Gene therapy for the treatment of cngb1-associated retinal pigmentosa

Abstract

The present invention relates to a polynucleotide comprising a promoter comprising a human photoreceptor cell-specific promoter element, a core promoter and at least one transgene. Furthermore, the present invention provides a plasmid comprising the polynucleotide, a viral vector comprising the polynucleotide and a pharmaceutical composition comprising the polynucleotide. The present invention also relates to the plasmid, the viral vector or the pharmaceutical composition for use as a medicament, in particular for use in the treatment of a retinal disease.

Description

Background of the Invention

[0002] Retinitis pigmentosa (RP) is a term used to describe a group of genetically diverse hereditary retinal degenerative diseases affecting photoreceptor cells. Genetic mutations involve genes specifically or primarily expressed in rod photoreceptor cells. Therefore, the disease is characterized by major impairment or loss of rod cell function and structure. Degeneration of rod cells is followed by secondary degeneration of cone cells. Early-onset and rapidly progressive changes, as well as late-onset and slowly progressive retinal degeneration, differ in their onset and temporal progression. The most common symptoms of RP are night blindness, progressive visual field narrowing, and abnormal accumulation of retinal pigment deposits. Clinical features include characteristic pigment deposition and progressive attenuation of retinal vessels. In many cases, RP eventually leads to legal blindness. The overall prevalence of RP is estimated at 1 in 4000. RP is genetically highly heterogeneous, with approximately 50 genes identified (Daiger Sp et al. (1998) Investigative Ophthalmology and Visual Science (Supplement) 39:S295). Many disease genes encode proteins required for light detection and processing (e.g., rhodopsin) or for maintaining rod cell morphology (e.g., peripheralin-2). 10% to 25% of RP cases show autosomal dominant inheritance (adRP), 6% to 18% show X-linked inheritance (xRP), and 20% to 30% show autosomal recessive inheritance (arRP). Another 40% to 50% are intermittent arRP, and this is the most genetically diverse subgroup of RP, with the relative frequencies of known disease genes not exceeding 15%. The most prevalent arRP genes are EYS (5% to 12%), USH2A (5% to 15%), CRB1 (approximately 5%), and PDE6B (4% to 10%). However, these values ​​vary between different subgroups and regions, likely due to the founder effect.

[0003] CNGB1 encodes the β subunit of the rod-ring nucleotide-gated (CNG) channel (RP45 locus). Mutations at the RP45 locus, causing so-called CNGB1-associated RP or type 45 RP, have been found in 2% to 4% of cases of arterial renal impairment (arRP) (Hartong DT et al. (2006) Lancet 368(9549):1795-1809). Therefore, the estimated number of patients with CNGB1-associated arRP is approximately 900 in Germany and approximately 5000 in the European Union. Visual impairment is considered one of the most important non-fatal impairments with significant clinical and socioeconomic implications. Patients with RP experience severe loss of quality of life throughout their lives. Unfortunately, there is no cure or symptomatic treatment for RP. Clinical experts and health organizations consider RP one of the best candidates for gene therapy. Previously, gene replacement therapy has been shown to restore vision and delay degeneration in a CNGB1(- / -) mouse model of retinitis pigmentosa using a recombinant AAV2 / 8 vector containing the mouse Cngb1 gene, controlled by the mouse rhodopsin (Rho) promoter: AAV2 / 8(Y733F)-Rho-Cngb1 (Koch S et al. (2012) Hum Mol. Genet. 21(20):4486-96). Injection of this vector into the eyes of mice with a gene deletion (Cngb1 KO) in exon 26 of the gene encoding Cngb1 enhanced photoreceptor survival and improved retinal function. However, the following issues make this approach less promising for treating retinal degeneration caused by CNGB1-associated RP:

[0004] (a) The size of the rAAV cis-vector genome (5.0 kb) is larger than that of the wild-type AAV genome (<4.7 kb);

[0005] (b) The mouse rhodopsin (Rho) gene promoter was used; and

[0006] (c) The mouse Cngb1 gene sequence was used.

[0007] Petersen-Jones et al. (2016) Invest. Ophthalmol. 57:1842 described an rAAV2 / 5 vector containing the coding sequence of the canine Cngb1 gene (cCngb1) under the control of the human rhodopsin kinase 1 (hGRK1) promoter: AAV5-hGRK1-cCngb1. This vector was injected into the eyes of dogs with a mutation in exon 26 of the Cngb1 gene. The injection improved retinal function. However, the following issues make this approach unpromising for treating retinal degeneration caused by CNGB1-related RP in humans:

[0008] (a) The hGRK1 promoter used in this method can drive not only expression in rod photoreceptors but also off-target expression in cone photoreceptors. This off-target expression may have negative effects on retinal function and morphology; and

[0009] (b) The canine Cngb1 gene sequence was used.

[0010] Therefore, there is a need in the art to identify transgenic elements that are small in size and do not produce negative effects or lose activity in vivo. Summary of the Invention

[0011] This invention is based on the surprising discovery that a small fraction of the human rod promoter enables rod-receptor cell-specific expression of a transgene operably linked to this promoter element in vivo. Stable expression of the transgene was observed when the promoter element defined herein was used in vivo. The expression levels were suitable for improving visual abilities in experimental animals infected with an adeno-associated virus vector containing the transgene. This surprising discovery provides, in particular, the following advantages over the prior art: (i) reduced size of the construct introduced into cells; (ii) improved packaging efficiency of the transgene in the viral vector; (iii) reduced chance of recombination events occurring in vivo; (iv) improved efficiency of transgene delivery to target cells, particularly to the target cell nucleus; (v) providing appropriate expression levels for treating rod-related diseases in human patients; (vi) protection and / or improvement of retinal function; and (vii) protection and / or improvement of visual acuity.

[0012] In a first aspect, the present invention relates to polynucleotides comprising, in the following order:

[0013] a) A promoter comprising a human rod photoreceptor cell-specific promoter element (hRPSPE) and a core promoter (CP), wherein the promoter element comprises a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof, is substantially composed of a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof, or is composed of a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof; and

[0014] b) Transgenic (TG) operatively linked to the promoter of a);

[0015] The variant of SEQ ID NO:1 contains one or more nucleic acid substitutions other than positions 6 to 13, 32 to 40, 70 to 83 and 87 to 94 of the nucleotide of SEQ ID NO:1, and the promoter length is specifically 350 bases or less.

[0016] In some exemplary embodiments, the 5' end of hRPSPE is located at positions 1 to 160 of the nucleic acid of SEQ ID NO: 2 or a variant thereof, while the 3' end is located at positions 290 to 310 of the nucleic acid of SEQ ID NO: 2 or a variant thereof.

[0017] In some exemplary embodiments, the CP includes a TATA box and / or a starter (Inr).

[0018] In some exemplary embodiments, the 5' end of the promoter is located at positions 1 to 160 of the nucleic acid of SEQ ID NO: 2 or a variant thereof, while the 3' end is located at positions 340 to 350 of the nucleic acid of SEQ ID NO: 2 or a variant thereof.

[0019] In some exemplary embodiments, the transgene contains nucleic acids encoding proteins that maintain or improve the physiological function of rod cells.

[0020] In some exemplary embodiments, the transgene: (i) comprises a functional fragment or variant thereof encoding a human rod-shaped nucleotide-gated β subunit (hCNGB1), ABCA4, AIPL1, BEST1, CACNA1F, CLN3, CLRN1, CNGA1, CEP290, CRB1, CRB2, CRX, GPR98, GUCA1A, GUCA1B, MYO7A, NRL, PDE6A, PDE6B, PRPH2, PROM1, RHO, ROM1, RP1, RP2, RPE65, RPGR, SAG, USH1C, USH1G, USH2A, or thereof. The nucleic acid; a nucleic acid encoding a miRNA or shRNA that targets the mRNA, the mRNA encoding a dominant inactivating mutant thereof; and / or a nucleic acid encoding an antibody or antibody-binding fragment that specifically binds to the dominant inactivating mutant thereof; or (ii) a nucleic acid containing a protein, preferably a toxin, that inhibits rod cell proliferation; a prodrug converting enzyme, such as thymidine kinase; a cell cycle inhibitor, such as retinoblastoma protein (pRB), p53, p21CIP1, p27KIP1, and p57KIP2; a nucleic acid containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof and / or a nucleic acid containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof.

[0021] In some exemplary embodiments, hCNGB1 comprises an amino acid sequence according to SEQ ID NO: 3, SEQ ID NO: 40 or SEQ ID NO: 41, or a variant thereof.

[0022] In some exemplary embodiments, the polynucleotide includes one or more other nucleotide sequence elements selected from: (i) a polyadenylation signal (PAS); and / or (ii) one or two inverted terminal repeat (ITR) sequences; and / or (iii) a viral nucleotide sequence necessary for forming an infectious viral vector, preferably an adenovirus, retrovirus, lentivirus, vaccinia virus, or herpesvirus vector, particularly a herpes simplex virus (HSV) vector.

[0023] In some exemplary embodiments, the polyadenylation signal comprises, is substantially composed of, or is composed of simian virus 40PAS.

[0024] In some exemplary embodiments, the polyadenylation signal comprises a nucleic acid according to SEQ ID NO: 4 or a functional variant thereof, consists substantially of a nucleic acid according to SEQ ID NO: 4 or a functional variant thereof, or consists of a nucleic acid according to SEQ ID NO: 4 or a functional variant thereof.

[0025] In some exemplary embodiments, the ITR sequence is adeno-associated virus (AAV) ITR.

[0026] In some exemplary embodiments, AAV is AVV serotype 2, 5, 8, or 9.

[0027] In some exemplary embodiments, the promoter and transgene are side-linked with an L-ITR at their 5' end and with an R-ITR at their 3' end.

[0028] In some exemplary embodiments, the L-ITR comprises a sequence according to SEQ ID NO: 5 or a variant thereof, consists substantially of a sequence according to SEQ ID NO: 5 or a variant thereof, or consists of a sequence according to SEQ ID NO: 5 or a variant thereof, and / or the R-ITR comprises a sequence according to SEQ ID NO: 6 or a variant thereof, consists substantially of a sequence according to SEQ ID NO: 6 or a variant thereof, or consists of a sequence according to SEQ ID NO: 6 or a variant thereof.

[0029] In some exemplary embodiments, the total length of the polynucleotide is 5200 bases or less, preferably 5100 bases or less, and more preferably 5000 bases or less.

[0030] In a second aspect, the present invention also relates to plasmids comprising the polynucleotides of the first aspect.

[0031] In some exemplary embodiments, the plasmid comprises a nucleic acid sequence or a variant thereof according to SEQ ID NO: 7, SEQ ID NO: 42 to SEQ ID NO: 44.

[0032] A third aspect of the invention relates to a viral vector comprising the polynucleotides of the first aspect of the invention.

[0033] In some exemplary embodiments, the virus is selected from AAV2, AAV5, AAV8, AVV9 or variants thereof.

[0034] The fourth aspect of the invention relates to polynucleotides according to the first aspect of the invention, plasmids according to the second aspect of the invention, and / or viral vectors according to the third aspect of the invention, which are used as pharmaceuticals.

[0035] The fifth aspect of the invention relates to a pharmaceutical composition comprising a polynucleotide according to the first aspect of the invention, a plasmid according to the second aspect of the invention, and / or a viral vector according to the third aspect of the invention, and a pharmaceutically acceptable carrier.

[0036] The sixth aspect of the invention relates to polynucleotides according to the first aspect of the invention, plasmids according to the second aspect of the invention, and / or viral vectors according to the third aspect of the invention, for the treatment of retinal diseases, particularly retinal degeneration.

[0037] In some exemplary embodiments, the route of administration is selected from intraocular, intraocular, intravitreal, or subretinal.

[0038] In some exemplary implementations, retinal degeneration is associated with genetic mutations, replacements, and / or deletions.

[0039] In some exemplary embodiments, retinal degeneration is selected from night blindness, blindness, retinal degeneration, retinal dystrophy, and retinitis pigmentosa.

[0040] In some exemplary embodiments, the retinitis pigmentosa is CNGB1-associated retinitis pigmentosa or retinitis pigmentosa type 45 (RP45).

[0041] The seventh aspect of the present invention relates to a polynucleotide comprising, in the following order:

[0042] a) A human rhodopsin promoter comprising a nucleic acid sequence according to SEQ ID NO: 9 or a variant thereof; and

[0043] b) At least one transgene (TG) operatively linked to the promoter of a).

[0044] In some exemplary embodiments, the transgene contains nucleic acids encoding proteins that maintain or improve the physiological function of rod cells.

[0045] In some exemplary embodiments, the transgene: (i) comprises a functional fragment or variant thereof encoding a human rod-shaped nucleotide-gated β subunit (hCNGB1), ABCA4, AIPL1, BEST1, CACNA1F, CLN3, CLRN1, CNGA1, CEP290, CRB1, CRB2, CRX, GPR98, GUCA1A, GUCA1B, MYO7A, NRL, PDE6A, PDE6B, PRPH2, PROM1, RHO, ROM1, RP1, RP2, RPE65, RPGR, SAG, USH1C, USH1G, USH2A, or thereof. The nucleic acid; a nucleic acid encoding a miRNA or shRNA that targets the mRNA, the mRNA encoding a dominant inactivating mutant thereof; and / or a nucleic acid encoding an antibody or antibody-binding fragment that specifically binds to the dominant inactivating mutant thereof; or (ii) a nucleic acid containing a protein, preferably a toxin, that inhibits rod cell proliferation; a prodrug converting enzyme, such as thymidine kinase; a cell cycle inhibitor, such as retinoblastoma protein (pRB), p53, p21CIP1, p27KIP1, and p57KIP2; a nucleic acid containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof and / or a nucleic acid containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof.

