RP has early and late-onset forms, with typical symptoms developing gradually over time (References [1-3]).

[0003] Retinitis pigmentosa (RP) is caused by mutations in any of at least 50 genes (https: / / rarediseases.info.nih.gov / diseases / 5694 / retinitis-pigmentosa). The modes of inheritance include autosomal dominant (ad), autosomal recessive (ar), X-linked (xl), and unknown inheritance. Studies on the incidence of non-syndrome RP in different populations indicate an incidence of 1 in 4000, with 30% to 40% of cases reported as autosomal dominant. The causative genes for autosomal dominant retinitis pigmentosa (adRP) include Rhodopsin (RHO), PRRF31, PRPH2, RP1, IMPDH1, and PRPF8. PRRF31 mutations have been reported as a common cause of secondary adRP following RHO, accounting for 2.5% to 6.7% of all cases (References [4,5]).

[0004] As is well known, PRPF31 is a messenger RNA precursor (pre-mRNA) splicing factor and a component of the U4 / U6 / U5 trimer. Although PRPF31 is widely expressed in human tissues, its mutations only cause pathological effects in rod photoreceptors and retinal pigment epithelium (RPE) cells (RP11 type). Most pathogenic mutations in PRPF31 are single-base alterations or small deletions, resulting in premature stop codons and nonsense mRNA decay (NMD) (see Table 1), suggesting that RP11 is caused by haploid insufficiency. Interestingly, about 5%–10% of individuals carrying PRPF31 mutations are asymptomatic (Reference [6]). The incomplete penetrance of RP11 supports that its molecular pathological cause is the loss of function of a single allele and haploid insufficiency. Since PRPF31 mutations lead to retinitis pigmentosa through haploid insufficiency, AAV-based gene replacement therapy is the most desirable treatment strategy.

[0005] Table 1. Pathogenic mutation sites of PRPF31 in RP11 patients

[0006] (Data source: https: / / rddc.tsinghua-gd.org / disease / RTN041)

[0007] The PRPF31 mRNA levels in the retina, nucleated blood cells, and lymphoblasts of normal individuals and non-carriers are similar, approximately 1.21 × 10⁻⁶. 8 ±1.39×10 7 Copy number / µg total RNA. Therefore, the PRPF31 mRNA level in lymphoblasts can represent the expression of PRPF31 in the retina (reference [7]). The mean expression levels of PRPF31 mRNA in symptomatic (S, carrying PRPF31 with deletion of 1115-1125), asymptomatic (AS, carrying PRPF31 with deletion of 1115-1125) and normal / non-carrier (N) lymphoblasts were 2.58 × 10⁻⁶ mRNAs, respectively. 7 ±3.09×10 6 4.26×10 7 ±9.02×10 6 and 1.09×10 8 ±6.71×10 6Copy number / µg total RNA (Reference [7]). In a large cohort of 200 healthy individuals, 3 asymptomatic individuals, and 7 symptomatic individuals, PRPF31 mRNA expression in lymphoblasts showed similar levels. PRPF31 mRNA expression was normalized to the median expression level, which was arbitrarily set to 1.0. The normalized PRPF31 mRNA levels in symptomatic patients were 0.29–0.50, in asymptomatic patients 0.53–0.67, and in healthy individuals 0.53–2.48 (Reference [8]). Therefore, restoring PRPF31 mRNA levels in the retinas of symptomatic patients to normal levels may salvage retinal cell function.

[0008] This hypothesis has been confirmed in several in vitro and in vivo models. Mutant PRPF31-induced pluripotent cell-derived RPE (iPSC-RPE) cells reproduced the cell phenotypes associated with PRPF31 pathology, including structural defects, impaired phagocytosis, defective ciliation, and impaired barrier function. Treatment of PRPF31 mutant iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and ciliation, and partially restored structural and barrier function (Reference [9]). Gene knockout (such as treatment with prpf31morphants or prpf31 knockout) resulted in severe impairment of zebrafish retinal progenitor cells (RPCs) and / or embryonic development, while injection of PRPF31 mRNA rescued these defects (References 10-12).

[0009] Animal models (naturally occurring or genetically engineered) are crucial tools for studying the function of target genes, disease pathogenesis, and the effectiveness of treatments. It is known in the art that vector tools used for gene expression in cells and gene therapy products that actually exert therapeutic effects in animals are entirely different concepts; the latter cannot be readily predicted from the former. Cellular-level drug efficacy cannot be extrapolated to animal-level efficacy because, compared to the relatively simple cellular system, animals are complex living organisms influenced by complex factors such as the cellular microenvironment and immune system—a fact evidenced by numerous examples in the development of oncology drugs and gene therapy drugs. However, currently, there are no effective animal models that can mimic the RP11 disease phenotype. Therefore, the construction and study of suitable animal models are essential for effectively exploring the efficacy and pharmacology of RP11 therapeutics.

[0010] Adeno-associated virus (AAV) is currently the most widely used delivery vector for gene therapy due to its good tissue specificity, low immunogenicity, and high safety. However, gene therapy using AAV vectors still faces challenges. First, not all AAV types are suitable for treating specific diseases. Second, expression cassettes and delivery vectors contain various components. Depending on the design, the results can vary greatly. Given the numerous options available for each important component, well-designed and efficient expression cassettes and delivery vectors are necessary for the success of PRPF31 gene therapy. Finally, the design needs to be tested and validated in appropriate disease models. Summary of the Invention

[0011] Through in-depth research, the inventors have for the first time provided an RP11 mouse model for in vivo efficacy testing of gene replacement therapy for autosomal dominant retinitis pigmentosa caused by PRPF31 mutation, and a method for constructing the model.

[0012] Furthermore, the inventors designed a series of AAV-based gene therapy vectors for PRPF31 gene replacement therapy, and studied the effects of different serotypes, injection methods, and promoter expression on the expression distribution and level of PRPF31 protein. The vectors were validated in cell lines, RP11-3D organoid models, wild-type mice, and especially the RP11 mouse model constructed above, thereby obtaining optimized PRPF31 gene therapy vector drugs with clinical application potential.

[0013] Therefore, according to one aspect of the invention, an adeno-associated virus (AAV) vector is provided, comprising a nucleotide sequence encoding the PRPF31 protein and a promoter operatively linked thereto.

[0014] In some embodiments, the adeno-associated virus vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, as well as any AAV variant, including AAV2.7m8, AAV2-tYF, and AAV8-Y447,733F, preferably AAV2.7m8 and AAV8.

[0015] In some embodiments, the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter, wherein the tissue-specific promoter is optionally selected from RPE cell-specific promoters, photoreceptor cell-specific promoters, retinal cell-specific promoters, corneal cell-specific promoters, ocular cell-specific promoters, or combinations thereof, preferably a combination of RPE cell-specific promoters (e.g., RPE65 promoter or VMD2 promoter) and photoreceptor cell-specific promoters (e.g., RK promoter).

[0016] In some embodiments, the promoter is selected from the group consisting of: CAG promoter, elongation factor 1α short (EFS) promoter, VMD2 (also known as BEST1) promoter, RPE65 promoter, BIP promoter, RK promoter, Rho promoter, TFPT promoter, human β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1α (EF-1α) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, OPEFS promoter, hGRK1 promoter, smCAG promoter or combinations thereof, preferably CAG promoter, EFS promoter and BIP promoter, more preferably CAG promoter and BIP promoter, or a combination of RK promoter and VMD2 promoter (SEQ ID NO:31 or SEQ ID NO:32).

[0017] In some embodiments, the amino acid sequence of the PRPF31 protein is the amino acid sequence encoded by SEQ ID NO:11, preferably encoded by a wild-type nucleotide sequence (SEQ ID NO:11) or a codon-optimized nucleotide sequence (SEQ ID NO:30), i.e., the vector contains a codon-optimized nucleotide sequence (SEQ ID NO:30) encoding the PRPF31 protein.

[0018] In some embodiments, the vector is AAV8 serotype or AAV2.7m8 serotype and the promoter is a CAG promoter (SEQ ID NO:10) or a BIP promoter (SEQ ID NO:22), preferably the vector is AAV8 serotype and the promoter is a CAG promoter (SEQ ID NO:10), more preferably the nucleotide sequence encoding the PRPF31 protein is a wild-type nucleotide sequence (SEQ ID NO:11), the vector is AAV8 serotype and the promoter is a CAG promoter (SEQ ID NO:10).

[0019] In some embodiments, the 3' end of the nucleotide sequence encoding the PRPF31 protein further includes a transcription terminator sequence, preferably a bGH poly(A) signal (SEQ ID NO:12), and more preferably the vector includes the sequence shown in SEQ ID NO:19.

[0020] In some embodiments, the vector further includes an inverted terminal repeat (ITR) sequence, preferably AAV2 ITR, at the 5' end of the promoter and the 3' end of the transcription terminator sequence.

[0021] In some embodiments, the vector further comprises a Kozak sequence (SEQ ID NO:20) downstream (3' end) of the promoter.

[0022] In some embodiments, the vector further comprises a WPRE (SEQ ID NO:28) or WPRE3 (SEQ ID NO:29) element downstream (3' end) of the nucleotide sequence encoding the PRPF31 protein.

[0023] In some embodiments, the vector further comprises the SV40 intron (SEQ ID NO:27).

[0024] In some embodiments, the vector comprises the nucleotide sequence (CAG-hPRPF31-BGH) shown in SEQ ID NO:19, and the vector is of serotype AAV2.7m8.

[0025] In some embodiments, the carrier is formulated for subretinal or intravitreal administration, preferably for subretinal administration, and more preferably for subretinal administration when the carrier is of the AAV8 serotype.

[0026] According to another aspect of the invention, a cell is provided that comprises the carrier described in the invention.

[0027] According to another aspect of the invention, a pharmaceutical composition is provided comprising: a carrier or cell as described in the invention; and a pharmaceutical excipient.