[0046] In some exemplary embodiments, the polynucleotide comprises one or more other nucleotide sequence elements selected from the following:

[0047] (i) Polyadenylation signal (PAS);

[0048] (ii) One or two inverted terminal repeat (ITR) sequences; and

[0049] (iii) Forming an infectious viral vector, preferably an adenovirus, retrovirus, lentivirus, vaccinia virus or herpesvirus vector, especially the viral nucleotide sequence necessary for a herpes simplex virus (HSV) vector.

[0050] In some exemplary embodiments, the polyadenylation signal comprises simian virus 40PAS.

[0051] In some exemplary embodiments, the ITR sequence is adeno-associated virus (AAV) ITR.

[0052] In some exemplary embodiments, AAV is AVV serotype 2, 5, 8, or 9.

[0053] The eighth aspect of the present invention relates to a viral vector comprising a polynucleotide according to the seventh aspect of the present invention.

[0054] In some exemplary embodiments, the virus is selected from AAV2, AAV5, AAV8, AVV9 or variants thereof.

[0055] The ninth aspect of the invention relates to a method for treating retinal degeneration in a subject in need, comprising administering to the subject a therapeutically effective amount of a polynucleotide according to the seventh aspect of the invention or a viral vector according to the eighth aspect of the invention.

[0056] In some exemplary embodiments, the polynucleotide or viral vector contains the nucleic acid sequence shown in SEQ ID NO: 43.

[0057] The tenth aspect of the invention relates to a method for treating retinitis pigmentosa in a subject in need, comprising administering to the subject a therapeutically effective amount of a polynucleotide according to the seventh aspect of the invention or a viral vector according to the eighth aspect of the invention.

[0058] In some exemplary embodiments, the polynucleotide or viral vector contains the nucleic acid sequence shown in SEQ ID NO: 43.

[0059] The eleventh aspect of the present invention relates to a method for treating retinal degeneration in a subject in need, wherein the retinal degeneration is characterized by a defect or absence of CNGB1 in the retinal cells of the subject, the method comprising administering to the subject a therapeutically effective amount of a viral vector containing the nucleic acid sequence shown in SEQ ID NO: 43.

[0060] In some exemplary embodiments, the retinal degeneration is CNGB1-associated retinitis pigmentosa or retinitis pigmentosa type 45 (RP45).

[0061] The twelfth aspect of the present invention relates to a method for treating CNGB1-associated retinitis pigmentosa or retinitis pigmentosa type 45 (RP45) in a subject in need, the method comprising administering a therapeutically effective amount of a viral vector containing the nucleic acid sequence shown in SEQ ID NO: 43 under the subject's retina.

[0062] The thirteenth aspect of the present invention relates to a polynucleotide comprising, in the following order:

[0063] a) A promoter comprising a human rod photoreceptor cell-specific promoter element (hRPSPE) and a core promoter (CP), wherein the promoter element comprises a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof; and

[0064] b) A transgene encoding the human rod-shaped nucleotide-gated channel β subunit (hCNGB1), which is operatively linked to the promoter of a).

[0065] The variant of SEQ ID NO:1 contains one or more nucleic acid substitutions other than positions 6 to 13, 32 to 40, 70 to 83, and 87 to 94 of the nucleotide in SEQ ID NO:1.

[0066] The fourteenth aspect of the present invention relates to a pharmaceutical composition comprising a polynucleotide and a pharmaceutically acceptable carrier, wherein the polynucleotide comprises, in the following order:

[0067] a) A promoter comprising a human rod photoreceptor cell-specific promoter element (hRPSPE) and a core promoter (CP), wherein the promoter element comprises a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof; and

[0068] b) A transgene encoding the human rod-loop nucleotide-gated channel β subunit (hCNGB1), which is operatively linked to the promoter of a);

[0069] The variant of SEQ ID NO:1 contains one or more nucleic acid substitutions other than positions 6 to 13, 32 to 40, 70 to 83, and 87 to 94 of the nucleotide in SEQ ID NO:1.

[0070] The fifteenth aspect of the present invention relates to a pharmaceutical composition comprising a viral vector and a pharmaceutically acceptable carrier, the viral vector comprising the nucleic acid sequence shown in SEQ ID NO: 43. Attached Figure Description

[0071] The contents of the accompanying drawings included in this specification are described below. In the context, please also refer to the detailed description of the invention above and / or below.

[0072] Figure 1 The structure of the rAAV hRHO194.hCNGB1 vector genome was shown.

[0073] Figure 2 The image shows the pGL2.0-hRHO194-hCNGB1a-SV40 cis vector plasmid.

[0074] Figures 3A to 3B Representative confocal images depicting native eGFP fluorescence in wild-type mice treated with a vector expressing eGFP instead of hCNGB1 were created. These representative confocal images show native eGFP fluorescence in cross-sections of the retina of 8-week-old wild-type mice treated subretinally with the rAAV.hRHO194.eGFP vector at week 4. Strong rod-specific eGFP signal was observed in the treated animals. Figure 3A However, this was not observed in the uninjected control group. Figure 3B ).

[0075] Figures 4A to 4B Representative ERG measurements of CNGB1(- / -) mice treated with the vector according to the invention are shown; electroretinal regography (ERG) measurements of CNGB1(- / -) mice treated with the vector according to the invention in one eye are also shown. Figure 4A Representative ERG traces obtained under 4.4 cd / m² single flash stimulation. Shaded lines are from the treated eyes of CNGB1(- / -) mice 4 months post-treatment, and black lines are from the untreated eyes. Figure 4B The graph shows a summary of ERG b-wave amplitudes measured under the same conditions in wild-type mice (gray), treated CNGB1(- / -) mice (dark gray), and untreated CNGB1(- / -) mice (black). *p<0.05, Student's t-test, N=4.

[0076] Figures 5A to 5C Optical coherence tomography (OCT) measurements of the photoreceptor layer thickness in a CNGB1(- / -) mouse treated in one eye with the carrier according to the invention are shown. Figures 5A to 5B ) from processed ( Figure 5A ) and untreated eyes ( Figure 5B A representative OCT scan of the photoreceptor layer. The thickness of the photoreceptor layer is marked with vertical black bars. The thickness of the photoreceptor layer is quantified using OCT. Figure 5C ). ***p<0.001, 1-factor ANOVA, N=9.

[0077] Figures 6A to 6B Depicting the treatment using a carrier according to the invention ( Figure 6A ) or not processed ( Figure 6B Representative confocal images of immunohistochemical staining of hCNGB1 in CNGB1(- / -) mice.

[0078] Figure 7 A schematic diagram illustrating a conventional carrier design according to the present invention is depicted.

[0079] Figures 8A to 8B Representative ERG measurements of CNGB1(- / -) mice treated with the vector according to the present invention are shown. Figure 8A Representative ERG measurements in wild-type and CNGB1(- / -) mice before treatment (). Figure 8B ).

[0080] Figures 9A to 9B The text describes the effects of treating CNGB1(- / -) mice with the vector according to the present invention. Figure 9A ) and untreated mice ( Figure 9BRepresentative confocal images of hCNGB1 immunohistochemical staining in ().

[0081] Figures 10A to 10C An OCT analysis revealing a significant delay in retinal degeneration was described. The conventional injection schedule of the vector according to the invention (…) Figure 10A In CNGB1(- / -) mice treated with the vector of the present invention ( Figure 10B ) and untreated mice ( Figure 10C OCT images collected at 9 months.

[0082] Figures 11A to 11E The recovery of rod cell function was depicted at two months in CNGB1(- / -) mice treated with the vector according to the invention. Representative ERG B-wave measurements were performed in CNGB1(- / -) mice treated with the vector of the invention and untreated mice. Figure 11A A summary plot showing the ERG b-wave amplitude measured in response to -0.5 log light stimulation (cd / m2) in CNGB1(- / -) mice treated with the carrier of the present invention and untreated mice is displayed. Figure 11B CNGB1(- / -) mice treated with the vector according to the present invention ( Figure 11C ) and untreated mice ( Figure 11D OCT measurements of the thickness of the photoreceptor layer. OCT was used to quantify the thickness of the photoreceptor layer. Figure 11E N = 6.

[0083] Figures 12A to 12B Depicting the use of rod cell-specific stimulation ( Figure 12A ) and flicker response ( Figure 12B In the case of CNGB1(- / -) dogs treated with the carrier of the present invention and untreated dogs, significant ERG rescue was observed.

[0084] Figures 13A to 13B Visual acuity test data are depicted, showing that CNGB1(- / -) dogs treated with the carrier according to the invention possess rod-mediated visual acuity and improved visual acuity test performance. Correct Exit Selection ( Figure 13A ) and exit time ( Figure 13B The improved performance indicates the recovery of rod vision.

[0085] Figure 14 depicts the ERG measurements, showing the improvement in A-wave and B-wave amplitudes in CNGB1(- / -) dogs treated with the carrier according to the invention. An improvement of more than 1.5 log units in the response threshold indicated by A-wave amplitude was found in the treated eyes. Figure 14A It was found that, in the eyes of the material, the response threshold indicated by the B-wave amplitude improved by more than 2 log units. Figure 14B ).

[0086] sequence list

[0087] The sequence of a 99-nucleotide-long fragment of the human rhodopsin promoter SEQ ID NO:1, which contains core tissue-specific elements;

[0088] The sequence of a 350-nucleotide-long fragment of the human rhodopsin promoter SEQ ID NO:2, which contains tissue-specific elements and transcription initiation sites;

[0089] The sequence of the human CNGB1 protein, SEQ ID NO:3;

[0090] The sequence of SEQ ID NO:4, the polyadenylation signal SV40.

[0091] SEQ ID NO:5 Left-inverted terminal repeat (L-ITR);

[0092] SEQ ID NO:6 Right-inverted terminal repeat (R-ITR);

[0093] SEQ ID NO:7 Vector construct: sequence of pGL2.0-hRho194-hCNGB1a-SV40;

[0094] The sequence of the CNGB1 gene in SEQ ID NO:8;

[0095] The sequence of the 194bp fragment of the human rhodopsin promoter SEQ ID NO:9.

[0096] SEQ ID NO:10 Sequence of human Abca4 protein;

[0097] The sequence of human AIPL1 protein, SEQ ID NO:11;

[0098] The sequence of human BEST1 protein, SEQ ID NO:12;

[0099] The sequence of human CACNA1F protein, SEQ ID NO:13;

[0100] SEQ ID NO:14 Sequence of human CLN3 protein;

[0101] SEQ ID NO:15 Sequence of human CLRN1 protein;

[0102] The sequence of human CNGA1 protein, SEQ ID NO:16;

[0103] The sequence of human CEP290 protein, SEQ ID NO:17;

[0104] SEQ ID NO:18 Sequence of human CRB1 protein;

[0105] SEQ ID NO:19 Sequence of human CRB2 protein;

[0106] SEQ ID NO:20 Sequence of human CRX protein;

[0107] The sequence of human GPR98 protein, SEQ ID NO:21;

[0108] The sequence of human GUCA1A protein, SEQ ID NO:22;

[0109] The sequence of human GUCA1B protein, SEQ ID NO:23;

[0110] SEQ ID NO:24 Sequence of human MYO7A protein;

[0111] The sequence of human NRL protein, SEQ ID NO:25;

[0112] The sequence of human PDE6A protein, SEQ ID NO:26;

[0113] The sequence of human PDE6B protein, SEQ ID NO:27;

[0114] The sequence of human PRPH2 protein, SEQ ID NO:28;

[0115] SEQ ID NO:29 Sequence of human PROM1 protein;

[0116] SEQ ID NO:30 Sequence of human RHO protein;

[0117] The sequence of human ROM1 protein, SEQ ID NO:31;

[0118] The sequence of human RP1 protein, SEQ ID NO:32;

[0119] The sequence of human RP2 protein, SEQ ID NO:33;

[0120] The sequence of human RPGR protein, SEQ ID NO:34;

[0121] The sequence of human SAG protein, SEQ ID NO:35;

[0122] The sequence of the human USH1C protein, SEQ ID NO:36;

[0123] The sequence of human USH1G protein, SEQ ID NO:37;

[0124] The sequence of the human USH2A protein, SEQ ID NO:38;

[0125] The sequence of human NR2E3 protein, SEQ ID NO:39;

[0126] SEQ ID NO:40 Sequence of human CNGB1 protein (next-generation sequencing; NGS);

[0127] The sequence of human CNGB1 protein (SEQ ID NO:41) (GenBank NG_016351);

[0128] SEQ ID NO:42 The sequence of the 5'ITR-hRHO promoter-CNGB1a-SV40polyA-3'ITR;

[0129] SEQ ID NO:43 The sequence of the 5'ITR-hRHO promoter-CNGB1a-SV40polyA-3'ITR(NGS);

[0130] The sequence of SEQ ID NO:44 5'ITR-hRHO promoter-CNGB1a-SV40polyA-3'ITR (GenBank); and

[0131] SEQ ID NO:45 Sequence of human RPE65 protein. Detailed Implementation

[0132] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as they can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0133] Several references are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions for use, etc.) is incorporated herein by reference in its entirety, both above and below. Nothing herein should be construed as an admission that the invention is not entitled to claim prior invention over such disclosure. Some of the references cited herein are characterized as "incorporated by reference." In the event of any conflict between the definitions or teachings in such incorporated references and those cited herein, the text of this specification shall prevail.