[0028] According to another aspect of the invention, a kit is provided comprising the vector or cells described in the invention.

[0029] According to another aspect of the invention, the use of the carrier or cell described in the invention in the preparation of a medicament for treating retinitis pigmentosa caused by a mutation in the PRPF31 gene (i.e., RP11 type).

[0030] In some embodiments, the drug is formulated in a form suitable for injection.

[0031] In some embodiments, the drug is formulated for subretinal or intravitreal injection, preferably for subretinal injection.

[0032] According to another aspect of the invention, a method for constructing an animal model of retinitis pigmentosa (RP) is also provided, the method comprising introducing a PRPF31 gene mutation into the animal genome, the mutation affecting PRPF31 gene expression in the animal, causing PRPF31 dysfunction and / or producing the RP11 disease phenotype. Preferably, the mutation results in autosomal dominant retinitis pigmentosa (adRP). In a preferred embodiment, the mutation is a pathogenic mutation of PRPF31 in RP11 patients listed in Table 1.

[0033] In one embodiment, the animal is a non-human mammal, preferably a ruminant, canine, rabbit, feline, or rodent, more preferably a mouse, pig, monkey, bear, sheep, goat, horse, donkey, rabbit, cat, cow, fox, or dog, even more preferably a mouse or rat, even more preferably a C57BL / 6 mouse or BALB / c mouse, even more preferably a C57BL / 6J or C57BL / 6N mouse, and most preferably a C57BL / 6J mouse.

[0034] In one embodiment, the RP11 disease phenotype is selected from the group consisting of: cellular structural defects, impaired phagocytic function, ciliary formation defects, impaired barrier function, impaired retinal function, thinning of the photoreceptor layer, photoreceptor apoptosis, and combinations thereof.

[0035] In one embodiment, the mutation is a deletion mutation, comprising the deletion of one, two, or more exons of the PRPF31 gene, the exons being selected from exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7, preferably exon 4, exon 5, or combinations thereof. In a preferred embodiment, the mutation is the deletion of exon 4 and optionally one or more other exons. In a preferred embodiment, the mutation is the deletion of exon 5 and optionally one or more other exons. In a preferred embodiment, the mutation is the deletion of exons 4 and 5 and optionally one or more other exons.

[0036] In one embodiment, the deletion is achieved by gene knockout technology, gene editing technology, or a combination thereof, preferably by conditional gene knockout technology or systemic gene knockout technology, wherein the conditional gene knockout technology is preferably Cre-loxP gene knockout technology, and the gene editing technology is selected from CRISPR / Cas technology, zinc finger nuclease technology (ZFN) and transcription activator-like effector nuclease technology (TALEN), preferably CRISPR / Cas technology.

[0037] In one embodiment, the Cre-loxP gene knockout technology includes estrogen-inducible Cre-loxP gene knockout technology, preferably including the step of inducing Cre activity using tamoxifen or an analogue thereof.

[0038] In one implementation, the method includes the following steps:

[0039] 1) Two loxP sites are inserted into the PRPF31 gene of the animal using conditional Cre-loxP gene knockout technology. Preferably, the two loxP sites are inserted into intron 3 and intron 5 of the PRPF31 gene, respectively; and

[0040] 2) The Cre enzyme is induced to enter the cell nucleus by tamoxifen or its analogues, resulting in the knockout of a segment of the PRPF31 gene between the two loxP sites.

[0041] In one implementation, in the method:

[0042] 1) The animal is a mouse, preferably a C57BL / 6J mouse;

[0043] 2) The mice obtained in step 1) were C57BL / 6JCya-Prpf31 em1flox / Cya mice, and compared them with C57BL / 6JCAGGCre-ER TM Mice were hybridized, passaged, and bred to obtain C57BL / 6JCya-Prpf31. em1flox / em1flox Mice (loxP mice) and C57BL / 6JCya-Prpf31em1flox / em1flox CAGGCre-ER TM In step 2), Cre mice (Cre mice) were induced to knock out the PRPF31 gene by tamoxifen.

[0044] 3) The method further includes a step of genotyping the obtained mouse model to determine if the PRPF31 gene has been knocked out. In some embodiments, this identification is performed by PCR, Southern blot, or Sanger sequencing; and / or

[0045] 4) The method further includes the step of performing phenotypic detection on the obtained mouse model to determine whether it has the RP11 disease phenotype.

[0046] In one embodiment, the RP11 disease phenotype is detected by examining photoreceptor degeneration using fundus photography (FP), optical coherence tomography (OCT), and hematoxylin and eosin (HE) staining, and by assessing visual function using ERG. The RP11 disease phenotype includes, but is not limited to, impaired retinal function, thinning of the photoreceptor layer, and / or photoreceptor apoptosis.

[0047] In one implementation, tamoxifen or its analogues are induced by intraperitoneal injection.

[0048] In one implementation, each mouse was intraperitoneally injected with 25-75 mg tamoxifen / kg mouse body weight daily for 3 to 5 consecutive days.

[0049] In one embodiment, tamoxifen or its analogues are induced by intravitreal injection of a higher concentration (e.g., 1 to 50 mg / ml, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 mg / ml) of tamoxifen solution (with corn oil as a solvent) (e.g., 1-10 μl, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μl) to induce a disease model of RP11 (tamoxifen dosage, such as 1-500 μg, such as 5, 10, 20, 50, 60, 80, 100, 200, 300, 400, and 500 μg).

[0050] According to another aspect of the invention, an RP animal model, preferably a mouse model, prepared by the method according to the invention is provided.

[0051] According to another aspect of the present invention, a method is provided for identifying and / or testing gene therapy drugs for treating RP11, comprising:

[0052] 1) Administering the gene therapy drug to an RP animal model prepared by the method according to the present invention; and

[0053] 2) Detect whether the RP11 disease phenotype in the animal model has improved;

[0054] If the RP11 disease phenotype in the animal model is improved, it indicates that the gene therapy drug can be effectively used to treat RP11.

[0055] In one implementation, the animal in step 1) is a mouse; the administration in step 1) is an intraocular administration (e.g., subretinal injection, intravitreal injection); and the detection of the disease phenotype of the RP in the animal model in step 2) includes one or more of the following steps: a) detecting fundus changes in the model animal by fundus photography abnormalities (FP) and optical coherence tomography (OCT); and b) measuring changes in visual function in the model animal by electroretinography (ERG).

[0056] In one embodiment, the gene therapy drug includes an AAV-based gene replacement drug or gene editing drug, preferably in the form of a recombinant AAV (rAAV) viral vector, more preferably in the form of AAV8 serotype and AAV2.7m8 serotype rAAV, and most preferably in the form of AAV8 serotype rAAV.

[0057] According to another aspect of the present invention, the use of the RP animal model described in the present invention for in vivo evaluation of the efficacy and / or safety of gene therapy drugs for treating RP is provided.

[0058] In a preferred embodiment, the RP11 mouse model obtained by this invention is a tamoxifen-induced conditional gene knockout (CKO) Cre-loxP mouse model. The CKO mouse model is designed to mimic PRPF31 deficiency. Identification results of this mouse model indicate that tamoxifen-induced retinal function is impaired, the photoreceptor cell layer is thinned, and photoreceptor cells undergo apoptosis, exhibiting a disease phenotype similar to that of RP11 patients caused by PRPF31 mutations. Furthermore, by administering gene replacement drugs subretinally, the fundus abnormalities and retinal function in the tamoxifen-induced mouse model were restored to some extent. These results demonstrate that this mouse model can be used as an in vivo pharmacodynamic study model for gene therapy (replacement) drugs. Attached Figure Description

[0059] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0060] Figure 1. Schematic diagram of PRPF31 CKO mouse model construction;

[0061] Figure 2. Genotype identification strategy;

[0062] Figure 3. Genotypes of model mice detected by PCR;

[0063] Figure 4. Fundus photography and OCT of mice 3 weeks after intravitreal injection of tamoxifen-corn oil. Fundus photography shows that the oil droplets have not spread completely; OCT results show abnormal retinal structure in the right eye of Cre mice, while no significant changes were found in the retinal structure of the left eye of Cre mice and both eyes of loxP mice.

[0064] Figure 5. Fundus photography and OCT of mice 6 weeks after intravitreal injection of tamoxifen-DMSO. A. Lox mice injected intravitreally with 100 ng / 3 μl tamoxifen; B. Cre mice injected intravitreally with 100 ng / 3 μl tamoxifen; C. Lox mice injected intravitreally with 300 ng / 3 μl tamoxifen; D. Cre mice injected intravitreally with 300 ng / 3 μl tamoxifen.

[0065] Figure 6. Fundus photography and OCT of mice 6 weeks after intravitreal injection of tamoxifen-ethanol. A. Lox mice injected intravitreally with 100 ng / 3 μl tamoxifen; B. Cre mice injected intravitreally with 100 ng / 3 μl tamoxifen; C. Lox mice injected intravitreally with 600 ng / 3 μl tamoxifen; D. Cre mice injected intravitreally with 600 ng / 3 μl tamoxifen.

[0066] Figure 7. Survival curves of Cre mice and loxP mice after intraperitoneal injection of tamoxifen;

[0067] Figure 8. FP and OCT of Cre and loxP mice before and after intraperitoneal tamoxifen induction;

[0068] Figure 9. HE staining of the retinas of Cre and loxP mice after intraperitoneal injection of tamoxifen;

[0069] Figure 10. TUNEL staining of the retinas of Cre and loxP mice after intraperitoneal injection of tamoxifen;

[0070] Figure 11. ERG amplitude of Cre and loxP mice after intraperitoneal injection of tamoxifen;

[0071] Figure 12. Changes in ERG amplitude before and after intraperitoneal injection of tamoxifen in Cre mice;

[0072] Figure 13. Genomic cleavage after intraperitoneal injection of tamoxifen in Cre mice;

[0073] Figure 14. Color images of fundus tissue in groups G1-G6;

[0074] Figure 15. Fluorescence images of frozen retinal sections from groups G1-G6;

[0075] Figure 16. Color images of fundus tissue in groups G7-G10;

[0076] Figure 17. Fluorescence images of frozen retinal sections from groups G7-G10;

[0077] Figure 18. Design of PRPF31 gene replacement drugs;

[0078] Figure 19. Expression of gene replacement drugs with different structures in 293A, ARPE19 and 661W cell lines;

[0079] Figure 20. mRNA expression of gene replacement drugs with different structures in the mouse retina;

[0080] Figure 21. Protein expression of gene replacement drugs with different structures in mouse retina;

[0081] Figure 22. Timeline of RP11 drug administration, induction, and detection in mice;

[0082] Figure 23. Effects of loxP deletion in the mPrpf31 genome in the retina of RP11 mice;

[0083] Figure 24. Expression of transgenic hPRPF31 mRNA in the retina of RP11 mice;

[0084] Figure 25. Expression of transgenic hPRPF31 protein in the retina of RP11 mice;

[0085] Figure 26. FP and OCT in Cre mice 6 weeks after AAV injection;

[0086] Figure 27. Percentage of ERG amplitude at 6 weeks after AAV injection in RP11 mice compared to ERG amplitude at 3 weeks after AAV injection;

[0087] Figure 28. ERG results of mice in each group after 3 weeks of tamoxifen induction;

[0088] Figure 29. Fundus photographs and OCT results of mice after tamoxifen induction in each group;

[0089] Figure 30. CRE cuts in mouse eye tissue (retina and RPE) and brain tissue;

[0090] Figure 31. mRNA expression of transgenic PRPF31 in mouse retina and RPE after intravitreal injection of AAV2.7m8-PRPF31;

[0091] Figure 32. Expression of GFP and hPRPR31 proteins in the mouse retina and RPE after intravitreal injection of AAV2.7m8-GFP (OS, left eye) or AAV2.7m8-PRPF31 (OD, right eye);

[0092] Figure 33. ERG detection results of mice in each group after tamoxifen induction;

[0093] Figure 34. Fundus photography and OCT results of mice in each group after tamoxifen induction. A. Fundus photography and OCT results of mice in groups G1-G4 after tamoxifen induction. B. Fundus photography and OCT results of mice in group G4 after tamoxifen induction;

[0094] Figure 35. TUNEL staining results of the retina of mice in each group after tamoxifen induction;

[0095] Figure 36. CRE cleavage of mouse eye tissue (retina and RPE) and brain tissue after tamoxifen induction in each group of mice;

[0096] Figure 37. mRNA expression of transgenic PRPF31 in mouse retina and RPE after subretinal injection of AAV2.7m8-BIP-PRPF31 or AAV8-BIP-PRPF31;

[0097] Figure 38. Protein expression of GFP in mouse retina and RPE after subretinal injection of AAV2.7m8-GFP or AAV8-GFP;

[0098] Figure 39. Protein expression of PRPF31 in mouse retina and RPE after subretinal injection of AAV2.7m8-BIP-PRPF31 or AAV8-BIP-PRPF31;

[0099] Figure 40. Expression and localization of ZO-1 and BEST1 in IPSC-RPE cells from normal individuals and RP11 patients;

[0100] Figure 41. PRPF31 protein expression in IPSC-RPE cells derived from normal individuals and RP11 patients;

[0101] Figure 42. PRPF31 mRNA expression in IPSC-RPE cells from normal individuals and RP11 patients;

[0102] Figure 43. Expression and localization of Rhodopsin and NRL in IPSC-3D organoids from normal individuals and RP11 patients;

[0103] Figure 44. Expression of GFP and hPRPR31 proteins in the retina and RPE of mice after drug administration;

[0104] Figure 45. ERG detection results of mice in each group before tamoxifen induction;

[0105] Figure 46. ERG detection results of mice in each group after tamoxifen induction;

[0106] Figure 47. Fundus photographs and OCT results of mice in each group before and after tamoxifen induction;

[0107] Figure 48. TUNEL staining results of the retina of mice in each group after tamoxifen induction;

[0108] Figure 49. Expression of GFP and hPRPR31 proteins in the retina and RPE of mice after drug administration;

[0109] Figure 50. ERG results of mice in each group before and after tamoxifen induction; and

[0110] Figure 51. Fundus photographs and OCT results of mice in each group before and after tamoxifen induction. Detailed Implementation

[0111] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this field, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0112] Terminology Definition

[0113] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0114] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transferring inserted nucleic acid molecules into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0115] In some cases, the vector may include a viral vector, such as AAV, lentivirus, retrovirus, adenovirus, herpesvirus, and hepatitis virus. Methods for generating viral vectors containing nucleic acid molecules (e.g., the isolated nucleic acid molecules described in this application) as part of the vector genome are well known in the art and can be performed by those skilled in the art without extensive experimentation. In other cases, the vector may be a recombinant AAV viral particle packaged with the nucleic acid molecules described in this application. Methods for generating recombinant AAV may include introducing the nucleic acid molecules described in this application into a packaging cell line, generating AAV infection, auxiliary functions of the AAV cap and rep genes, and recovering the recombinant AAV from the supernatant of the packaging cell line. Various types of cells can be used as packaging cell lines. For example, usable packaging cell lines include, but are not limited to, HEK 293 cells, HeLa cells, and Vero cells.

[0116] In some cases, the vector may be an adeno-associated virus (AAV) vector. In this application, the term "adeno-associated virus vector" generally refers to a vector derived from naturally occurring and available adeno-associated viruses and artificial AAVs. The AAV may include different serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, as well as any AAV variant (e.g., AAV2.7m8, AAV2-tYF, and AAV8-Y447,733F) or mixtures. The AAV genome typically has terminal inverted repeats (ITRs) at both ends. The term "ITR" or "terminal inverted repeat" refers to a nucleic acid sequence segment present in AAV and / or recombinant AAV that forms a T-shaped palindromic structure required to complete the AAV lysis and latency life cycle. Techniques for generating AAV vectors are standard in the art, involving providing cells with the polynucleotides to be delivered, the rep and cap genes, and the AAV genome to be packaged for helper viral functions. The production of AAV vectors typically requires the presence of the following components within a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from the rAAV genome (e.g., not present therein), and a helper virus. The AAV rep and cap genes can originate from any AAV serotype, or from an AAV serotype that differs from the AAV genomic ITR, including but not limited to the AAV serotypes described herein.

[0117] The AAV vector of this application can be derived from various species. For example, the AAV can be avian AAV, bovine AAV, or goat AAV. In some embodiments, the vector is AAV2.7m8, AAV2-tYF, or AAV8-Y447,733F.

[0118] The method of this application may include generating packaging cells, i.e., generating cell lines that can be used to stably express all the essential components of AAV. For example, integrating the AAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes isolated from the AAV genome, and plasmids (or plasmids) carrying selection markers such as neomycin resistance genes into the genome of the cell. The AAV genome has been introduced into bacterial plasmids via methods such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. ETSA, 79: 2077-2081). The packaging cell lines can then be infected with helper viruses (e.g., adenoviruses). In addition to plasmids, adenoviruses or baculoviruses may also be used to introduce the AAV genome and / or the rep and cap genes into the packaging cells.

[0119] In this application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene. A promoter can be recognized by RNA polymerase, which initiates transcription to synthesize RNA. During RNA synthesis, the promoter can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription). A promoter comprises a core promoter region and a regulatory region, located in the regulatory sequence controlling gene expression, upstream of the gene transcription start site (at the 5' direction of the DNA antisense strand), and does not itself have a coding function. Based on their mode of action and function, promoters are classified into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue-specific or developmental stage-specific), and inducible promoters (regulated by external chemical or physical signals). The vector described in this application may contain a promoter. In this application, the promoter may comprise an RPE cell-specific promoter, a photoreceptor cell-specific promoter, a retinal cell-specific promoter, a corneal cell-specific promoter, an ocular cell-specific promoter, a constitutive promoter, or a combination thereof, preferably a combination of an RPE cell-specific promoter (e.g., the RPE65 promoter or the VMD2 promoter) and a photoreceptor cell-specific promoter (e.g., the RK promoter). The promoter may also comprise a mammalian β-actin promoter or a viral promoter. The promoter may also include the CAG promoter, the short elongation factor 1α (EFS) promoter, the VMD2 (also known as BEST1) promoter, the RPE65 promoter, the BIP promoter, the RK promoter, the Rho promoter, the TFPT promoter, the human β-actin promoter, the small CBA (smCBA) promoter, the CBS promoter or the CBh promoter, the elongation factor 1α (EF-1α) promoter, the CMV promoter, the PGK promoter, the UBC promoter, the GUSB promoter, the UCOE promoter, the OPEFS promoter, the hGRK1 promoter or the smCAG promoter, preferably the CAG promoter, the EFS promoter and the BIP promoter, more preferably the CAG promoter and the BIP promoter, or a (tandem) combination of the RK promoter and the VMD2 promoter (SEQ ID NO:31 or SEQ ID NO:32).

[0120] In this application, the term "operable link" generally refers to placing a regulatory sequence necessary for the expression of a coding sequence in an appropriate position relative to the coding sequence to achieve the expression of the coding sequence. For example, the first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. In some embodiments, this may refer to the arrangement of coding sequences and transcriptional control elements in an expression vector. The control elements may include promoters, enhancers, and termination elements. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. In some embodiments, "operable link" may also refer to the linking of a target gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the target gene.

[0121] In this application, the term "cell" can generally refer to a single cell, cell line, or cell culture that is or is already a recipient of a nucleic acid molecule or vector. Cells can include the nucleic acid molecules described in this application or the vectors described in this invention. Cells can include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells can include cells transfected in vitro using the vectors described in this application. Cells can be bacterial cells (e.g., *E. coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are HEK293T cells.