[0134] The elements of the invention will be described below. These elements are listed along with specific embodiments, but it should be understood that they can be combined in any manner and in any number to create additional embodiments. The embodiments and preferred embodiments described differently should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, it should be considered that any permutation and combination of the elements described herein is disclosed in the description of this application.

[0135] definition

[0136] To carry out this invention, unless otherwise stated, conventional methods of chemical, biochemical, and recombinant DNA techniques are used, as explained in the literature in this field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0137] The following sections provide definitions for some terms that are frequently used in this specification. In the remainder of the specification, these terms have their defined and preferred meanings in each instance of their use.

[0138] As used in this specification and the appended claims, the absence of a quantifier before an element includes plural references, unless otherwise expressly stated in the text.

[0139] As used in this specification, the term "nucleic acid" includes polymers or oligomers, or large biomolecules, essential for all known forms of life. Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are composed of monomers called nucleotides. Most naturally occurring DNA molecules consist of two intertwined, complementary biopolymer chains forming a double helix. DNA chains are also called polynucleotides, which are composed of nucleotides. Each nucleotide consists of a nitrogenous nucleotide base and a monosaccharide called deoxyribose or ribose, and a phosphate group. Naturally occurring nucleotide bases include guanine (G), adenine (A), thymine (T), uracil (U), or cytosine (C). Nucleotides are linked together in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, forming an alternating sugar-phosphate backbone. If the sugar is deoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Typically, polynucleotides are formed by phosphodiester bonds between the individual nucleotide monomers. In the context of this invention, the term "nucleic acid" includes, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as RNA-DNA hybrids (intra-stranded), as well as cDNA, genomic DNA, recombinant DNA, cRNA, and mRNA. Nucleic acids can consist of an entire gene or a portion thereof, and can also be miRNA, siRNA, piRNA, or shRNA. miRNAs are short ribonucleic acid (RNA) molecules with an average length of 22 nucleotides, but may be longer than 22 nucleotides, and are present in all eukaryotic cells, i.e., plants, animals, and some viruses. They play a role in the transcriptional and post-transcriptional regulation of gene expression. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNA), typically leading to translational repression and gene silencing. Small interfering RNAs (siRNAs), sometimes also called short interfering RNAs or silencing RNAs, are short ribonucleic acid (RNA) molecules with a length of 20 to 25 nucleotides. They participate in the RNA interference (RNAi) pathway, in which they interfere with the expression of specific genes. Short hairpin RNA (shRNA), also known as small hairpin RNA, is an artificial RNA molecule with a tight hairpin loop that can be used to silence target gene expression via RNA interference (RNAi). shRNA expression in cells is typically accomplished through plasmid delivery or via viral vectors.

[0140] When used in the context of this invention, the term "polynucleotide" refers to a nucleic acid, not limited to a specific number of nucleotides.

[0141] In the context of this invention, the term "human rod photoreceptor" refers to a special type of cell, namely, a photoreceptor cell. The retina of the human eye contains two types of photoreceptor cells: rod cells and cone cells. On average, there are approximately 90 million rod cells in the human retina. Rods are more sensitive than cones. However, they are not sensitive to color. They are responsible for dark adaptation, or scotopic vision. Rod cells are typically found concentrated at the outer edge of the retina and are used for peripheral vision. Therefore, peripheral vision is more sensitive to light, enabling people to see darker objects in peripheral vision. Rod cells are more sensitive than cone cells and are almost entirely responsible for night vision. Rod cells use a photosensitive pigment called rhodopsin. Photoreceptor cells are highly specialized, light-sensitive neurons designed to capture light quanta, thereby triggering changes in cell membrane potential. Rod photoreceptors enable dim vision, while cone photoreceptors mediate color vision and high visual acuity under brighter light conditions. In the retinas of vertebrates, including mice and humans, only one type of rod photoreceptor cell exists, carrying the visual pigment rhodopsin. When in an "activated" state, each opsin molecule covalently binds to the photosensitive chromophore 11-cis-retinal. Upon photon capture, the chromophore isomerizes to all-trans-retinal, leading to a conformational change in rhodopsin and activation into interstitial rhodopsin II. This initiates phototransduction, a series of biochemical events ultimately resulting in the closure of ion channels in the cell membrane, causing hyperpolarization of the photoreceptor cell and transmitting signals to secondary neurons in the inner retina by regulating the release of neurotransmitters at synaptic terminals. The integrity and function of photoreceptor cells are absolutely crucial for vision; mutations affecting photoreceptor function or survival disrupt phototransduction, leading to vision loss.

[0142] In the context of this invention, the term "promoter" refers to a nucleotide sequence containing elements necessary for transcriptional control (including binding sites for transcriptional activators and repressors) and elements that initiate transcription. The binding sites for transcriptional activators and / or repressors are typically located directly upstream of or at the 5' end of the transcription start site within the core promoter. Thus, RNA polymerase and necessary transcription factors bind to the promoter sequence and initiate transcription. The promoter sequence determines the direction of transcription and indicates which DNA strand is transcribed; this strand is called the sense strand. The promoter of this invention specifically transfers rod photoreceptor cells to a transgene located downstream, i.e., at the 3' end of the promoter.

[0143] The term “core promoter” (CP), used herein in its usual sense, refers to a nucleotide region of DNA containing a regulatory sequence derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Thus, the core promoter is the smallest part of the promoter required to properly initiate gene transcription and contains the binding site for RNA polymerase (RNA polymerase I, RNA polymerase II, or RNA polymerase III). The RNA polymerase binding site of the CP is located approximately 25 to 35 bases upstream (5') of the transcription start site. The core promoter may contain a so-called TATA box (also known as the Goldberg-Hogness box), a DNA sequence (cis-regulatory element) commonly found in the promoter regions of archaea and eukaryotic genes. The TATA box has a core DNA sequence 5'-TATAAA-3' or a variant thereof, typically accompanied by three or more adenine bases. The TATA box is usually located 25 to 35 base pairs upstream of the transcription start site. Core promoters can also be without a TATA box. Genes lacking the TATA box use initiator elements or downstream core promoters instead. Core promoters may also contain an initiator (Inr). The Inr consists of an initiator motif and functions similarly to the TATA box. Inr elements promote binding to transcription factor IID (TFIID).

[0144] In the context of this invention, the term "human rod photoreceptor-specific promoter element" (hRPSPE) refers to a promoter element that mediates downstream transgene transcription only in rod cells, particularly human rod cells. The use of tissue-specific promoters allows for tissue-specific expression of proteins or functional RNAs in the retinal cells of the human eye. hRPSPEs contain only portions or fragments of naturally occurring human rod photoreceptor promoter sequences.

[0145] In the context of this invention, the terms "gene," "coding sequence," or "sequence encoding a specific protein or peptide" refer to a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo. The boundaries of a gene are determined by a start codon at the 5' (i.e., amino) end and a translation stop codon at the 3' (i.e., carboxyl) end. The term gene includes, but is not limited to, prokaryotic or eukaryotic mRNA, cDNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is typically located at the 3' end of the gene sequence.

[0146] In the context of this invention, the term "transgenic" refers to a gene removed from its natural background and placed under the expression control of a heterologous promoter. An example of a transgenic gene of this invention is the "rod-cyclic nucleotide-gated channel β" (CNGB1) gene, which encodes a subunit of the rod-cyclic nucleotide-gated channel β. In other embodiments, the transgenic may include transgenes of the following human proteins: ATP-binding box subfamily A member 4 (ABCA4), aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), vitrectomycin 1 (BEST1), calcium voltage-gated channel subunit α1F (CACNA1F), neuronal ceroid lipofuscin deposition 3 (CLN3), Clarin1 (CLRN1), cyclic nucleotide-gated channel α1 (CNGA1), centrosome protein 290 (CEP290), Crumbs 1 (CRB1), and Crumbs... 2 (CRB2), cone-rod homeobox (CRX), G protein-coupled receptor 98 (GPR98), guanylate cyclase activator 1A (GUCA1A), guanylate cyclase activator 1B (GUCA1B), myosin VIIA (MYO7A), nuclear receptor subfamily 2 group E member 3 (NR2E3), neuroretinal leucine zipper (NRL), phosphodiesterase 6A (PDE6A), phosphodiesterase 6B (PDE6B), peripheral protein 2 (PRPH2), prominin 1. PROM1, rhodopsin (RHO), retinal outer segment membrane protein 1 (ROM1), retinitis pigmentosa protein 1 (RP1), retinitis pigmentosa protein 2 (RP2), retinal pigment epithelium-specific protein 65 (RPE65), retinitis pigmentosa GTPase regulatory protein (RPGR), S antigen visual repressor protein (SAG), Usher syndrome type 1C protein (USH1C), Usher syndrome type 1G protein (USH1G), Usher syndrome type 2A protein (USH2A), or functional fragments or variants thereof. The amino acid sequences of specific embodiments of the above proteins are indicated in SEQ ID NO: 10 to SEQ ID NO: 41 and SEQ ID NO: 45. Functional fragments are those that maintain the function of the corresponding proteins in the normal function of rod photoreceptors. Similarly, variants also maintain the function of the individual proteins in rod photoreceptors. Proteins with long amino acid sequences, such as human CACNA1F, CEP290, GPR98, MYO7A, RP1, and USH2A, are too long to be encoded by transgenes. These transgenes can be delivered via specially selected vector systems, particularly AAV vectors. To accommodate the size limitations of AAV vectors, a "split vector" technique can be used, which employs an intepitide-mediated splitting system developed for gene therapy. By using split intepitides, the packaging limitations of AAVs can be bypassed.Therefore, each half of the target transgene can fuse to the corresponding splitting integrin moiety, and integrin-mediated transsplicing only occurs during co-expression, allowing for the reconstruction of the complete transgene protein. Thus, it is possible to construct vectors encoding two transgene protein fragments that assemble into a full-length functional protein in target cells during co-transduction.

[0147] As used in the context of this application, the term "CNGB1" refers to the gene or the protein encoded by the CNGB1 gene, namely a cGMP-gated cation channel in rod photoreceptors that helps regulate ion inflow into the outer segment of rod photoreceptors in response to light-induced changes in intracellular cGMP levels. This channel consists of two subunits, α and β, with the protein encoded by the gene representing the β subunit. Diseases associated with CNGB1 and defects in this gene include retinitis pigmentosa and CNGB1-associated retinitis pigmentosa. The CNGB1 subunit of the cyclic nucleotide-gated channel plays an important role in both visual and olfactory signal transduction. When bound to CNGA1, it participates in regulating ion inflow into the outer segment (ROS) of rod photoreceptors in response to light-induced changes in intracellular cGMP levels.

[0148] As used herein, the term "proliferation" refers to an increase in the number of cells, manifested as cell growth and cell division that may result in increased or decreased cell proliferation. Hyperproliferative disorders are characterized by widespread cell proliferation, in which cell division is increased relative to normal tissue. Such disorders are characterized by abnormal proliferation (production), i.e., excessive cell production. Hyperproliferative disorders include neoplastic diseases. Neoplastic diseases can include benign or malignant tumors, with malignant tumors being referred to as cancer. The term hyperproliferative disease includes both cancer and precancerous diseases. In a particular embodiment, a hyperproliferative disease is a disease of excessive proliferation of rod cells, particularly retinoblastoma.

[0149] The term "amino acid" generally refers to any monomeric unit containing a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups, or analogs of any of these groups. As used herein, the term "amino acid" includes the following twenty naturally encoded or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). Where the “X” residue is undefined, these should be defined as “any amino acid”. The structures of these twenty natural amino acids are shown, for example, in Strayer et al., Biochemistry, 5th edition, Freeman and Company (2002). Other amino acids, such as selenocysteine ​​and pyrrolysine, can also be genetically encoded (Stadtman (1996) “Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducing pyrrolysine,” CurrBiol. 12(13):R464-R466). Amino acids can be linked by peptide bonds to form peptides or polypeptides.

[0150] In the context of this invention, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins, but is typically shorter. The shortest peptide is a dipeptide, consisting of two amino acids linked by a single peptide bond. Tripeptides, tetrapeptides, pentapeptides, etc., can also exist. Typically, peptides have a length of up to 8, 10, 12, 15, 18, or 20 amino acids. Peptides have an amino terminus and a carboxyl terminus, unless they are cyclic peptides.

[0151] In the context of this invention, the term "peptide" refers to a single linear chain of amino acids linked together by peptide bonds and typically contains at least about 21 amino acids. A polypeptide can be one chain of a protein composed of more than one chain, or if a protein is composed of a single chain, the polypeptide can be the protein itself.