[0122] In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, or human patient. For example, a pharmaceutical composition described in this application may comprise the carrier and / or cells described in this application, and optionally pharmaceutically acceptable excipients. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition may be non-toxic to the recipient at the dosage and concentration used. Pharmaceutical compositions of this application include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0123] In this application, the term "treatment" generally refers to a clinical intervention used to alter the natural processes of an individual or cell in a clinicopathological process. It may include improving disease status, eliminating lesions, or improving prognosis.

[0124] In this application, the terms "application" and "administration" are used interchangeably and refer to the introduction of cells and / or vectors into a subject, or a desired site of the subject, by a method or route. The cells and / or vectors may express the nucleic acid molecules of this application (e.g., sequences encoding gRNAs and / or gRNAs) at the desired site (e.g., sites of damage or repair) to produce the desired effect. Cells (or their differentiated progeny) and / or vectors may be applied via any suitable route that delivers the cells (or their differentiated progeny) and / or vectors to the desired site in the subject, with at least a portion of the implanted cells (or cellular components) and / or vectors remaining viable. After application to the subject, cell survival can range from short periods of time, such as twenty-four hours, or days, to long periods of time, even consistent with the patient's lifespan. In some cases, the application includes injection. For example, the vectors may be administered via systemic routes such as intraperitoneal or intravenous routes. For example, the application may include subretinal injection.

[0125] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0126] In this application, the term "retinitis pigmentosa (RP)" generally refers to a hereditary blinding eye disease characterized by the progressive, selective loss of retinal photoreceptor cells (cones and rods) and retinal pigment epithelial cells. RP can be inherited in autosomal recessive (arRP), autosomal dominant (adRP), and X-linked (xlRP), with xlRP having an early onset and causing the most severe damage. Clinical manifestations of RP may include night blindness, progressive visual field defects, and central vision loss after macular involvement, ultimately leading to blindness. The main fundus changes in RP are equatorial retinal pigment disturbances, with osteocyte-like pigment deposition gradually progressing towards the posterior pole and ora serrata. Retinal pigment epithelial cells (RPE), photoreceptor cells, and the choroidal capillary layer gradually atrophy, revealing the large choroidal vessels. The retina appears bluish-gray, the retinal arteries become thinner, and the optic disc becomes waxy yellow and atrophied. Methods for assessing retinal function and morphology may include best-corrected visual acuity (BCVA), fundus autofluorescence, visual field testing, electroretinography (ERG), fundus color photography, optical coherence tomography (OCT), and fluorescein angiography (FFA).

[0127] In this application, the term "PRPF31" refers to a proribonucleic acid splicing factor whose mutations cause pathological effects (RP11 type) in rod photoreceptor and retinal pigment epithelium (RPE) cells. The human PRPF31 gene is located on the positive strand of chromosome 19, NC_000019.10 (54115754..54131713); the mouse PRPF31 gene is located on the positive strand of chromosome 7, NC_000073.7 (3632984..3645484). The PRPF31 protein mainly contains three major functional domains: the NOSIC domain (92-144 aa), the NOP domain (186-334 aa), and the C-terminal domain (336-465 aa), with the NOP domain, which is the RNA-binding domain, being the most conserved.

[0128] In this application, the term "RP11 type" refers to retinitis pigmentosa 11, also known as RP11. The gene associated with retinitis pigmentosa 11 is PRPF31 (pre-mRNA processing factor 31). Affected tissues include the retina and eye, and associated phenotypes include macular degeneration and macular atrophy.

[0129] In this application, the term "gene mutation" refers to a change that occurs in a DNA sequence, which may be a single base substitution, insertion, or deletion in the gene sequence, or a larger gene deletion, duplication, or inversion.

[0130] In this application, the term "gene knockout (KO)" refers to the removal of certain important exons or functional domains, or even all exons, of a target gene, resulting in the loss of expression of the target gene.

[0131] In this application, the term "systemic gene knockout technology" is also called complete gene knockout, which refers to gene knockout in all tissue cells.

[0132] In this application, the term "conditional knockout (CKO)" refers to restricting the modification of a gene to certain specific cell types or a specific stage of development, thereby achieving spatiotemporally specific modification of the genome.

[0133] In this application, the term "Cre-loxP gene knockout" refers to gene knockout achieved through site-specific recombination of the Cre-loxP system. Cre-loxP gene knockout can be divided into constitutive and inducible Cre-loxP gene knockout. Preferably, Cre-loxP gene knockout technology includes estrogen-induced Cre-loxP gene knockout technology, and more preferably includes the step of inducing Cre activity using tamoxifen or its analogues.

[0134] In this application, the term "gene replacement" refers to the replacement of a missing or abnormal gene in a patient's body by introducing a foreign gene. Common gene replacement techniques include gene repair, gene insertion, and gene splicing. For example, a normal foreign gene can be introduced into a patient's cells to replace an abnormal gene, which can be done using a vector (such as a viral vector like AAV or a plasmid vector).

[0135] The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. All reagents and materials used in the following experiments are commercially available products unless otherwise specified.

[0136] Example 1

[0137] Mouse model construction strategy

[0138] The strategy for constructing the mouse model is shown in Figure 1.

[0139] First, two loxP sites were inserted into introns 3 and 5 of the mouse PRPF31 gene (NCBI reference sequence: NC_000073.7). Therefore, when the Cre enzyme enters the nucleus, the sequence between the two loxP sites in the mouse PRPF31 genome is deleted, resulting in a 182bp deletion of mouse PRPF31 mRNA and downregulation of mouse PRPF31 protein expression. This step yielded C57BL / 6JCya-Prpf31. em1flox / Cya mice (commissioned by Cyagen Biotech Ltd.)

[0140] Secondly, C57BL / 6JCAGGCre-ER was purchased from Cyagen Biotech Ltd. TM In mice, without the action of the estrogen analogue tamoxifen, the CreER recombinase remains in the cytoplasm and cannot function; only when bound to estrogen can CreER enter the nucleus and drive recombination at the LoxP site. After induction with tamoxifen, the Cre protein enters the nucleus and cleaves and recombines the loxP site on the genome, resulting in the knockout of the target gene.

[0141] Then, we will use the above C57BL / 6JCya-Prpf31 em1flox / Cya mice and C57BL / 6JCAGGCre-ER TM Mice were crossbred to obtain C57BL / 6JCya-Prpf31em1flox / em1flox mice (hereinafter referred to as loxP mice) and C57BL / 6JCya-Prpf31em1flox / em1flox CAGGCre-ER mice. TMMice (hereinafter referred to as Cre mice). LoxP mice—which do not express Cre protein and do not have PRPF31 gene knockout after tamoxifen induction—serve as a control model of mouse phenotype; after tamoxifen induction, Cre protein enters the cell nucleus of Cre mice and cleaves and recombines the loxP site on the PRPF31 genome, resulting in PRPF31 gene knockout and downregulation of protein expression.

[0142] Specifically, based on the above construction strategy, targeting vectors for the mouse PRPF31 gene were designed, namely gRNA-1, gRNA-2, gRNA-3, and gRNA-4, with gRNA sequences shown in Table 2. The gRNAs, the donor vector containing the loxP site, and Cas9 mRNA were co-injected into mouse zygotes to generate targeted conditional gene knockout progeny, obtaining F0 generation mice. These F0 mice were then crossed with wild-type mice for germline propagation to generate F1 mice. The F1 targeted mice were then crossbred with C57BL / 6JCAGGCre-ER mice. TM Gene knockout mice were crossed to produce F2 generation mice.

[0143] Table 2. gRNA Sequences

[0144] Example 2

[0145] Genotyping of model mice

[0146] (1) Genotype identification strategy

[0147] The genotype identification strategy is shown in Figure 2, and the primer sequences are shown in Table 3.

[0148] Table 3. Primer sequences for genotype identification

[0149] (2) Genotyping of model mice

[0150] Table 4

[0151] DNA was extracted from the tails of the obtained F2 generation mice, and genotyping was performed using PCR with F1 / R1 and CAGGCre-ERTM-F / CAGGCre-ERTM-R primer pairs. Amplification with F1 / R1 primers showed a single 201 bp band for homozygotes, two bands (201 bp and 132 bp) for heterozygotes, and a single 132 bp band for the wild-type allele. Amplification with CAGGCre-ERTM-F / CAGGCre-ERTM-R primers showed a single 180 bp band. The PCR results are shown in Figure 3.

[0152] The identified F2 generation homozygous mice (Cre mice) were intraperitoneally injected with 75 mg / kg body weight of tamoxifen (dissolved in corn oil, Sigma, C8267) for 5 consecutive days. After 4 weeks, the tails were cut off for DNA extraction. PCR amplification was performed using F1 / R3 primers, and PRPF31 knockout was successful, resulting in a 157 bp band.

[0153] Example 3

[0154] Intraocular induction conditions in PRPF31 CKO mouse model

[0155] Since the RP11 disease phenotype is primarily ocular, and systemic induction with tamoxifen is expected to lead to systemic knockout of the PRPF31 gene and systemic toxicity, the inventors initially investigated possible intraocular induction conditions for tamoxifen. Tamoxifen is soluble in corn oil, chloroform, methanol, ethanol, DMSO, etc., but almost insoluble in water (solubility <0.01%, 20°C). Considering the significant in vivo toxicity of chloroform and methanol, we used corn oil, ethanol, and DMSO as solvents to dissolve tamoxifen, respectively, and induced Cre and loxP mice by intravitreal injection of tamoxifen.

[0156] (1) Tamoxifen 20 mg / ml was dissolved in corn oil. 3 μl of corn oil was injected into the vitreous cavity of the left eye, and 3 μl of tamoxifen was injected into the vitreous cavity of the right eye (tamoxifen dosage 60 μg). After 3 weeks, it was observed (Figure 4) that the oil droplets in the vitreous cavity did not dissolve, indicating that tamoxifen could not diffuse. OCT results showed abnormal retinal layer structure in the right eye of Cre mice, while no significant changes were observed in the retinal structure of the left eye of Cre mice and the left and right eyes of loxP mice.

[0157] (2) Tamoxifen 3.3 mg / ml was dissolved in DMSO, diluted 100-fold / 33-fold in PBS, and 3 μl was injected intravitreally (tamoxifen dosage 100 ng / 300 ng). Fundus photography and OCT were performed 6 weeks after induction. The results are shown in Figure 5. The results showed that after induction with 100 ng / 300 ng tamoxifen, both lox mice and Cre mice had highly reflective punctate distributions in their fundus, with no significant difference, which was caused by the drug or injection and was not related to Cre expression or PRPF31 knockout.

[0158] (3) Tamoxifen 20 mg / ml was dissolved in ethanol, diluted 600 times / 100 times in PBS, and 3 μl was injected intravitreally (tamoxifen dosage 100 ng / 600 ng). The results are shown in Figure 6. The results showed that after induction with 100 ng tamoxifen, there were no significant changes in the fundus of both lox and Cre mice; after induction with 600 ng tamoxifen, there were highly reflective punctate distributions in the fundus of both lox and Cre mice, with no significant difference, which was caused by the drug or injection and was not related to Cre expression or PRPF31 knockout.

[0159] The results above indicate that intravitreal injection of higher concentrations of tamoxifen (with corn oil as a solvent) can successfully induce the RP11 disease model, while injection of lower concentrations of tamoxifen (with ethanol or DMSO as a solvent) cannot successfully induce the RP11 disease model.

[0160] Example 4

[0161] Peritoneal induction and phenotypic study of PRPF31 CKO mouse model

[0162] Although systemic induction of tamoxifen may lead to systemic knockout of the PRPF31 gene and systemic toxicity, the inventors unexpectedly discovered through research that intraperitoneal injection of tamoxifen into Cre mice can successfully induce a disease model of RP11.

[0163] Cre mice were intraperitoneally injected with 75 mg / kg tamoxifen for 5 consecutive days. After 4 weeks of systemic induction, the mice experienced a significant decrease in body weight. Animals were euthanized if the weight loss exceeded 20%. The survival curves of Cre and loxP mice induced by intraperitoneal injection of tamoxifen are shown in Figure 7.

[0164] For patients with retinopathy of prematurity (RP), abnormal fundus photography (FP) and progressive photoreceptor degeneration are the main characteristic phenomena, leading to retinal structural abnormalities and visual function loss. To determine whether the PRPF31 CKO mouse model has a similar pathogenesis and disease phenotype to RP patients, we detected photoreceptor degeneration using fundus photography (FP), optical coherence tomography (OCT), and HE staining, and assessed visual function using ERG. Whitening of the fundus indicates photoreceptor apoptosis. After 3 weeks of tamoxifen induction, dense white spots appeared in the fundus of Cre mice, suggesting retinal degeneration, as shown in Figure 8.

[0165] Four weeks after tamoxifen induction, mice were euthanized and their eyeballs were harvested. Retinal sections were stained with hematoxylin and eosin (HE) and TUNEL. HE staining showed that, compared with loxP control mice, Cre mice had a significantly reduced density of photoreceptor cells in the outer nuclear layer (ONL) after tamoxifen induction, indicating that photoreceptor cell degeneration occurred, as shown in Figure 9.

[0166] TUNEL (TdT-mediated dUTP nick-end labeling) staining (staining technology provided by Beijing Yanyou Technology Co., Ltd.) is a widely used technique for monitoring apoptotic cells. It is based on in situ labeling of DNA breakage sites within intact, fixed cell nuclei. Intranuclear TUNEL signals characterize DNA breakage and apoptosis, while extranuclear TUNEL signals are false positives. In Cre mice, numerous strong positive signals co-localized with ONL cell nuclei, indicating widespread apoptosis in tamoxifen-induced ONL cells, as shown in Figure 10.

[0167] To examine retinal function in the PRPF31 CKO mouse model, ERG analysis was performed 3 weeks after systemic induction with intraperitoneal injection of tamoxifen, before significant weight loss was observed. The amplitudes of both dark-adapted a-wave and dark-adapted b-wave in Cre mice were significantly reduced, indicating impaired visual function following systemic tamoxifen induction, as shown in Figure 11. These results demonstrate that intraperitoneal injection of tamoxifen can successfully induce the RP11 disease model in Cre mice.

[0168] Example 5

[0169] Study on the induction method of PRPF31 CKO mouse model (selection of intraperitoneal induction drug dosage)

[0170] Tamoxifen was administered intraperitoneally to loxP and Cre mice at doses of 75 mg / kg, 225 mg / kg, and 675 mg / kg body weight, respectively, for five consecutive days. The survival status of the mice is shown in the table below.

[0171] Table 5. Survival status of mice after induction with tamoxifen intraperitoneal administration

[0172] Therefore, the highest tolerated dose of tamoxifen for intraperitoneal injection in mice is 75 mg / kg mouse body weight, administered for 5 consecutive days.

[0173] Example 6

[0174] Optimization of peritoneal induction conditions in PRPF31 CKO mouse model

[0175] We investigated the drug concentration induced by intraperitoneal injection of tamoxifen. Thirty Cre mice were selected and divided into 5 groups of 6 mice each. Group G1 received no induction; Group G2 received tamoxifen at a dose of 25 mg / kg body weight for 5 consecutive days; Group G3 received tamoxifen at a dose of 50 mg / kg body weight for 5 consecutive days; Group G4 received tamoxifen at a dose of 75 mg / kg body weight for 5 consecutive days; and Group G5 received tamoxifen at a dose of 75 mg / kg body weight for 3 consecutive days. ERG analysis was performed before induction and 3 weeks after induction (Figure 12), and the survival of the mice was observed. Genomic DNA was extracted from the tails of the mice 3 weeks after induction, and Cre cleavage was analyzed (Figure 13).

[0176] The results showed that, except for group G1, all groups G2-G5 experienced weight loss after induction and died 3-4 weeks after induction, with no significant differences among the groups. Figure 12 shows the ERG levels before and after intraperitoneal induction. Except for group G1, the ERG levels in all groups G2-G5 decreased significantly after induction, indicating that intraperitoneal injection of tamoxifen at doses of 25 mg / kg-75 mg / kg body weight for 3-5 consecutive days can successfully induce the RP11 disease model.

[0177] Example 7

[0178] Screening of AAV vectors by different serotypes and injection methods

[0179] We investigated the effects of different serotypes and injection methods of the AAV vector on the expression and distribution of the target protein in the eye (experimental design is shown in Table 6).

[0180] Different serotypes of AAV-EFS-GFP virus (all purchased from Paizhen Biotechnology) were injected subretinally or intravitreal into wild-type mice (C57 / BL6J, Beijing Vital River Laboratory Animal Technology Co., Ltd.). The groups are shown in the table below: groups G1-5 were subretinal injection (SR), groups G6-10 were intravitreal injection (IVT), and group G11 was untreated. Four weeks later, GFP expression was observed by fundus photography. Frozen sections of the eyes were then used for immunofluorescence analysis to observe GFP localization.

[0181] Table 6. Experimental Design

[0182] Following subretinal intraretinal injection, fundus photography (Figure 14) showed that AAV2.7m8 had the strongest expression intensity, AAV5 expression was relatively weak, and the expression intensities of AAV8, AAV9, AAV8-Y447F, Y733F, and AAV2-tYF were intermediate. Frozen section fluorescence images of the retina (Figure 15) showed that AAV5 was mainly expressed in the RPE cell layer, AAV8 and AAV8-Y447F, Y733F were mainly expressed in the photoreceptor cell layer (outer nuclear layer, inner and outer segments) and the RPE cell layer, and AAV9, AAV2.7m8, and AAV2-tYF were mainly expressed in the optic nerve cell layer and bipolar cell layer.

[0183] Following intravitreal injection, fundus photography (Figure 16) showed that AAV2.7m8 exhibited the strongest expression intensity, while AAV9, AAV8-Y447F, Y733F, and AAV2-tYF showed weaker expression. Frozen retinal fluorescence images (Figure 17) revealed that AAV2.7m8 and AAV2-tYF were primarily expressed in the optic nerve cell layer and bipolar cell layer, while AAV9, AAV8-Y447F, and Y733F were expressed in small amounts in the photoreceptor cell layer.

[0184] Example 8

[0185] Comparison of expression of gene replacement drugs with different structures in cell lines

[0186] We designed a gene replacement drug based on the mRNA splicing defect caused by insufficient PRPF31 in RP11 patients (see Figure 18). The CDS sequence of the human PRPF31 gene was inserted after the promoter of the pAV-CAG-CYP4V2 expression vector (Chinese Patent Application No. 202010520246.7, CN 113106124 A, AAV vector expressing CYP4V2 and its uses), replacing the CDS region of the CYP4V2 gene, to obtain pAV-CAG-hPRPF31-BGH (the sequences of CAG, hPRPF31 and BGH elements are shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively). Furthermore, we modified the vector based on pAV-CAG-hPRPF31-BGH by replacing the CAG promoter with various promoters (EFS, BIP, TFPT, RPE65, VMD2, or RK promoters), and / or adding WPRE or WPRE3 elements, and / or replacing the wild-type PRPF31 encoding gene hPRPF31 with a codon-optimized PRPF31 encoding gene (hPRPF31-CO), thereby obtaining pAV-EFS-hPRPF31-BGH, pAV-BIP- The gene sequences for the PRPF31-BGH, pAV-TFPT-hPRPF31-BGH, pAV-RPE65-hPRPF31-BGH, pAV-VMD2-hPRPF31-BGH, pAV-RK-hPRPF31-BGH, pAV-BIP-hPRPF31-WPRE-BGH (BW1), and pAV-BIP-hPRPF31-WPRE3-BGH (BW3) are provided, where the BIP and TFPT promoter sequences are from the literature (Rose, Shah et al. 2012); and the codon-optimized PRPF31 vectors pAV-BIP-hPRPF31-CO-BGH (BIP-CO) and pAV-BIP-hPRPF31-CO-WPRE3-BGH (COW3). The sequences of the aforementioned promoters, WPRE or WPRE3 elements, and the codon-optimized PRPF31 coding gene (hPRPF31-CO) are provided in Table 9.