[0152] As used herein, the term "fragment" refers to naturally occurring fragments (e.g., splice variants) as well as artificially constructed fragments, particularly those obtained through genetic engineering. Typically, compared to the parental polypeptide, the fragment is located at the N-terminus and / or C-terminus and / or internally, preferably at the N-terminus, at both the N- and C-terminus, or at the C-terminus with a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65. 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acids.

[0153] As used herein, the term "variant" should be understood as a polypeptide or polynucleotide that differs from a polypeptide or polynucleotide derived by one or more variations in length or sequence. Polypeptides or polynucleotides from which variants of the polypeptide or polynucleotide are derived are also called parental polypeptides or parental polynucleotides. The term "variant" includes "fragments" or "derivatives" of the parent molecule. Typically, a "fragment" is shorter or smaller than the parent molecule, while a "derivative" has one or more sequence differences compared to the parent molecule. Variants also include modified molecules, such as, but not limited to, post-translational modified proteins (e.g., glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. The term "variant" also includes mixtures of different molecules, such as, but not limited to, RNA-DNA hybrids. Typically, variants are artificially constructed, preferably by genetic technology, and the parent protein or parent polynucleotide is a wild-type protein or wild-type polynucleotide, or a common sequence thereof. However, naturally occurring variants should also be understood to be included in the term "variant" as used herein. Furthermore, variants that can be used in this invention may also be derived from homologs, orthologs, or paralogs of the parent molecule, or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent molecule, i.e., the variant has functional activity.

[0154] Specifically, the terms "peptide variant" or "polypeptide variant" should be understood as a peptide, polypeptide, or protein, distinct from peptides, polypeptides, or proteins derived through one or more changes in their amino acid sequence. Peptides, polypeptides, or proteins from which peptide, polypeptide, or protein variants are derived are also called parental peptides, parental polypeptides, or parental proteins. Furthermore, variants usable in this invention can also be derived from homologs, orthologs, or paralogs of parental peptides, parental polypeptides, or parental proteins, or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parental peptide, parental polypeptide, or parental protein. Changes in the amino acid sequence can be amino acid substitutions, insertions, deletions, N-terminal truncation, C-terminal truncation, or any combination of these changes, and can occur at one or more sites. Peptide, polypeptide, or protein variants may exhibit a total of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) amino acid sequence variations (i.e., substitutions, insertions, deletions, N-terminal truncation, and / or C-terminal truncation). Amino acid substitutions may be conserved and / or non-conserved. Alternatively or additionally, as used herein, a “variant” can be characterized by a degree of sequence identity with the parent peptide, parent polypeptide, or parent protein from which it is derived. More specifically, the peptide variants, polypeptide variants, or protein variants described in this invention exhibit at least 80% sequence identity with their parent peptide, parent polypeptide, or parent protein. The sequence identity of the peptide variants, polypeptide variants, or protein variants covers a continuous segment of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, or more than 100 amino acids.

[0155] The "sequence identity percentage" is determined by comparing two best-aligned sequences within a comparison window. This comparison window can include additions or deletions (i.e., gaps) compared to a reference sequence (excluding additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate a number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0156] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" means that two or more sequences or subsequences are identical, i.e., contain the same nucleotide or amino acid sequence. When using one of the following sequence comparison algorithms or measuring a specified region by manual alignment and visual inspection, or comparing and aligning maximum correspondences within a comparison window, sequences are considered "substantially identical" if they have a specific percentage of identical nucleotide or amino acid residues (e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity in the aligned region). These definitions also relate to the complementary sequence of the test sequence. Therefore, regarding polypeptide and polynucleotide sequence comparisons, the term "at least 80% sequence identity" is used throughout the specification. The expression preferably refers to having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding reference polypeptide or the corresponding reference polynucleotide.

[0157] The term "sequence comparison" refers to a method of comparing a test sequence to a reference sequence, where one sequence serves as the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. Default program parameters are typically used, but alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity or similarity between the test sequence and the reference sequence based on the program parameters. When comparing two sequences and no reference sequence is specified for calculating the percentage of sequence identity, the longer of the two sequences is referenced unless otherwise specified. If a reference sequence is specified, but no other parameters are specified, the sequence identity is determined based on the full length of the reference sequence indicated by the SEQ ID.

[0158] In the context of this invention, "operably linked" refers to the arrangement of elements in which the described components are configured to perform their usual function. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter affects the transcription of one or more transgenes, it is operably linked to one or more transgenes. Furthermore, control elements operably linked to a coding sequence enable the expression of the coding sequence. Control elements need not be adjacent to the coding sequence, as long as they serve to guide its expression. Therefore, for example, there may be an untranslated but still transcribed sequence between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0159] As used herein, the term "polyadenylation signal" (PAS) refers to a sequence involved in the production of mature messenger RNA (mRNA) for translation. Therefore, it constitutes part of a larger gene expression process. The process of polyadenylation begins with the termination of gene transcription. The 3' terminal fragment of the newly prepared precursor mRNA is first cleaved by a group of proteins; these proteins then synthesize a poly(A) tail at the 3' end of the RNA. In some genes, these proteins add a poly(A) tail at one of several possible sites. Thus, similar to alternative splicing, polyadenylation can produce more than one transcript from a single gene (selective polyadenylation). The poly(A) tail is important for nuclear export, translation, and stability of mRNA. The tail shortens over time, and when it is short enough, the mRNA is degraded by enzymes. However, in some cell types, mRNA with a short poly(A) tail is stored for later activation in the cytoplasm through re-polyadenylation. The PAS of the present invention may contain a nucleic acid encoding a polyadenylation signal (SV 40PAS) of simian virus 40 (SV40). This modification of the polynucleotide has the advantage of significantly enhancing the expression of a target gene, such as hCNGB1, in photoreceptor cells. The long-term expression achieved by containing the SV40 PAS qualifies this polynucleotide for use as an active gene therapeutic agent. Specifically, the PAS may contain the nucleic acid sequence according to SEQ ID NO: 4.

[0160] As used herein, the term "vector," also known as an expression construct, is typically a virus designed to express a protein in a cell. The term "vector" refers to a protein or polynucleotide or a mixture thereof capable of being introduced into a cell or capable of introducing the protein and / or nucleic acid contained therein into the cell. Examples of vectors include, but are not limited to, plasmids, phages, viruses, or artificial chromosomes. In particular, vectors are used to transfer the promoters and transgenes of the present invention into suitable host cells. Vectors may contain "replicon" polynucleotide sequences that facilitate autonomous replication of the vector within the host cell. Exogenous DNA is defined as heterologous DNA, which is DNA not naturally present in the host cell, such as replicating vector molecules, encoding selectable or screenable markers, or encoding transgenes. Once inside the host cell, the vector may replicate independently of or simultaneously with the host chromosomal DNA, and may produce several copies of the vector and its inserted DNA. Furthermore, the vector may contain essential elements that allow the inserted DNA to be transcribed into mRNA molecules or otherwise replicated into multiple copies of RNA. The vector may also contain "expression control sequences" that regulate the expression of the target gene. Typically, expression control sequences are polypeptides or polynucleotides, such as, but not limited to, promoters, enhancers, silencers, insulators, or repressors. In vectors containing more than one polynucleotide encoding one or more target gene products, expression can be controlled together or separately by one or more expression control sequences. More specifically, each polynucleotide contained in the vector can be controlled by a single expression control sequence, or all polynucleotides contained in the vector can be controlled by a single expression control sequence. Polynucleotides contained in a single vector controlled by a single expression control sequence can form an open reading frame. Some expression vectors additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and / or allow the mRNA to be translated into protein molecules. Therefore, many mRNA and polypeptide molecules encoded by the inserted DNA can be rapidly synthesized.

[0161] In the context of this invention, the term "AAV vector" refers to a complete viral particle, i.e., a straight-stranded single-stranded AAV nucleic acid genome comprising an AAV capsid protein shell. In this respect, complementary sense (i.e., "sense" or "antisense") single-stranded AAV nucleic acid molecules can be packaged into either AAV virion; both strands have the same infectivity. The AAV vector of this invention can also be an infectious and replication-defective virus composed of an AAV protein shell encapsulating a target heterologous DNA molecule (e.g., hCNGB1), which may have AAV ITRs flanking it. An exemplary AAV 5'ITR has the nucleic acid sequence according to SEQ ID NO: 5, and an exemplary AAV 3'ITR has a nucleic acid sequence complementary to SEQ ID NO: 6. The AAV vector of this invention can be generated in a suitable host cell in which the AAV vector, AAV helper functions, and additional functions have been introduced. In this way, the host cell is able to encode the AAV polypeptide required to package the AAV genome (i.e., containing the target recombinant nucleotide sequence) into a recombinant virion particle for subsequent gene delivery.

[0162] Multiple natural serotypes of adenovirus-associated virus (AAV) have been identified, including 12 human serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12) and several non-human primate serotypes. The genomic sequences of different AAV serotypes also differ, for example, in the presence of different inverted terminal repeat (ITR) sequences or sequences encoding the capsid. In the context of this invention, the term "AAV genome" refers to any nucleic acid sequence derived from an adenovirus-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-9, AAV-7, etc. An AAV genome may have one or more AAV wild-type genes, preferably with all or part of the Rep and / or Cap genes deleted, but retaining functionally flanking inverted terminal repeat ("ITR") sequences. Typically, functional ITR sequences are essential for the rescue, replication, and packaging of the AAV genome. Therefore, this document defines the AAV genome as containing at least those sequences (e.g., functional ITRs) required for cis-replication and viral packaging. The ITR need not be a wild-type nucleotide sequence and can be altered (e.g., by nucleotide insertion, deletion, or substitution) as long as the sequence provides functional rescue, replication, and packaging. The ITR may include sequences according to SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0163] In the context of this invention, "antibody" refers to a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably. Antibodies are protein molecules produced by plasma cells and used by the immune system to recognize and neutralize foreign substances, such as bacteria and viruses. Antibodies recognize a unique portion of a foreign target, i.e., an antigen.

[0164] As used herein, the term "antibody-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments covered by the term "antibody-binding fragment" include antigen-binding fragments (Fab), Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single-domain antibodies (sdAb), single-chain variable fragments (scFv), variable fragments (Fv), and V... H Structural domain, V L Domains, single-domain antibodies, nanobodies, IgNAR (immunoglobulin neoantigen receptor), dual scFv, bispecific T cell adaptor (BITE), dual affinity retargeting (DART) molecules, triantibodies, biantibodies, single-chain biantibodies, selective scaffold proteins, and their fusion proteins.

[0165] The term "pharmaceutical composition" as used in this application includes active compounds or ingredients, namely formulations of the polynucleotides, plasmids, and / or carriers of the present invention, and refers to one or a combination of substances for identifying, preventing, maintaining, or treating a tissue state or disease. Pharmaceutical compositions are formulated to be administered to a patient to prevent and / or treat a disease and / or maintain a physiological state. Additionally, a pharmaceutical composition refers to a combination of an active agent with an inert or active carrier that makes the composition suitable for therapeutic use. Pharmaceutical compositions can be formulated according to their chemical and physical properties for routes of administration such as oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous, or vaginal administration. Pharmaceutical compositions include solid, semi-solid, liquid, and transdermal therapy systems (TTS). Solid compositions are selected from tablets, coated tablets, powders, granules, pills, capsules, effervescent tablets, or transdermal therapy systems. Pharmaceutical compositions also include liquid compositions selected from solutions, syrups, infusion solutions, extracts, solutions for intravenous administration, solutions for infusion, or solutions of the carrier systems of the present invention. Semi-solid compositions that can be used in the context of this invention include emulsions, suspensions, creams, lotions, gels, beads, buccal tablets, and suppositories.

[0166] "Pharmaceutical acceptable" refers to a drug approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more particularly in humans.

[0167] The term "carrier" as used in this specification includes components capable of delivering reagents to desired human compartments, such as components of a specific cell type, and which, upon administration via a selected route and regimen, can be used to deliver and control the release of the drug.

[0168] As used herein, routes of administration describe the absorption of exogenous substances in the human body and are categorized by the site of administration of the biological exogenous substance. In particular, in therapeutic applications, pharmaceutical compositions containing polynucleotides and / or viral vectors are administered via sites selected from the intraocular, intraocular, vitreous, or subretinal regions.

[0169] The term "disease" refers to an abnormal state, particularly an abnormal physical condition, such as illness or injury, in which cells, tissues, organs, or individuals are no longer able to perform their functions effectively. Often, but not always, a disease is associated with specific symptoms or signs that indicate its presence. Therefore, the presence of these symptoms or signs can indicate that a cell, tissue, organ, or individual is diseased. Changes in these symptoms or signs can indicate the progression of the disease. The progression of a disease is typically characterized by an increase or decrease in these symptoms or signs, which can indicate a "worsening" or "improving" state of the disease. A "worsening" state of disease is characterized by a reduced ability of cells, tissues, organs, or individuals / patients to perform their functions effectively, while a "improving" state of disease is typically characterized by an increased ability of cells, tissues, organs, or individuals / patients to perform their functions effectively. Cells, tissues, organs, or individuals that are "sensitive" to disease are particularly susceptible to disease even when they are otherwise healthy, for example, due to genetic predisposition, lack of vaccination, underdeveloped or immature immune systems, poor nutritional status, etc.

[0170] In the context of this invention, "retinal disease" refers to, but is not limited to, any kind of retinal degeneration. Retinal dystrophy is a type of retinal degeneration, a group of hereditary retinal diseases with varying degrees of severity and different inheritance patterns. Retinal dystrophy belongs to the category of retinitis pigmentosa. Retinitis pigmentosa is the most common type of retinal dystrophy, characterized by retinal pigment deposition on fundus examination, primary loss of rod photoreceptor cells, followed by secondary loss of cone photoreceptor cells. Patients typically experience night vision loss and loss of mid-peripheral visual field. As the disease progresses, patients with this disease lose the outermost peripheral visual field and eventually lose central visual field. Retinal degeneration may be associated with genetic mutations, replacements, and / or deletions. Retinal degeneration is selected from night blindness, blindness, retinal degeneration, retinal dystrophy, and retinitis pigmentosa. Retinitis pigmentosa can be CNGB1-associated retinitis pigmentosa or retinitis pigmentosa type 45 (RP45).

[0171] Other examples of retinal diseases include, but are not limited to, RPE65-mediated retinal diseases, macular degeneration (e.g., age-related macular degeneration), hereditary macular degeneration (e.g., Sturges disease), rod-cone dystrophy, cone-rod dystrophy, microstomia, Malattia Leventinese, etc.

[0172] As used in this article, “CNGB1-associated retinitis pigmentosa” refers to a class of diseases involving progressive retinal degeneration that typically begins in the mid-periphery of the retina and progresses toward the macula and fovea (Ferrani et al. (2011) Curr. Genomics 12(4): 238). Typical phenotypic symptoms include night blindness, followed by visual field reduction, leading to visual field narrowing, and eventually legal blindness, or in many cases, even complete blindness. At the cellular level, this is associated with the rod photoreceptor system, which is primarily affected. In later stages, the disease may further affect cone photoreceptors, eventually leading to complete blindness. Affected photoreceptors undergo apoptosis, which is reflected in reduced outer nuclear layer thickness within the retina, as well as fundus lesions and / or retinal pigment deposition. Patients may lose most of their photoreceptor cells before vision loss. Clinical phenotypic features include, but are not limited to: (i) fundus abnormalities with osteophyte deposits and narrowing of retinal vessels; (ii) abnormal, weakened, or absent a-waves and b-waves in electroretinal spectroscopy (ERG); and (iii) visual field narrowing. Symptoms typically begin in early adolescence and can progress to severe vision impairment by age 40 to 50.

[0173] An example of a known pathogenic genetic variant for retinitis pigmentosa is a homozygous splicing mutation (3444+1G-A) at the donor site of exon 32 of the CNGB1 gene, resulting in a frameshift and truncation of the last 28 amino acids. Another example of a known pathogenic genetic variant for retinitis pigmentosa is a homozygous 2978G-T conversion of exon 30 of the CNGB1 gene, which is expected to result in the replacement of glycine at position 993 of the protein with valine (G993V). Position 993 of the glycine residue in CNGB1 is a conserved residue. Yet another example of a known pathogenic genetic variant for retinitis pigmentosa is a homozygous c.1589C-G conversion of the CNGB1 gene, resulting in the replacement of proline at position 530 of the CNGB1 protein with arginine (P530R). Another known genetic variant pathogenic to retinitis pigmentosa includes the c.2128C-T change in the CNGB1 gene, resulting in the replacement of glutamine at position 710 of the CNGB1 protein with a terminator (Q710Stop). Other genetic variants pathogenic to retinitis pigmentosa can be found in the online Mendelian Male Genetics (OMIM) database and the ClinVar database maintained by the National Center for Biotechnology Information, and are incorporated herein by reference in their entirety for all purposes.

[0174] CNGB1-associated retinitis pigmentosa can be identified using methods known in the art, by detecting one or more phenotypic symptoms described herein and / or one or more genetic variations of the CNGB1 gene. Any genetic variation that causes changes in conserved residues of CNGB1 can lead to retinitis pigmentosa.

[0175] As used herein, “treatment,” “management,” “nursing care,” or “therapeutic treatment” means achieving one or more of the following: (a) reducing the severity of the condition; (b) limiting or preventing the development of symptoms of the treated condition; (c) suppressing the worsening of symptoms of the treated condition; (d) limiting or preventing the recurrence of the condition in an individual who previously had the condition; and (e) limiting or preventing the recurrence of symptoms in an individual who previously had symptoms of the disease.

[0176] Implementation Plan

[0177] In the following sections, different aspects of the invention will be defined in more detail. Each aspect thus defined may be combined with any other one or more aspects unless expressly indicated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0178] In gene therapy and / or gene correction therapies that introduce nucleic acids into cells to, for example, increase expression, replace defective genes, and / or suppress the expression of defective genes, it is generally desirable that all transgenic elements be small. However, it is often difficult to identify transgenic elements that can be reduced in size in vivo without negatively impacting or losing their activity. In vitro experiments are typically unsuitable for indicative of the in vivo behavior of elements, making the determination of possible size reduction difficult and unpredictable. In the work forming this invention, it has been surprisingly shown that short portions of the human rod promoter, i.e., elements of less than 200 base pairs, can achieve rod-receptor-specific expression of transgenes operatively linked to this promoter element in vivo. Stable integration and expression of the transgene were observed when the promoter element defined herein was used in an in vivo setting. The expression levels were suitable for improving the visual abilities of test animals transfected with an adeno-associated virus vector containing the transgene.

[0179] This surprising discovery offers the following advantages over existing technologies: (i) reducing the size of the construct introduced into cells; (ii) improving the packaging efficiency of transgenes in viral vectors; (iii) reducing the chance of recombination events in vivo; (iv) improving the efficiency of transgene delivery to target cells, particularly to the target cell nucleus; (v) providing appropriate expression levels for treating rod-related diseases in human patients; (vi) maintaining and / or improving retinal function in vivo and / or (vii) maintaining and / or improving vision in vivo.

[0180] In a first aspect, the present invention relates to a polynucleotide comprising, in the following order:

[0181] a) A promoter comprising a human rod photoreceptor cell-specific promoter element (hRPSPE) and a core promoter (CP), the promoter element comprising a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof, substantially consisting of a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof, or consisting of a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof; and

[0182] b) At least one transgene (TG) operatively linked to the promoter of a);

[0183] The variant of SEQ ID NO:1 comprises one or more nucleic acid substitutions other than positions 6 to 13, 32 to 40, 70 to 83, and 87 to 94 of the nucleotide in SEQ ID NO:1, and wherein the promoter length is particularly 350 bases or less. A promoter providing one or more of the above advantages may also be longer than 350 bases, for example, 600 bp or less, 500 bp or less, or 400 bp or less. In a particular embodiment, the promoter length is 300 bases or less; in other embodiments, it is 300 bases or less; in other embodiments, it is 250 bases or less; in other embodiments, it is 200 bases or less; and in other embodiments, it is 194 bases or less.

[0184] To minimize the total length of heterologous bases introduced into the patient, the polynucleotide does not contain any other rod promoter and / or gene nucleotide sequences other than those explicitly defined in a) above.

[0185] Since the inventors believe that these nucleotide sequences contribute to the specific expression of hRPSPE on rod photoreceptor cells, the indicated nucleotides are retained in the variant of SEQ ID NO:1. One or more nucleotides may be mutated or inserted outside the putative transcription factor binding sequence (TFB). If inserting nucleotides, an insertion of 1 to 70 nucleotides is advantageous, with multiples of seven being particularly advantageous because this number preserves the relative rotational position of the TFBs. However, it is advantageous not to alter the distance between the TFBs to avoid rotational shift of the transcription factor binding to the promoter element. Therefore, within the 99 bp long sequence according to SEQ ID NO:1, one or more nucleotides may be mutated at positions 1 to 5, 14 to 31, 41 to 69, 84 to 86, and 95 to 99. Thus, a maximum of 50 nucleotides may be mutated in SEQ ID NO:1. Therefore, a specific variant contains 1 to 50 mutations, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mutations. Other specific variants contain 5 to 40, 10 to 30, or 20 to 25 mutations. Promoters containing hRPSPE variants exhibit rod-receptor-specific expression levels as promoters containing the sequence according to SEQ ID NO: 1, preferably exhibiting rod-receptor-specific expression levels as promoters composed of nucleotides 155 to 350 or 155 to 348 of SEQ ID NO: 2. It is advantageous if the variant exhibits at least 10% of the expression level of a promoter composed of nucleotides 155 to 350 or 155 to 348 of SEQ ID NO: 2. Other advantageous expression levels are at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. Different expression levels may be advantageous depending on the respective therapeutic approach, particularly if higher expression levels of the transgene overcompensate for defects or lead to detrimental effects; lower expression levels may be advantageous compared to expression levels obtained with promoters composed of nucleotides 155 to 350 of SEQ ID NO: 2.

[0186] It is also foreseeable that the polynucleotide of the present invention comprises two or more transgenes operatively linked to the promoter of a). In this case, the separate expression of two or more transgenes can be obtained by inserting a nucleotide sequence between the two transgenes that allows for the separate translation of the two transgenes, such as encoding an internal ribosome entry site (IRES).

[0187] In an embodiment of the first aspect of the invention, the 5' end of the hRPSPE contained in the promoter of a) is located at positions 1 to 160 of the nucleic acid of SEQ ID NO:2 or a variant thereof, while the 3' end is located at positions 290 to 310 of the nucleic acid. In a particular embodiment, the 5' end of the hRPSPE is located at one of the following nucleic acid positions: position 1, position 10, position 20, position 30, position 40, position 50, position 60, position 70, position 80, position 90, position 100, position 110, position 120, position 130, position 140, position 145, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, or position 160 of SEQ ID NO:2. In one particular implementation, the 3' end of hRPSPE is located at one of the following nucleic acid positions: position 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, or 310 of SEQ ID NO: 2. Therefore, according to a specific implementation, the promoter of a) contains hRPSPE spanning positions 1 to 310, 10 to 309, 20 to 308, 30 to 307, 40 to 306, 50 to 305, 60 to 304, 70 to 303, 80 to 302, 90 to 301, 100 to 300, 110 to 299, 120 to 298, 130 to 297, 140 to 296, 150 to 295, 151 to 294, 152 to 293, 153 to 292, 154 to 291, or 155 to 290 of the nucleic acid of SEQ ID NO: 2. The term "variant of hRPSPE" has the meaning outlined above. Therefore, variants of the fragments indicated in this paragraph have the indicated 5' and 3' ends, respectively, and may additionally include mutations beyond those indicated above for the sequence indicated for SEQ ID NO: 1.

[0188] In an embodiment of the first aspect of the invention, CP includes a TATA box and / or a promoter (Inr). In a specific embodiment, a TATA box and Inr include a human rho promoter. In one particular implementation, the 5' end of the CP contained in the promoter of a) is located at positions 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, and 314 of the nucleotide in SEQ ID NO: 2, and the 3' end is located at positions 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 342, 343, 344, 345, 346, 347, 348, 349, or 350 of the nucleic acid in SEQ ID NO: 2. Therefore, according to a specific embodiment, the promoter of a) includes CP spanning positions 300 to 350, 301 to 350, 302 to 350, 303 to 350, 304 to 349, 305 to 349, 306 to 349, 307 to 349, 308 to 348, 309 to 348, 310 to 348, 311 to 348, 312 to 348, 313 to 348, or 314 to 348 of the nucleic acid of SEQ ID NO:2 or a variant thereof. In one embodiment of the first aspect of the invention, the 5' end of the promoter is located at positions 1 to 160 of the nucleic acid of SEQ ID NO:2 or a variant thereof, and the 3' end is located at positions 340 to 350 of the nucleic acid of SEQ ID NO:2 or a variant thereof. In one particular implementation, the 5' end of the promoter is located at one of the following nucleic acid positions: position 1, position 10, position 20, position 30, position 40, position 50, position 60, position 70, position 80, position 90, position 100, position 110, position 120, position 130, position 140, position 145, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, or position 160. In one particular implementation, the 3' end of the promoter is located at one of the following nucleic acid positions: position 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 342, 343, 344, 345, 346, 347, 348, 349, or 350 of SEQ ID NO: 2.Therefore, according to a specific implementation scheme, the promoter of a) spans positions 1 to 350, 10 to 350, 20 to 350, 30 to 350, 40 to 350, 50 to 350, 60 to 350, 70 to 350, 80 to 350, 90 to 349, 100 to 349, 110 to 349, 120 to 349, 130 to 349, 140 to 348, 150 to 348, 151 to 348, 152 to 348, 153 to 348, 154 to 348, or 155 to 348 of the nucleic acid of SEQ ID NO: 2. In one particular implementation, the promoter contains SEQ ID NO: 9, is substantially composed of SEQ ID NO: 9, or is composed of SEQ ID NO: 9.

[0189] In one embodiment of the first aspect of the invention, the transgene comprises, or is substantially composed of, a nucleic acid encoding a protein that maintains or improves the physiological function of rod cells and / or inhibits rod cell proliferation. Typically, such genes are naturally expressed in healthy rod cells. Those skilled in the art know that a large number of genes expressed in rod cells are involved in the physiological functions of rod cells. These functions include, in particular, the detection of photons and the generation of neural impulses in response to the detection of one or more photons.