[0187] We transfected the above vectors into the 293A, ARPE19, and 661W cell lines, respectively. The results (Figure 19) showed that, except for TFPT and specific promoters (RPE65 and VMD2 are RPE-specific promoters, and RK is a photoreceptor-specific promoter), the vectors with the other structures were expressed.

[0188] Example 9

[0189] Comparison of expression of gene replacement drugs with different structures in wild-type mice

[0190] The vectors described in Example 8 were packaged into AAV8 viruses (virus packaging was performed by Paizhen Biotechnology). WT mice were injected subretinally with a dose of 1e9 / μl. Six weeks later, samples were collected to detect the mRNA and protein expression of hPRPF31. The results are shown in Figures 20 and 21: In the retina, vectors such as CAG, RK, BIP, BW1, BW3, and BIP-CO showed high expression levels; in the RPE, vectors such as RPE65, BIP-CO, VMD2, BW1, BW3, BIP, and CAG showed high expression levels.

[0191] Example 10

[0192] Gene replacement drugs can restore PRPF31 protein expression in RP11 disease model mice and improve retinal structure and function (1)

[0193] We used the tamoxifen-induced RP11 mouse model constructed above to detect the in vivo efficacy of the gene replacement therapy vector drug designed above. Following the steps described in CN113106124A, pAV-CAG-hPRPF31-BGH was packaged as AAV8 virus, with the drug code AAV8-PRPF31, and a single subretinal injection efficacy verification experiment was conducted in PRPF31 CKO model mice. The experiment consisted of four groups: Group G1: without tamoxifen induction, subretinal injection of 1e9vg / μl / eye AAV8-EGFP (AAV8-CAG-EGFP, purchased from Paizhen Biotechnology); Group G2: induced with 75mg / kg mice using tamoxifen, subretinal injection of 1e9vg / μl / eye AAV8-EGFP; Group G3-LD / HD: induced with 75mg / kg mice using tamoxifen, subretinal injection of 1e8vg / μl / eye (AAV-PRPF31-LD) or 1e9vg / μl / eye (AAV-PRPF31-HD) AAV8-PRPF31. The timeline for drug injection, tamoxifen induction, in vivo testing, and animal euthanasia in the experiment is shown in Figure 22. AAV injection was performed 3 weeks prior to tamoxifen induction; FP, OCT, and ERG tests were performed 6 weeks prior to AAV injection to evaluate the structure and function of the mouse retina; after the in vivo data testing was completed, the mice were euthanized as planned to detect the distribution and expression of gene therapy drugs.

[0194] All mice were euthanized as planned after in vivo data analysis was completed. Genomic DNA, mRNA, and protein were extracted after retinal separation. The genomic DNA was used for PCR with specific primers (SEQ ID NO: 5-9), and the results further confirmed the Cre mouse genotype, showing that mPrpf31 was effectively deleted, as shown in Figure 23.

[0195] After reverse transcription of mRNA, specific primers (SEQ ID NO: 13-18) were used for qPCR to detect the mRNA level of the transgenic hPRPF31. Proteins extracted from the primer sequences were used for Western blotting with specific antibodies (anti-PRPF31, Atlas, HPA041939; anti-GFP, Abclonal, AE012; anti-actin, Abclonal, AC026). The results showed that the effective expression of the transgenic hPRPF31 was dose-dependent, as shown in Figures 24 and 25. These results indicate that this model can effectively detect the distribution and expression of gene-altering drugs.

[0196] While detecting the distribution and expression of gene therapy drugs, we evaluated the retinal structure and function of mice before obtaining animal samples. As shown in Figure 26, OCT and FP results showed that 6 weeks after AAV injection, G2 mice (induced by tamoxifen, subretinal injection of AAV-EGFP) had denser leukoplakia in their fundus than G1 mice (not induced by tamoxifen, subretinal injection of AAV-EGFP). However, the dense leukoplakia in the fundus of G3 mice (induced by tamoxifen, subretinal injection of AAV-PRPF31) was reduced, consistent with OCT results. Furthermore, changes in mouse retinal function were further examined using ERG, performed before and after systemic tamoxifen induction. The b-wave amplitude (i.e., the ratio of dark-adapted b-wave amplitude at week 6 to that at week 3) in the ERG was calculated to reflect retinal function in the Cre mouse model. The results showed that the dark-adapted b-wave amplitude of G1 (without tamoxifen induction, subretinal injection of AAV-EGFP) remained almost unchanged; while the dark-adapted b-wave amplitude of G2 (with tamoxifen induction, subretinal injection of AAV-EGFP) decreased to 53%; however, the dark-adapted b-wave amplitude of G3 (with tamoxifen induction, subretinal injection of AAV-PRPF31) decreased to 78%, as shown in Figure 27. This indicates that the AAV-PRPF31 injection group retained more visual function after tamoxifen induction.

[0197] The above results indicate that after gene replacement therapy, the fundus abnormalities and retinal function of RP11 mice were restored to a certain extent.

[0198] Example 11

[0199] Gene replacement drugs can restore PRPF31 protein expression in RP11 disease model mice and improve retinal structure and function (2)

[0200] We packaged pAV-CAG-hPRPF31-BGH into an AAV2.7m8 virus, with the drug code AAV2.7m8-PRPF31 (AAV2.7m8-CAG-PRPF31). RP11 mouse models (i.e., Cre mice marked in the chart) or control mice (Lox mice, without Cre expression, and not subject to genome cleavage after tamoxifen induction) were injected intravitreally at a dose of 1 μl. The left eye was injected with AAV2.7m8-EFS-GFP virus (purchased from Paizhen Biotechnology), and the right eye was injected with AAV2.7m8-CAG-PRPF31 (virus packaging completed by Paizhen Biotechnology). The experimental groups are as follows:

[0201] Table 7

[0202] Twelve weeks after intravitreal injection of gene replacement drug, mice were induced with tamoxifen intraperitoneally at a dose of 75 mg / kg for three consecutive days. Three weeks post-induction (W15), ERG was used to assess visual function; fundus photography and OCT were performed before induction (W10) and four weeks post-induction (W16) to examine retinal structure. After all tests were completed, tissue samples were collected to analyze drug distribution and expression.

[0203] The ERG results (Figure 28) showed that, compared with group G1, the overall ERG levels in groups G2-G4 were decreased, indicating that the RP11 animal model was successfully induced. In groups G2-G4, compared with the control eye injected with AAV2.7m8 virus, intravitreal injection of AAV2.7m8-PRPF31 increased the ERG amplitude in mice in a dose-dependent manner. When the dose was 1e9vg / eye, the increase in ERG amplitude was statistically different from that in the control eye (paired t-test, *p<0.05; **p<0.01; ***p<0.001), indicating that intravitreal injection of AAV2.7m8-PRPF31 significantly improved the visual function of RP11 model mice.

[0204] Fundus photography and OCT results (Figure 29) showed no significant changes in fundus photography before and after induction in Lox mice in group G1; fundus photography of Cre mice in groups G2-G5 after induction showed large-area retinal degeneration. There was no significant difference between the right eye (AAV2.7m8-PRPF31) and the left eye (AAV2.7m8-GFP).

[0205] We performed CRE cleavage analysis on mouse eye tissue (retina and RPE) and brain tissue, as shown in Figure 30. The results showed that the mouse mPRPF31 genome was effectively cleaved in groups G2-G4.

[0206] We examined the expression of the drug, and the mRNA expression levels are shown in Figure 31. The mRNA expression level of endogenous mPRPF31 in mice was higher than that in RPE cells. After intraperitoneal induction in Cre mice, the mRNA expression level of mPRPF31 decreased by approximately half. The mRNA level of transgenic hPRPF31 in retinal cells was higher than that in RPE cells. At an injection dose of 1e6, the mRNA expression level was below the detection limit; at injection doses of 1e7-1e9, the transgenic mRNA was expressed at high levels in a dose-dependent manner. At an injection dose of 1e8, the transgenic mRNA level in both retinal cells and RPE cells was closest to the mRNA expression level of endogenous mPRPF31 in mice.

[0207] Protein expression is shown in Figure 32. The expression levels of GFP protein in retinal cells and the transgenic hPRPF31 protein were high, both in a dose-dependent manner. Protein expression levels were weak in RPE cells.

[0208] The experimental results of Examples 10 and 11 demonstrate that the combination of the CAG promoter with the wild-type encoding nucleotide sequence of PRPF31 protein (SEQ ID NO: 11), expressed in AAV8 serotype or AAV2.7m8 serotype vectors, can not only restore PRPF31 protein expression in RP11 disease model mice, but also improve retinal structure and function, and therefore can be used as a gene replacement drug.

[0209] Example 12

[0210] Gene replacement drugs can restore PRPF31 protein expression in RP11 disease model mice and improve retinal structure and function (3)

[0211] The pAV-BIP-hPRPF31-BGH vector was packaged into AAV2.7m8 virus (AAV2.7m8-BIP-PRPF31) and AAV8 virus (AAV8-BIP-PRPF31), respectively (virus packaging was performed by Paizhen Biotechnology). A subretinal injection PRPF31-CKO mouse model was established, with an injection dose of 1 μl. The left eye was injected with either AAV2.7m8-EFS-GFP or AAV8-EFS-GFP virus (purchased from Paizhen Biotechnology), and the right eye was injected with either AAV2.7m8-BIP-PRPF31 or AAV8-BIP-PRPF31. The experimental groupings are shown in the table below:

[0212] Table 8

[0213] Twelve weeks after subretinal injection of gene replacement drug, mice were induced with tamoxifen intraperitoneally at a dose of 75 mg / kg for three consecutive days. Three weeks post-induction (W15), ERG was used to assess visual function. Fundus photography and OCT were performed before induction (W10) and four weeks post-induction (W16) to examine retinal structure. After all tests were completed, tissue samples were collected to analyze drug distribution and expression.

[0214] ERG results (Figure 33) showed that after tamoxifen induction, there was no statistically significant difference in ERG amplitude between the two eyes of Lox mice (G1-G2 groups) compared to the control eyes. However, the treated eyes in the G3-G4 groups showed increased ERG amplitude levels, with statistically significant differences at certain stimulation intensities (paired t-test, *p<0.05; **p<0.01; ***p<0.001). These ERG results indicate that subretinal injection of AAV2.7m8-BIP-PRPF31 or AAV8-BIP-PRPF31 can improve visual function in RP11 model mice.

[0215] Fundus photography and OCT results showed that in the G1-G2 group of Lox mice, there were no significant changes in fundus photography and OCT results before and after induction. In the G3-G4 group of Cre mice, after induction, fundus photography of the left control eye showed numerous white spots, and OCT showed blurred retinal structures and disappearance of the outer nuclear layer and RPE junction, indicating large-area retinal degeneration. Conversely, fundus photography and OCT of the right treatment eye did not show significant changes (Figure 34, A). There was no significant difference between the right and left eyes. These differences were observed in both eyes of all mice in the G4 group (Figure 34, B).

[0216] TUNEL staining of mouse retinal sections, as shown in Figure 35, revealed no significant TUNEL signal in the retina of loxP mice (G1-2) after tamoxifen induction. In Cre mice (G3-4), strong TUNEL signal was observed in the left eye (injected with GFP) after tamoxifen induction, and the signal was co-localized with some nuclei of cells in the outer nuclear layer, indicating that a large number of photoreceptor cells underwent apoptosis. The right eye (injected with PRPF31) showed no TUNEL signal or very little TUNEL, indicating that PRPF31 replacement therapy can effectively reduce tamoxifen-induced apoptosis of retinal photoreceptor cells.

[0217] We performed CRE cleavage analysis on mouse eye tissue (retina and RPE) and brain tissue, as shown in Figure 36. The results showed that the mouse mPRPF31 genome was effectively cleaved in groups G2-G4.

[0218] We examined the expression of the drug, and the mRNA expression is shown in Figure 37. The mRNA expression level of endogenous mPRPF31 in mouse retinal cells was higher than that in RPE cells. After intraperitoneal induction in Cre mice, the mPRPF31 mRNA expression level decreased by approximately half. After subretinal administration, the transgenic mRNA levels of AAV8 serotype and AAV2.7m8 serotype virus in retinal cells were comparable, ranging from 3.6 to 4.7 x 10⁻⁶. 5 Copy / ng RNA; however, in RPE, the expression level of the AAV8 serotype was significantly higher than that of the AAV2.7m8 serotype, at 3.9–5.7 x 10^6 copies / ng RNA, respectively. 4 Copy / ng RNA, and 2.9–3.3 x 10 5 The difference between the copies / ng RNA was approximately 6-8 times.

[0219] Protein expression is shown in Figures 38 and 39. In retinal cells and RPE cells, the protein expression level of the transgenic hPRPF31 from the AAV8 serotype was higher than that from AAV2.7m8, consistent with RNA levels. These results indicate that subretinal administration is more effective in infecting RPE cells than intravitreal administration; and the AAV8 serotype has a stronger infectivity for RPE cells than the AAV2.7m8 serotype.

[0220] Example 13

[0221] Gene replacement drugs can restore PRPF31 protein expression in the RP11-RPE cell model and improve RPE cell structure.

[0222] iPSC-induced retinitis pigmentosa (iPSC-RPE) and retinal organoids (iPSC-3D) are important tools for in vitro retinal function studies. Insufficient PRPF31 expression leads to structural and functional abnormalities in iPSC-RPE and iPSC-3D, while restoring PRPF31 expression can improve these abnormalities (Rodrigues, A., et al. (2022). "Modeling PRPF31retinitis pigmentosa using retinal pigment epithelium and organoids combined with gene augmentation rescue." NPJ Regen Med 7(1):39.). We reprogrammed urinary epithelial cells from RP11 patients (obtained from Peking University Third Hospital) into iPSCs, and then further induced differentiation to obtain iPSC-RPE-RP11 and iPSC-3D-RP11. The cell preparation and culture of iPSCs and iPSC-RPE were based on patent PCT / CN2020 / 102292.

[0223] We selected iPSC-RPE cells from two healthy individuals and two patients. iPSC-RPE-RP11 cells from the two patients were infected with AAV2.7m8-CAG-PRPF31 at different MOIs (MOI = 0, 6E4, 3E5) and cultured for 4 weeks. After 4 weeks, the cells were fixed with 4% paraformaldehyde (Solarbio, P1110) and stained with DAPI (Solarbio, C0060), ZO-1 (Abcam, AB221547), and BEST1 antibodies (Abcam, ab2182), goat anti-rabbit AF488 secondary antibody (Invitrogen), and goat anti-mouse AF568 secondary antibody (Invitrogen). Compared with iPSC-RPE-WT cells from healthy individuals, iPSC-RPE-RP11 cells showed abnormal morphology, unclear ZO-1 expression, and abnormal BEST1 expression location. After infection with AAV2.7m8-BIP-PRPF31, cell morphology was restored, ZO-1 expression was regular, and BEST1 expression was in a normal position (Figure 40).

[0224] The results of protein and mRNA detection (Figures 41 and 42) showed that compared with iPSC-RPE-WT from normal human sources, the expression levels of PRPF31 protein and mRNA in iPSC-RPE-RP11 cells were lower. After infection with AAV2.7m8-CAG-PRPF31, the expression levels of PRPF31 protein and mRNA were significantly increased.

[0225] Example 14

[0226] Gene replacement drugs can improve the structure of RP11-3D organoid models

[0227] We selected iPSC-3D organoids from two healthy individuals and one patient. Approximately 100 days after infection, each organoid was infected with 5E10 vg / organoid of AAV2.7m8-CAG-PRPF31 or AAV2.7m8-CAG-GFP. Thirty days later, section staining was performed to analyze the expression of RHO and NRL proteins in the iPSC-3D organs. The results (Figure 43) showed that NRL was expressed in the nucleus in both healthy and patient-derived iPSC-3D organs, with no significant difference in expression location or intensity. Rhodopsin length was significantly shortened in patient-derived iPSC-3D organs, representing rod cell shortening or apoptosis. Infection with AAV2.7m8-CAG-GFP did not affect the length of Rhodopsin in patient-derived iPSC-3D organs, but infection with AAV2.7m8-CAG-PRPF31 significantly prolonged the length of Rhodopsin in patient-derived iPSC-3D organs, making it comparable to the length of Rhodopsin in healthy human iPSC-3D organs. This indicates that gene replacement drugs can improve the structure of the RP11-3D organoid model.

[0228] Example 15

[0229] PRPF31 replacement therapy vectors with RPE cell-specific or photoreceptor cell-specific promoters could not simultaneously restore PRPF31 protein expression and improve retinal structure and function in RP11 disease model mice.

[0230] We packaged AAV8 virus into pAV-RPE65-hPRPF31-BGH, pAV-VMD2-hPRPF31-BGH, and pAV-RK-hPRPF31-BGH (virus packaging was performed by Paizhen Biotechnology). A PRPF31-CKO mouse model was established by subretinal injection, while control eyes were injected with AAV8-EFS-GFP virus (purchased from Paizhen Biotechnology). The injection dose was 1e9vg / eye. The experimental groups are shown in the table below:

[0231] Table 9

[0232] Twelve weeks after subretinal injection of gene replacement drug, mice were induced with tamoxifen intraperitoneally at a dose of 75 mg / kg for three consecutive days. Three weeks post-induction (W15), ERG was used to assess visual function. Fundus photography and OCT were performed before induction (W10) and four weeks post-induction (W16) to examine retinal structure. After all tests were completed, tissue samples were collected to analyze drug distribution and expression.

[0233] Protein detection results (Figure 44) showed that PRPF31 protein was effectively expressed in retinal samples from mice injected with AAV8-RK-PRPF31 virus or AAV8-RPE65-PRPF31 virus, while PRPF31 protein was effectively expressed in RPE samples from mice injected with AAV8-RPE65-PRPF31 virus or AAV8-VMD2-PRPF31 virus. ERG results showed that before tamoxifen induction, the ERG amplitude of mice injected with AAV8-RPE65-PRPF31 virus was significantly decreased compared to the control eye (Figure 45), indicating that this promoter has some toxicity to the mouse retina. After tamoxifen induction, the ERG amplitude of mice injected with AAV8-VMD2-PRPF31 virus was significantly increased compared to the control eye (Figure 46), indicating that the VMD2 promoter can improve retinal function in mice.

[0234] Fundus photography and OCT results showed that after tamoxifen induction, fundus photography of the control eye and mice injected with AAV8-RK-PRPF31 virus showed a large number of white spots, and OCT showed that the structure of each layer of the retina was blurred and the junction between the outer nuclear layer and the RPE layer disappeared, indicating large-area retinal degeneration. In contrast, fundus photography and OCT of mice injected with AAV8-RPE65-PRPF31 virus or AAV8-VMD2-PRPF31 virus did not show significant changes, indicating that the RPE65 promoter or VMD2 promoter can improve the retinal degeneration in the RP11 disease model (Figure 47).