[0190] In one embodiment of the first aspect of the invention, the genetic modification:

[0191] (i) Nucleic acids comprising a functional fragment or variant thereof encoding a human rod-shaped nucleotide-gated channel β subunit (hCNGB1), ABCA4, AIPL1, BEST1, CACNA1F, CLN3, CLRN1, CNGA1, CEP290, CRB1, CRB2, CRX, GPR98, GUCA1A, GUCA1B, MYO7A, NRL, PDE6A, PDE6B, PRPH2, PROM1, RHO, ROM1, RP1, RP2, RPE65, RPGR, SAG, USH1C, USH1G, USH2A, or the target mRNA; a miRNA or shRNA encoding a dominant inactivating mutant thereof; and / or an antibody or antibody-binding fragment encoding an antibody that specifically binds to the dominant inactivating mutant thereof; or

[0192] (ii) Contains a protein encoding an inhibitor of rod cell proliferation, preferably a toxin; a prodrug converting enzyme, such as thymidine kinase; a cell cycle inhibitor, such as retinoblastoma protein (pRB), p53, p21CIP1, p27KIP1 and p57KIP2; mRNA containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof and / or nucleic acid containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof.

[0193] Some rod cell diseases are characterized by recessive mutations in one or more genes, particularly those encoding proteins shown in (i) or (ii), that maintain or improve rod cell function or prevent excessive proliferation. In such cases, introducing a transgene encoding a functional protein into rod cells, particularly using a vector, is often sufficient to cure or at least alleviate the disease. However, if the disease is caused by a dominant-inactivating mutation, providing a transgene encoding a functional protein or a functional fragment thereof is often insufficient to cure or alleviate the disease. In such cases, it is preferable to reduce, i.e., knock down, the expression or function of the dominant-inactivating mutant protein in the cell. This knockdown can be performed by expressing a transgene encoding a repressive RNA that specifically reduces the expression of the dominant-inactivating mutant protein, or by expressing one or more transgenes encoding an antibody or a fragment thereof that specifically binds to and inactivates the dominant-inactivating mutant protein without significantly binding to the corresponding functional protein. Those skilled in the art are well aware of how to design repressive RNAs that are specific to the mRNA encoding the corresponding dominant-inactivating mutant protein. Similarly, those skilled in the art know how to generate antibodies that specifically bind only to the dominant-inactivating mutant protein and not to the wild-type protein. The natural form of an antibody contains two distinct protein chains. Therefore, if both protein chains of the antibody are expressed to knock down the protein, one transgene may contain nucleotides encoding the light chain of another transgene that links to the heavy chain via the internal ribosome entry site (IRES). In this way, both antibody chains can be expressed from a single mRNA. It will be apparent to those skilled in the art that the order of the light and heavy chains can be reversed without affecting antibody expression within rod cells. Alternatively, a single-chain antibody may be encoded by the transgene.

[0194] In one particular embodiment, the disease to be treated is characterized by a dominant inactivation mutation in one or more genes that maintain or improve rod cell function or promote excessive proliferation, particularly one or more of AIPL1, BEST1, NR2E3, NRL, PRPH2, RHO, ROM1, and / or RP1. In this case, it is preferable to knock down the expression and / or function of the protein encoded by the dominant inactivation mutant gene and to introduce a transgene encoding the functional protein or a functional fragment thereof. If size limitations on the individual vectors are permissible, it is preferable to have both a transgene encoding a functional protein or a functional fragment thereof and a transgene encoding a repressive RNA or a repressive antibody or a fragment thereof. If both transgenes encode proteins, they can be used under the control of the same promoter, such as the IRES sequence between the two transgenes, or if one transgene encodes a protein and the other encodes a repressive RNA, each transgene can be operatively linked to a separate promoter according to i) of the first aspect of the invention.

[0195] The term "functional fragment" refers to the deletion of the N-terminus and / or C-terminus of a corresponding protein, which does not result in the loss of the rod-cell-specific function of the corresponding protein. The term "variant thereof" refers to a protein having at least 70% sequence identity with the human wild-type protein respectively indicated, particularly the proteins according to SEQ ID NO:3, SEQ ID NO:10 to SEQ ID NO:41 and SEQ ID NO:45. In a particular embodiment, the variant has at least 70% sequence identity with SEQ ID NO:3, particularly 75%, more particularly 80%, more particularly 85%, more particularly 90%, and more particularly 95% sequence identity. In a particular embodiment, the variant has at least 70% sequence identity with SEQ ID NO:10, particularly 75%, more particularly 80%, more particularly 85%, more particularly 90%, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 11, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 12, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 13, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 14, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 15, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity.In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 16, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 17, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 18, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 19, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 20, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 21, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 22, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 23, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 24, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity.In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 25, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 26, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 27, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 28, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 29, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 30, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 31, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 32, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 33, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity.In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 34, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 35, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 36, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 37, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 38, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 39, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 40, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 41, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity. In one particular embodiment, the variant shares at least 70% sequence identity with SEQ ID NO: 45, particularly 75% sequence identity, more particularly 80% sequence identity, more particularly 85% sequence identity, more particularly 90% sequence identity, and more particularly 95% sequence identity.

[0196] The functional segments of the aforementioned proteins are those that maintain the function of their respective proteins in normally functioning rod photoreceptor cells, particularly in the detection of photons and / or the transmission of information about photon detection. Similarly, variants also maintain the function of their respective proteins in normally functioning rod photoreceptor cells.

[0197] In one embodiment of the first aspect of the invention, hCNGB1 encoded by the transgene comprises the amino acid sequence according to SEQ ID NO: 3 or a variant thereof. In one embodiment of the first aspect of the invention, hCNGB1 encoded by the transgene comprises the amino acid sequence according to SEQ ID NO: 40 or a variant thereof. In one embodiment of the first aspect of the invention, hCNGB1 encoded by the transgene comprises the amino acid sequence according to SEQ ID NO: 41 or a variant thereof.

[0198] In one embodiment of the first aspect of the invention, the polynucleotide comprises one or more other nucleotide sequence elements selected from:

[0199] (i) Polyadenylation signal (pA); and / or

[0200] (ii) One or two inverted terminal repeat (ITR) sequences; and / or

[0201] (iii) Forming an infectious viral vector, preferably an adeno-associated virus, adenovirus, retrovirus, lentivirus, vaccinia virus or herpesvirus vector, especially the viral nucleotide sequence necessary for a herpes simplex virus (HSV) vector.

[0202] The viral nucleotide sequences necessary for forming the corresponding type of infectious viral vector are well known in the art. Any of these elements can be included in the polynucleotide of the first aspect of the invention.

[0203] In one embodiment of the first aspect of the invention, the polyadenylation signal comprises simian virus 40PAS, is substantially composed of simian virus 40PAS, or is composed of simian virus 40PAS.

[0204] In one embodiment of the first aspect of the invention, the polyadenylation signal comprises a nucleic acid according to SEQ ID NO: 4 or a functional variant thereof, substantially composed of a nucleic acid according to SEQ ID NO: 4 or a functional variant thereof, or composed of a nucleic acid according to SEQ ID NO: 4 or a functional variant thereof.

[0205] In one embodiment of the first aspect of the invention, the ITR sequence is an adeno-associated virus (AAV) ITR.

[0206] In one embodiment of the first aspect of the invention, the AAV ITR is an ITR of AVV serotype 2, 5, 8 or 9.

[0207] In one embodiment of the first aspect of the invention, the promoter and transgene have an L-ITR on their 5' side and an R-ITR on their 3' side. In one particular embodiment, these elements are arranged in the 5' to 3' direction in the following order: L-ITR-promoter-transgene-R-ITR, L-ITR-transgene-promoter-R-ITR, R-ITR-promoter-transgene-L-ITR, or R-ITR-transgene-promoter-L-ITR. In another particular embodiment, these elements are arranged in the 5' to 3' direction in the following order: L-ITR-promoter-transgene-PAS-R-ITR, L-ITR-PAS-transgene-promoter-R-ITR, R-ITR-promoter-transgene-PAS-L-ITR, or R-ITR-PAS-transgene-promoter-L-ITR.

[0208] In one embodiment of the first aspect of the invention, the L-ITR comprises a sequence according to SEQ ID NO: 5 or a variant thereof, consists substantially of a sequence according to SEQ ID NO: 5 or a variant thereof, or consists of a sequence according to SEQ ID NO: 5 or a variant thereof; and / or the R-ITR comprises a sequence according to SEQ ID NO: 6 or a variant thereof, consists substantially of a sequence according to SEQ ID NO: 6 or a variant thereof, or consists of a sequence according to SEQ ID NO: 6 or a variant thereof.

[0209] Depending on the viral vector used, the length of nucleic acid that can be effectively packaged in the viral vector varies considerably. Some vectors, such as adenovirus vectors, can accommodate larger nucleic acid inserts, while others, such as adenovirus-associated vectors, can effectively package polynucleotides of 4700 bases or less. Regardless of the nucleic acid packaging capacity of the vector, it is generally desirable to minimize the length of any heterologous nucleic acid introduced into the patient, especially when the heterologous nucleic acid is stably introduced into the genome. Therefore, in one embodiment of the first aspect of the invention, the total length of the polynucleotide is 5200 bases or less, particularly 5100 bases or less, particularly 5000 bases or less, particularly 4900 bases or less, particularly 4800 bases or less, and more particularly 4700 bases or less.

[0210] In a specific embodiment of the first aspect of the invention, the polynucleotide comprises, or is substantially composed of, the following nucleic acid elements in the 5' to 3' direction: L-ITR-promoter-transgenic-SV40PAS-R-ITR, L-ITR-SV40PAS-transgenic-promoter-R-ITR, R-ITR-promoter-transgenic-SV40 PAS-L-ITR, or R-ITR-SV40 PAS-transgenic-promoter-L-ITR, wherein the transgenic element comprises, substantially comprises, or is composed of the nucleotide sequence encoding the hCNGB1 protein of SEQ ID NO: 3, or is composed of the nucleotide sequence encoding the hCNGB1 protein of SEQ ID NO: 3, PAS comprises, substantially comprises, or is composed of the nucleotide sequence SEQ ID NO: 4, L-ITR comprises, substantially comprises, or is composed of the nucleotide sequence SEQ ID NO: 5, and R-ITR comprises, substantially comprises, or is composed of the nucleotide sequence SEQ ID NO: 5. NO: 6, substantially comprising or consisting of the nucleotide sequence SEQ ID NO: 6, and the promoter comprising, substantially comprising, or consisting of, the nucleotide sequence spanning positions 155 to 348 of SEQ ID NO: 1. Also in this embodiment, the total length of the polynucleotide is 5200 bases or less, particularly 5100 bases or less, particularly 5000 bases or less, particularly 4900 bases or less, particularly 4800 bases or less, and more particularly 4700 bases or less.

[0211] A second aspect of the invention relates to plasmids comprising the polynucleotides of the first aspect of the invention. Plasmids are circular DNA molecules that can replicate in bacteria.

[0212] In one embodiment of the second aspect of the invention, the plasmid comprises a nucleic acid sequence according to SEQ ID NO: 7 or a variant thereof, is substantially composed of a nucleic acid sequence according to SEQ ID NO: 7 or a variant thereof, or is composed of a nucleic acid sequence according to SEQ ID NO: 7 or a variant thereof. In one embodiment of the second aspect of the invention, the plasmid comprises a nucleic acid sequence according to SEQ ID NO: 42 or a variant thereof, is substantially composed of a nucleic acid sequence according to SEQ ID NO: 42 or a variant thereof, or is composed of a nucleic acid sequence according to SEQ ID NO: 42 or a variant thereof. In one embodiment of the second aspect of the invention, the plasmid comprises a nucleic acid sequence according to SEQ ID NO: 43 or a variant thereof, is substantially composed of a nucleic acid sequence according to SEQ ID NO: 43 or a variant thereof, or is composed of a nucleic acid sequence according to SEQ ID NO: 43 or a variant thereof. In one embodiment of the second aspect of the invention, the plasmid comprises a nucleic acid sequence according to SEQ ID NO: 44 or a variant thereof, is substantially composed of a nucleic acid sequence according to SEQ ID NO: 44 or a variant thereof, or is composed of a nucleic acid sequence according to SEQ ID NO: 44 or a variant thereof.

[0213] A third aspect of the invention relates to a viral vector comprising the polynucleotides of the first aspect of the invention. In one specific embodiment, the viral vector is an AAV, adenovirus, retrovirus, lentivirus, vaccinia virus, or herpesvirus vector, particularly a herpes simplex virus (HSV) vector. In one specific embodiment, the viral vector is AAV.

[0214] In one embodiment of the third aspect of the invention, the virus is selected from AAV2, AAV5, AAV8, AVV9 or variants thereof.

[0215] The fourth aspect of the invention relates to polynucleotides according to the first aspect of the invention, plasmids according to the second aspect of the invention, and / or viral vectors according to the third aspect of the invention, which are used as pharmaceuticals.

[0216] The fifth aspect of the invention relates to a pharmaceutical composition comprising a polynucleotide according to the first aspect of the invention, a plasmid according to the second aspect of the invention, and / or a viral vector according to the third aspect of the invention, and a pharmaceutically acceptable carrier.