[0235] TUNEL staining of mouse retinal sections, as shown in Figure 48, revealed strong TUNEL signals in the control eye and the retinas of mice injected with AAV8-RPE65-PRPF31 virus or AAV8-VMD2-PRPF31 virus after tamoxifen induction. These signals were co-localized with some nuclei in the outer nuclear layer, indicating extensive apoptosis of photoreceptor cells. In contrast, no TUNEL signal was observed in the retinas of mice injected with AAV8-RK-PRPF31 virus, suggesting that the RK promoter can reduce apoptosis of retinal photoreceptor cells in the RP11 disease model.

[0236] The conclusions are as follows:

[0237] Table 10

[0238] Example 16

[0239] The RPE cell-specific promoter, when tandemly linked with the photoreceptor cell-specific promoter, can restore PRPF31 protein expression in RP11 disease model mice and improve retinal structure and function.

[0240] We modified the pAV-VMD2-hPRPF31-BGH and pAV-RK-hPRPF31-BGH vectors to obtain pAV-VMD2-RK-hPRPF31-BGH and pAV-RK-VMD2-hPRPF31-BGH, respectively. The promoter sequences (VMD2-RK tandem promoter and RK-VMD2 tandem promoter) are shown in SEQ ID NO:31 and SEQ ID NO:32, respectively, and were packaged with AAV8 virus (virus packaging was performed by Paizhen Biotechnology). A PRPF31-CKO mouse model was established by subretinal injection, while the control eye was injected with the excipient. The experimental groupings are shown in the table below:

[0241] Table 11

[0242] Twelve weeks after subretinal injection of gene replacement drug, mice were induced with tamoxifen intraperitoneally at a dose of 75 mg / kg for three consecutive days. Three weeks post-induction (W15), ERG was used to assess visual function. Fundus photography and OCT were performed before induction (W10) and four weeks post-induction (W16) to examine retinal structure. After all tests were completed, tissue samples were collected to analyze drug distribution and expression.

[0243] Protein detection results (Figure 49) showed that mice simultaneously injected with AAV8-RK-PRPF31 virus or AAV8-VMD2-PRPF31 virus, as well as retinal and RPE samples injected with AAV8-VMD2-RK-PRPF31 and AAV8-RK-VMD2-PRPF31 respectively, could effectively express PRPF31 protein.

[0244] ERG results showed that after tamoxifen induction, the ERG amplitude of all three groups of mice was increased compared with the control eye (Figure 50), indicating that both tandem promoters can improve retinal function in mice.

[0245] Fundus photography and OCT results showed that after tamoxifen induction, the control eyes showed a large number of white spots in fundus photography, and OCT showed that the structure of each layer of the retina was blurred and the junction between the outer nuclear layer and the RPE was lost, indicating large-area retinal degeneration. In contrast, fundus photography and OCT of the three groups of treated eyes did not show significant changes, indicating that both groups of tandem promoters can improve the retinal degeneration in the RP11 disease model (Figure 51).

[0246] The above results indicate that the tandem tandem of the RPE cell-specific promoter and the photoreceptor cell-specific promoter can simultaneously restore PRPF31 protein expression in RP11 disease model mice and improve retinal structure and function.

[0247] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

[0248] References

[0249] 1.O'Neal, TB and EELuther, Retinitis Pigmentosa, in Stat Pearls. 2021: Treasure Island (FL).

[0250] 2. Verbakel, SK, et al., Non-syndromic retinitis pigmentosa. Prog Retin Eye Res, 2018.66: p.157-186.

[0251] 3.Hartong, DT, ELBerson, and TPDryja, Retinitis pigmentosa. Lancet, 2006.368(9549): p.1795-809.

[0252] 4.Audo, I., et al., Prevalence and novelty of PRPF31 mutations in French autosomal dominant rod-cone dystrophy patients and a review of published reports. BMC Med Genet, 2010.11:p.145.

[0253] 5. Hafler, BP, et al., Course of Ocular Function in PRPF31 Retinitis Pigmentosa. Semin Ophthalmol, 2016.31(1-2):p.49-52.

[0254] 6.Rose,A.M.,et al.,Dominant PRPF31 mutations are hypostatic to a recessive CNOT3 polymorphism in retinitis pigmentosa:a novel phenomenon of"linked trans-acting epistasis".Ann Hum Genet,2014.78(1):p.62-71.

[0255] 7.Vithana,E.N.,et al.,Expression of PRPF31 mRNA in patients with autosomal dominant retinitis pigmentosa:a molecular clue for incomplete penetrance?Invest Ophthalmol Vis Sci,2003.44(10):p.4204-9.

[0256] 8.Rio Frio,T.,et al.,Two trans-acting eQTLs modulate the penetrance of PRPF31 mutations.Hum Mol Genet,2008.17(20):p.3154-65.

[0257] 9.Brydon,E.M.,et al.,AAV-Mediated Gene Augmentation Therapy Restores Critical Functions in Mutant PRPF31(+ / -)iPSC-Derived RPE Cells.Mol Ther Methods Clin Dev,2019.15:p.392-402.

[0258] 10.Li,J.,et al.,Prpf31 is essential for the survival and differentiation of retinal progenitor cells by modulating alternative splicing.Nucleic Acids Res,2021.

[0259] 11.Linder,B.,et al.,Systemic splicing factor deficiency causes tissue-specific defects:a zebrafish model for retinitis pigmentosa.Hum Mol Genet,2011.20(2):p.368-77.

[0260] 12.Yin,J.,et al.,Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for Retinitis pigmentosa.Mol Neurodegener,2011.6:p.56.

Claims

1. An adeno-associated virus (AAV) vector containing a nucleotide sequence encoding the PRPF31 protein and an operable promoter thereto.

2. The vector according to claim 1, wherein the adeno-associated virus vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, and any AAV variant, including AAV2.7m8, AAV2-tYF and AAV8-Y447,733F, preferably AAV2.7m8 and AAV8.

3. The vector according to claim 1 or 2, wherein the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter, wherein optionally the tissue-specific promoter is selected from RPE cell-specific promoters, photoreceptor cell-specific promoters, retinal cell-specific promoters, corneal cell-specific promoters, ocular cell-specific promoters, or combinations thereof, preferably a combination of RPE cell-specific promoters (e.g., RPE65 promoter or VMD2 promoter) and photoreceptor cell-specific promoters (e.g., RK promoter).

4. The vector according to claim 3, wherein the promoter is selected from the group consisting of: CAG promoter, elongation factor 1α short (EFS) promoter, VMD2 (also known as BEST1) promoter, RPE65 promoter, BIP promoter, RK promoter, Rho promoter, TFPT promoter, human β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1α (EF-1α) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, OPEFS promoter, hGRK1 promoter, smCAG promoter or combinations thereof, preferably CAG promoter, EFS promoter and BIP promoter, more preferably CAG promoter and BIP promoter, or a combination of RK promoter and VMD2 promoter (SEQ ID NO:31 or SEQ ID NO:32).

5. The vector according to any one of claims 1 to 4, wherein the amino acid sequence of the PRPF31 protein is the amino acid sequence encoded by SEQ ID NO:11, preferably the nucleotide sequence encoding the PRPF31 protein is a wild-type nucleotide sequence (SEQ ID NO:11) or a codon-optimized nucleotide sequence (SEQ ID NO:30).

6. The vector according to claim 5, wherein the vector is AAV8 serotype or AAV2.7m8 serotype and the promoter is a CAG promoter (SEQ ID NO:10) or a BIP promoter (SEQ ID NO:22), preferably the vector is AAV8 serotype and the promoter is a CAG promoter (SEQ ID NO:10), more preferably the nucleotide sequence encoding PRPF31 protein is a wild-type nucleotide sequence (SEQ ID NO:11), the vector is AAV8 serotype and the promoter is a CAG promoter (SEQ ID NO:10).

7. The vector according to any one of claims 1 to 6, wherein the 3' end of the nucleotide sequence encoding the PRPF31 protein further comprises a transcription terminator sequence, preferably a bGH poly(A) signal (SEQ ID NO:12), more preferably the vector comprises the sequence shown in SEQ ID NO:

19.

8. The vector according to any one of claims 1 to 7, wherein the vector further comprises an inverted terminal repeat (ITR) sequence, preferably AAV2 ITR, at the 5' end of the promoter and the 3' end of the transcription terminator sequence.

9. The vector according to any one of claims 1 to 8, wherein the vector further comprises a Kozak sequence (SEQ ID NO: 20) downstream (3' end) of the promoter.

10. The vector according to any one of claims 1 to 9, wherein the vector further comprises a WPRE (SEQ ID NO:28) or WPRE3 (SEQ ID NO:29) element downstream (3' end) of the nucleotide sequence encoding the PRPF31 protein.

11. The vector according to any one of claims 1 to 10, wherein the vector further comprises an SV40 intron (SEQ ID NO:27).

12. The vector according to any one of claims 1 to 11, wherein the vector comprises the nucleotide sequence shown in SEQ ID NO:19, and the vector is of serotype AAV2.7m8.

13. The carrier according to any one of claims 1 to 12, wherein the carrier is formulated for subretinal or intravitreal administration, preferably for subretinal administration, and more preferably for subretinal administration when the carrier is of the AAV8 serotype.

14. A cell comprising the vector according to any one of claims 1-13.

15. A pharmaceutical composition comprising: a carrier according to any one of claims 1-13 or a cell according to claim 14; and a pharmaceutical excipient.

16. A kit comprising the vector according to any one of claims 1-13 or the cells according to claim 14.

17. Use of the carrier according to any one of claims 1-13 or the cell according to claim 14 in the preparation of a medicament for treating retinitis pigmentosa caused by PRPF31 gene mutation.

18. The use according to claim 17, wherein the said drug is formulated in a form suitable for injection.

19. The use according to claim 18, wherein the drug is formulated for subretinal or intravitreal injection, preferably for subretinal injection.

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