[0217] The sixth aspect of the present invention relates to a polynucleotide according to the first aspect of the present invention, a plasmid according to the second aspect of the present invention, and / or a viral vector according to the third aspect of the present invention, for the treatment of retinal diseases.

[0218] Advantageously, the polynucleotides according to the first aspect of the invention, the plasmids according to the second aspect of the invention, and / or the viral vectors according to the third aspect of the invention can be used in diseases associated with loss or abnormality of rod photoreceptor function, particularly in retinal degeneration or excessive proliferation of rod cells, especially in retinoblastoma. Although tissue-specific expression of the transgene is achieved via the promoter of the polynucleotide of the first aspect of the invention, and therefore systemic administration of the therapeutic polynucleotide, plasmid, or viral vector can be systemic, without requiring but still limited to rod photoreceptor cells, it is more effective if the therapeutic polynucleotide, plasmid, or viral vector of the invention is administered directly to the patient's eye. Therefore, specific routes of administration are selected from intraocular, intraocular, intravitreal, or subretinal administration.

[0219] In one embodiment of the sixth aspect of the invention, retinal degeneration is associated with genetic mutations, replacements, and / or deletions.

[0220] In one embodiment of the sixth aspect of the invention, retinal degeneration is associated with genetic mutations, replacements, and / or deletions.

[0221] In one embodiment of the sixth aspect of the invention, the degeneration is selected from night blindness, blindness, retinal degeneration, retinal dystrophy, and retinitis pigmentosa.

[0222] In one embodiment of the sixth aspect of the invention, the retinitis pigmentosa is CNGB1-associated retinitis pigmentosa or retinitis pigmentosa type 45 (RP45).

[0223] A seventh aspect of the present invention relates to a polynucleotide comprising, in the following order:

[0224] a) A human rhodopsin promoter comprising a nucleic acid sequence according to SEQ ID NO: 9 or a variant thereof; and

[0225] b) At least one transgene (TG) operatively linked to the promoter of a).

[0226] In one embodiment of the seventh aspect of the invention, the transgene comprises a nucleic acid encoding a protein that maintains or improves the physiological function of rod cells.

[0227] In one embodiment of the seventh aspect of the invention, the genetic modification:

[0228] (i) Nucleic acids comprising a functional fragment or variant thereof encoding a human rod-shaped nucleotide-gated channel β subunit (hCNGB1), ABCA4, AIPL1, BEST1, CACNA1F, CLN3, CLRN1, CNGA1, CEP290, CRB1, CRB2, CRX, GPR98, GUCA1A, GUCA1B, MYO7A, NRL, PDE6A, PDE6B, PRPH2, PROM1, RHO, ROM1, RP1, RP2, RPE65, RPGR, SAG, USH1C, USH1G, USH2A, or the target mRNA; a miRNA or shRNA encoding a dominant inactivating mutant thereof; and / or an antibody or antibody-binding fragment encoding an antibody that specifically binds to the dominant inactivating mutant thereof; or

[0229] (ii) Contains a protein encoding an inhibitor of rod cell proliferation, preferably a toxin; a prodrug converting enzyme, such as thymidine kinase; a cell cycle inhibitor, such as retinoblastoma protein (pRB), p53, p21CIP1, p27KIP1 and p57KIP2; mRNA containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof and / or nucleic acid containing a cell cycle inhibitor encoding a dominant inactivating mutant thereof.

[0230] In one embodiment of the seventh aspect of the invention, the polynucleotide further comprises one or more nucleotide sequence elements selected from:

[0231] (i) Polyadenylation signal (PAS);

[0232] (ii) One or two inverted terminal repeat (ITR) sequences; and

[0233] (iii) Forming an infectious viral vector, preferably an adenovirus, retrovirus, lentivirus, vaccinia virus or herpesvirus vector, especially the viral nucleotide sequence necessary for a herpes simplex virus (HSV) vector.

[0234] In one embodiment of the seventh aspect of the invention, the polyadenylation signal comprises simian virus 40PAS.

[0235] In one embodiment of the seventh aspect of the invention, the ITR sequence is an adeno-associated virus (AAV) ITR.

[0236] In one embodiment of the seventh aspect of the invention, AAV is AVV serotype 2, 5, 8 or 9.

[0237] The eighth aspect of the present invention relates to a viral vector comprising the polynucleotides of the seventh aspect of the present invention.

[0238] In one embodiment of the eighth aspect of the invention, the virus is selected from AAV2, AAV5, AAV8, AVV9 or variants thereof.

[0239] The polynucleotides of the present invention comprising a human rod photoreceptor heterologous promoter element (hRPSPE) or a variant thereof operably linked to a transgene (e.g., CNGB1) and a core promoter (CP), or comprising a human rhodopsin promoter operably linked to a transgene (e.g., CNGB1), can be used for gene therapy and / or gene correction therapy. In such therapies, the polynucleotides are introduced into cells to enhance expression, replace defective genes, and / or suppress the expression of defective genes.

[0240] Therefore, the ninth aspect of the present invention relates to a method for treating retinal degeneration in a subject in need, comprising administering to the subject a therapeutically effective amount of a polynucleotide according to the seventh aspect of the present invention or a viral vector according to the eighth aspect of the present invention.

[0241] In one embodiment of the ninth aspect of the present invention, the polynucleotide or viral vector comprises the nucleic acid sequence shown in SEQ ID NO: 43.

[0242] The tenth aspect of the invention relates to a method for treating retinitis pigmentosa in a subject in need, comprising administering to the subject a therapeutically effective amount of a polynucleotide according to the seventh aspect of the invention or a viral vector according to the eighth aspect of the invention.

[0243] In one embodiment of the tenth aspect of the present invention, the polynucleotide or viral vector comprises the nucleic acid sequence shown in SEQ ID NO: 43.

[0244] The eleventh aspect of the present invention relates to a method for treating retinal degeneration in a subject in need, wherein the retinal degeneration is characterized by a defective deletion of CNGB1 in the retinal cells of the subject, the method comprising administering to the subject a therapeutically effective amount of a viral vector comprising the nucleotide sequence shown in SEQ ID NO: 43.

[0245] In one embodiment of the eleventh aspect of the present invention, the retinal degeneration is CNGB1-associated retinitis pigmentosa or retinitis pigmentosa type 45 (RP45).

[0246] The twelfth aspect of the invention relates to a method for treating CNGB1-associated retinitis pigmentosa or retinitis pigmentosa type 45 (RP45) in a subject in need, the method comprising administering a therapeutically effective amount of a viral vector comprising the nucleotide sequence shown in SEQ ID NO: 43 under the subject's retina.

[0247] The thirteenth aspect of the present invention relates to a polynucleotide comprising, in the following order:

[0248] a) A promoter comprising a human rod photoreceptor cell-specific promoter element (hRPSPE) and a core promoter (CP), wherein the promoter element comprises a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof; and

[0249] b) A transgene encoding the human rod-shaped nucleotide-gated channel β subunit (hCNGB1), which is operatively linked to the promoter of a).

[0250] The variant of SEQ ID NO:1 contains one or more nucleic acid substitutions other than the nucleotides at positions 6 to 13, 32 to 40, 70 to 83, and 87 to 94 of SEQ ID NO:1.

[0251] The fourteenth aspect of the present invention relates to a pharmaceutical composition comprising a polynucleotide, the polynucleotide comprising, in the following order:

[0252] a) A promoter comprising a human rod photoreceptor cell-specific promoter element (hRPSPE) and a core promoter (CP), wherein the promoter element comprises a nucleic acid sequence according to SEQ ID NO: 1 or a variant thereof; and

[0253] b) A transgene encoding the human rod-loop nucleotide-gated channel β subunit (hCNGB1), which is operatively linked to the promoter of a);

[0254] The variant of SEQ ID NO: 1 contains one or more nucleic acid substitutions other than nucleotides 6 to 13, 32 to 40, 70 to 83, and 87 to 94 of SEQ ID NO: 1.

[0255] Pharmaceutically acceptable carrier.

[0256] The fifteenth aspect of the present invention relates to a pharmaceutical composition comprising a viral vector and a pharmaceutically acceptable carrier, the viral vector comprising the nucleic acid sequence shown in SEQ ID NO: 43.

[0257] Table 1: List of sequences selected in this invention

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275] Example

[0276] Example 1: Nucleic Acid Vector

[0277] In this exemplary embodiment, the rAAV.hCNGB1 vector is a heterozygous AAV-based vector carrying cDNA of the human CNGB1 gene encoding the B subunit of the rod photoreceptor cyclic nucleotide-gated (CNG) channel. hCNGB1 cDNA expression is controlled by the rod-cell-specific rhodopsin promoter (hRHO) and enhanced using the SV40 pA sequence. The expression assembly is flanked by an AAV serotype 2 inverted terminal repeat (ITR), and the recombinant genome is packaged in an AAV serotype 8 capsid. The expression assembly includes the following elements:

[0278] • Promoter of the human rhodopsin gene: 0.194Kb

[0279] cDNA of the human CNGB1a subunit of cGMP in rod photoreceptor cells

[0280] Phosphodiesterase: 3.74Kb

[0281] • Polyadenylation signal of simian virus 40 (SV40): 0.23 Kb

[0282] • AAV serotype 2 inverted terminal repeat (ITR): 0.13K. The structure of the rAAV.hRHO194.hCNGB1 vector genome is as follows: Figure 1As shown.

[0283] Example 2: pGL2.0-hRHO194-hCNGB1a-SV40 cis-vector plasmid

[0284] In one exemplary embodiment, the pGL2.0-hRHO194-hCNGB1a-SV40 cis-vector plasmid having the nucleotide sequence shown in SEQ ID No. 7 is used. This plasmid contains an expression component comprising a 194 bp rod photoreceptor-specific human rhodopsin (hRHO) promoter and full-length (3738 bp) human CNGB1 cDNA. The expression component also contains a 227 bp simian virus 40 polyadenylation signal (SV40 pA). The 5591 bp vector backbone with kanamycin resistance (KanR) is 1943 bp from the L-ITR, 2853 bp from the R-ITR, and 2024 bp from the pUC18 ori. The rAAV.hCNGB1 vector is generated by transient co-transfection of a cis-vector plasmid and a trans-helper plasmid encoding the rep and cap sequences in human embryonic kidney 293T cells (HEK293T) and an adenovirus gene. rAAV.hRHO194.hCNGB1 was harvested from culture medium and / or cell lysates using standard purification methods, such as cesium chloride gradient ultracentrifugation, ion exchange chromatography, and / or tangential flow filtration. The resulting rAAV.hRHO194.hCNGB1 carrier suspension was then aseptically filtered, filled, and stored as a pharmaceutical product.

[0285] Example 3: Activity and specificity of the hRHO194 promoter

[0286] To verify the activity and specificity of the novel hRHO194 promoter, the inventors constructed an AAV cis-vector containing eGFP cDNA instead of hCNGB1 cDNA. The resulting pGL2.0-hRHO194-eGFP-SV40 cis-vector plasmid map is shown below. Figure 2 As shown. Delivery of the rAAV.hRHO194.eGFP vector into the subretinal space of 4-week-old wild-type mice resulted in strong eGFP protein expression 4 weeks after injection of rod photoreceptor cells only. Figure 3A This confirmed the retinal cell type specificity of the promoter. For representative results, see [link to relevant documentation]. Figures 3A to 3B Treatment with the rAAV.hRHO194.eGFP vector resulted in strong eGFP protein expression in the treated eye, only in rod photoreceptor cells, which was reflected by natural eGFP fluorescence.

[0287] Example 4: Bioactivity and transgenic expression conferred by rAAV.hRHO194.hCNGB1

[0288] To verify biological activity and transgene expression, the inventors delivered the AAV.hRHO194.hCNGB1 vector into the subretinal space of 4-week-old CNGB1(- / -) mice. The delivery process was similar to that described by Koch et al., gene therapy can restore vision and delay the degeneration of the CNGB1(- / -) mouse model of retinitis pigmentosa. Hum Mol Genet. 2012; 21(20):4486-96. PubMed PMID:22802073. Mice were injected subretinally into the treated eye (TE), while an untreated eye (UE) served as a control. The efficacy of the vector was evaluated by electroretinography (ERG, an objective in vivo functional assay) 4 months after injection. Figure 4A and Figure 4B CNGB1(- / -) mice lack normal rod photoreceptor function. Following the degeneration of rod cells, unaffected cone photoreceptors also degenerate, leading to loss of cone function in the later stages of the disease. Therefore, the ERG protocol, which specifically tests rod and cone function, is suitable as an indirect measure of CNGB1 function and for assessing the bioactivity (BAA) of the rAAVhRHO194.hCNGB1 vector.

[0289] Example 5: In vivo optical coherence tomography (OTC) for determining BAA

[0290] In another set of experiments, BAA was determined by in vivo optical coherence tomography (OCT) followed by quantification of photoreceptor layer thickness. For this purpose, mice were injected subretinally into the treated eye (TE), while an untreated eye (UE) served as a control. Photoreceptor layer thickness was measured by OCT 4 months post-injection. Figures 5A to 5C In CNGB1(- / -) mice, the rod photoreceptor cells degenerate over time, resulting in a thinning of the photoreceptor layer. Figure 5B and Figure 5C Therefore, the bioavailability (BAA) of the rAAV.hRHO194.hCNGB1 vector can be indirectly measured by the photoreceptor layer thickness in treated CNGB1 (- / -) mice determined using OCT. Treatment with the rAAV.hRHO194.hCNGB1 vector produced a significant therapeutic effect in the treated eyes, manifested in the preservation of photoreceptor layer thickness. Specifically, an increase in photoreceptor layer thickness of over 45% was observed. Figure 5C ).

[0291] Example 6: Cngb1 - / - CNGB1 gene amplification in mice

[0292] Use 1×10 10AAV8-I1RHO 194 -hCNGSi-SV40 or AAV5-hRHOi 94 Viral genome (1e 10 viral genome copies (vg)) of -hCNGSi-SV40 treated under the retina of 4-week-old dCngb1 - / - Mice. Structural results were measured at 1 and 3 months post-injection using SD-OCT, histology, and immunohistochemistry.

[0293] General carrier design such as Figure 7 As shown. 1 μL of 1e 10 total viral genomes were subretinally injected into 4-week-old Cngb1 cells. - / - In mice (4 weeks after birth; PW4).

[0294] Found in Cngb1 - / - Subretinal injection of AAV8-hRHOi in mice 94 -hCNGSi-SV40 gene amplification restored rod function. The effect was observed to persist for up to 8 months post-treatment. Significant improvement in dark-adapted ERG B-wave amplitude was also observed in treated mice. Figures 8A to 8B ). Figure 8A The results showed that at 9 months (8 months after treatment), the rod-specific stimulant Cngb1 was present. - / - Electroretinography under scotopic conditions. Before treatment, wild-type and Cngb1... - / - ERG in mice showed that Cngb1 was injected at the time of injection. - / - ERG B wave is absent in mice. Figure 8B It was found that CNGB1 channel expression was restored in the outer segment of rod cells. Figures 9A to 9B Rabbit polyclonal anti-CNGB1 antibody (Sigma-Aldrich) recognizing amino acid positions 1078 to 1168 of human CNGB1a was used for transgenic expression analysis. AAV8-hRHOi 94 Cngb1 processed by -hCNGS7-SV40 - / - Mice and untreated Cngb1 - / - mice ( Figure 9B In the 9th month, immunohistochemistry was performed (8 months after treatment); Figure 9A ), and showed the processing of Cngb1 - / - Recovery of CNGB1 channel protein expression in mice. OCT analysis showed a significant delay in retinal degeneration. Figures 10A to 10C A typical injection schedule is as follows: Figure 10A As shown. Using AAV8-hRHOi 94 Cngb1 processed by -hCNGS7-SV40 - / - Mice (8 months after treatment); Figure 10B ) and unprocessed Cngb1 - / - mice ( Figure 10C In this study, in vivo optical coherence tomography (OCT) images were collected at 9 months. As shown in Figure 10, at 9 months, it was found that, compared with untreated Cngb1... - / - Compared to mice, the treated Cngb1 - / - Mice have a thicker layer of photoreceptor cells.

[0295] Found in Cngb1 - / - Two months after subretinal injection in mice, AAV5-hRHOi 94 -hCNGSi-SV40 gene amplification led to the recovery of rod function. It was found that the amplitude of dark-adapted ERG B waves was significantly improved in treated mice. Figures 11A to 11E At 3 months of age (2 months after subretinal injection in treated mice), the levels of Cngb1 in treated and untreated mice were significantly different. - / - Dark-vision ERG was measured in mice, and the results were as follows: Figure 11A As shown. At 3 months of age (2 months after subretinal injection in treated mice), the levels of Cngb1 in treated and untreated mice were significantly different. - / - The response measured in mice (n=8) was -0.5log(cd s / m 2 The amplitude of the B wave stimulated by light is as follows: Figure 11B As shown. OCT analysis revealed a significant delay in retinal degeneration. Using AAV5-hRHOi 94 Cngb1 processed by -hCNGS7-SV40 - / - Mice (2 months after treatment); Figure 11C ) and unprocessed Cngb1 - / - mice ( Figure 11D In this study, in vivo optical coherence tomography (OCT) images were collected at month 3. Measurements of photoreceptor layer thickness showed that, compared with wild-type Cngb1... - / - Compared to mice, 3-month-old treated Cngb1 - / - Retinal degeneration was significantly delayed in mice (2 months after treatment) (n=6; Figure 11E ).

[0296] Example 7: Design of a mutant dog study

[0297] Cngb1 - / - A mutation in exon 26 in the dog resulted in truncation and the production of nonfunctional proteins, leading to loss of rod cell function and retinal degeneration. At 3 months of age, AAV5-hRHOi was administered to both eyes. 94 -hCNGB1a Retinal processing three Cngb1 - / -Dogs. For each animal, eye 1 was treated with a dose of 5e 11 vg (target 2 x 100 μL vesicles; "low dose"), and eye 2 was treated with a dose of 1e 12 vg (target 2 x 100 μL vesicles; "high dose"). Structural outcome measurements included SC-OCT, histology, and immunohistochemistry at 1 and 3 months post-injection. Functional outcome measurements included visual acuity testing and ERG at 1 and 3 months post-injection.

[0298] Example 8: Results of a study on mutant dogs

[0299] Found in Cngb1 - / - One month after subretinal injection in dogs, AAV5-hRHOi 94 -hCNGBia-SV40 gene amplification leads to the recovery of rod cell function.

[0300] At both assessment doses, dark-adapted ERG waveforms were significantly improved after treatment. Comparable injections were performed in both eyes. Compared to untreated dogs, those treated with rod-cell-specific stimulation (…) Figure 12A ) and flicker response ( Figure 12B Significant ERG relief was observed in both eyes of the treated dogs. Larger ERG amplitudes were observed in eyes treated with higher doses.

[0301] Vision tests showed that the treated dogs had rod-mediated vision and improved performance in a four-out-of-one vision testing device. Figures 13A to 13B The results of vision tests in treated and untreated dogs are shown one month after injection. A four-option vision test device was used. Untreated Cngb1 was found. - / - Dogs at this stage have normal cone vision but lack rod-mediated vision. Untreated Cngb1 - / - Dogs in low light levels (e.g., 5.7e-2 cd / m²) 2 Blindness resulted in fewer correct exit choices and longer exit times from the test device. It was found that both treatment groups (high dose and low dose) made correct exit choices at the lowest illumination level. Figure 13A ) and exit time ( Figure 13B The performance of the rod has been significantly improved, which indicates the recovery of visual acuity.

[0302] The mean ERG A and B wave amplitudes were found to be higher in the high-dose group than in the low-dose group. The A and B wave amplitudes in the treated eyes were found to be approximately 80% of the wild-type level. Figures 14A to 14BERG amplitude measurements are shown one month after injection in each treatment group and in the untreated group. A-wave amplitude was significantly increased in both treatment groups compared to the untreated control group. An improvement of greater than 1.5 log units was found in the response threshold in the treated eyes. Figure 14A Compared to the untreated control group, B-wave amplitude was significantly increased in all phases in the high-dose group and in all phases except for the second and third strongest stimuli in the low-dose group. An improvement in the response threshold was found to be greater than 2 log units in the treated eyes. Figure 14B ).

Claims

1. A polynucleotide comprising, in the following order: a) A promoter comprising a human rod photoreceptor cell-specific promoter element (hRPSPE), said promoter element comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 9; and b) At least one transgene operatively linked to the promoter of a).

2. The polynucleotide of claim 1, wherein the promoter further comprises a core promoter, the core promoter comprising a TATA box and / or an initiator.

3. The polynucleotide according to claim 1 or 2, wherein the transgene comprises a nucleic acid encoding a protein that maintains or improves the physiological function of rod cells.

4. The polynucleotide according to claim 1 or 2, wherein the transgene: (i) Nucleic acids containing a functional fragment or variant thereof encoding the β subunit of a human rod-loop nucleotide-gated channel, ABCA4, AIPL1, BEST1, CACNA1F, CLN3, CLRN1, CNGA1, CEP290, CRB1, CRB2, CRX, GPR98, GUCA1A, GUCA1B, MYO7A, NRL, PDE6A, PDE6B, PRPH2, PROM1, RHO, ROM1, RP1, RP2, RPE65, RPGR, SAG, USH1C, USH1G, USH2A, or thereof; nucleic acids encoding miRNA or shRNA that targets the mRNA encoding its dominant inactivating mutant; and / or nucleic acids encoding an antibody or antibody-binding fragment that specifically binds to its dominant inactivating mutant; or (ii) Nucleic acids containing proteins that inhibit rod cell proliferation, prodrug-converting enzymes, or cell cycle inhibitors; mRNAs containing cell cycle inhibitors encoding their dominant inactivating mutants; and / or nucleic acids containing cell cycle inhibitors encoding their dominant inactivating mutants.

5. The polynucleotide according to claim 4, wherein the protein inhibiting rod cell proliferation is a toxin, and / or the prodrug-converting enzyme is thymidine kinase, and / or the cell cycle inhibitor is retinoblastoma protein, p53, or p21. CIP1 p27 KIP1 or p57 KIP2 .

6. The polynucleotide of claim 4, wherein the human rod-shaped nucleotide-gated channel β subunit comprises an amino acid sequence according to SEQ ID NO: 3, SEQ ID NO: 40 or SEQ ID NO: 41, or a variant thereof.

7. The polynucleotide according to claim 1 or 2, comprising one or more additional nucleotide sequence elements selected from: (i) polyadenylation signal; and / or (ii) One or two inverted terminal repeat sequences; and / or (iii) The viral nucleotide sequence necessary for the formation of an infectious viral vector.

8. The polynucleotide according to claim 7, wherein the infectious viral vector is an adenovirus vector, a retrovirus vector, a poxvirus vector, or a herpesvirus vector.

9. The polynucleotide according to claim 7, wherein the infectious viral vector is a lentiviral vector.

10. The polynucleotide according to claim 7, wherein the infectious viral vector is a vaccinia virus vector.

11. The polynucleotide of claim 8, wherein the herpesvirus vector is a herpes simplex virus vector.

12. The polynucleotide of claim 7, wherein the polyadenylation signal comprises or is composed of simian virus 40 polyadenylation signal.

13. The polynucleotide of claim 12, wherein the polyadenylation signal comprises or consists of a nucleic acid according to SEQ ID NO: 4 or a functional variant thereof.

14. The polynucleotide of claim 7, wherein the inverted terminal repeat sequence is an adeno-associated virus inverted terminal repeat.

15. The polynucleotide of claim 14, wherein the adeno-associated virus is adeno-associated virus serotype 2, 5, 8 or 9.

16. The polynucleotide of claim 14, wherein the promoter and transgene are coupled to an L-reverse end at their 5' end and to an R-reverse end at their 3' end.

17. The polynucleotide of claim 16, wherein the L-reverse terminal repeat comprises or consists of the sequence according to SEQ ID NO: 5 or a variant thereof, and / or the R-reverse terminal repeat comprises or consists of the sequence according to SEQ ID NO: 6 or a variant thereof, or consists of the sequence according to SEQ ID NO: 6 or a variant thereof.

18. The polynucleotide according to claim 1 or 2, wherein the total length of the polynucleotide is 5200 bases or less.

19. The polynucleotide according to claim 1 or 2, wherein the total length of the polynucleotide is 5100 bases or less.

20. The polynucleotide according to claim 1 or 2, wherein the total length of the polynucleotide is 5,000 bases or less.

21. A plasmid comprising the polynucleotide of any one of claims 1 to 20.

22. The plasmid according to claim 21, comprising a nucleic acid sequence according to SEQ ID NO: 7, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO:

44.

23. A viral vector comprising the polynucleotide of any one of claims 1 to 20.

24. The viral vector of claim 23, wherein the virus is selected from adeno-associated virus 2, adeno-associated virus 5, adeno-associated virus 8, adeno-associated virus 9, or variants thereof.

25. A pharmaceutical composition comprising the polynucleotide of any one of claims 1 to 20, the plasmid of claim 21 or 22 and / or the viral vector of claim 23 or 24, and a pharmaceutically acceptable carrier.

26. Use of the polynucleotide according to any one of claims 1 to 20, the plasmid according to claim 21 or 22, the viral vector according to claim 23 or 24, and / or the pharmaceutical composition according to claim 25 in the preparation of a medicament for treating retinal diseases.

27. The use according to claim 26, wherein the retinal disease is retinal degeneration.

28. The use according to claim 26 or 27, wherein the route of administration is intraocular.

29. The use according to claim 26 or 27, wherein the route of administration is intraocular.

30. The use according to claim 26 or 27, wherein the route of administration is intravitreal or subretinal.

31. The use according to claim 27, wherein the retinal degeneration is associated with a genetic mutation.

32. The use according to claim 31, wherein the genetic mutation is a substitution and / or deletion.

33. The use according to claim 27, wherein the retinal degeneration is selected from night blindness, blindness, retinal dystrophy, and retinitis pigmentosa.

34. The use according to claim 33, wherein the retinitis pigmentosa is cyclic nucleotide-gated channel β subunit-associated retinitis pigmentosa or retinitis pigmentosa type 45.

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