Cell models of eye diseases and therapies for eye diseases

Abstract

Provided are cell models of eye diseases, methods and compositions for treating eye diseases.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Application No. 62 / 539,473, filed on July 31, 2017, entitled “CELLULAR MODELS OF AND THERAPIES FOR OCULAR DISEASES,” the entire contents of which are incorporated herein by reference. [Background Technology] Background of the Invention

[0004] Crystalline retinitis pigmentosa (also known as crystalline retinal degeneration or Bietti's crystalline dystrophy, Bietti's crystalline dystrophy, BCD)

[0005] Bietti Crystalline Retinitis Pigmentosa (BCD, also known as Bietti Crystalline Corneoretinal Dystrophy, Bietti Crystalline Retinopathy, and Bietti's Retinal Dystrophy (OMIM 210370)) is a rare, autosomal recessive, blinding retinal degeneration characterized by the presence of numerous small, iridescent, yellowish-white crystalline deposits at the posterior pole of the retina, associated with atrophy of the retinal pigment epithelium (RPE), pigment clumps, and choroidal sclerosis. It was first described by Dr. GB Bietti in 1937. Fundus photographs and SD-OCT images of BCD patients show that the crystalline deposits are primarily located on the retinal side of the retinal pigment epithelium (RPE). (H. Kojima, A. Otani, K. Ogino, et al., "Outer retinal circular structures in patients with Bietti crystalline retinopathy", British Journal of Ophthalmology, Vol. 96, pp. 390-393, 2012). Crystalline deposits in the corneal limbus are estimated to occur in one-quarter to one-third of people with BCD (Kaiser-Kupfer et al. Clinical biochemical and pathologic correlations in Bietti's crystalline dystrophy, Am J Ophthalmol., 1994, 118:569-82). In some cases, crystalline deposits have also been observed in the lens (Chung et al., J Ophthalmol. 57:447-450, 2013). In advanced stages, BCD patients have advanced choroidal sclerosis, reduced or absent crystalline deposits, and thinning of the retinal blood vessels (Wada et al. Am J Ophthalmol 2005; 139:894-9). Abnormal ERGs and retinal thinning are also present in BCD.

[0006] Clinically, BCD is progressive and associated with degeneration and degeneration of the RPE. Significant asymmetry is common between the eyes of the same patient. The age of disease onset and progression vary from patient to patient with BCD, even within the same family. Most patients develop night blindness, constricted visual field, poor color vision, macular degeneration, and decreased vision between the second and fourth decades of life, and progress to legal blindness between the third and sixth decades of life.

[0007] RPE is between the photosensitive outer section of choriocapillaris blood vessel and photoreceptor, and it is the monolayer pigment cell that closely interacts with photoreceptor (cone and rod) to maintain visual function.The main function of RPE is to supply nutrition, and removes waste from photoreceptor, and photoreceptor is sensory nerve retina.Other functions of RPE include but are not limited to: light absorption, epithelial transport, spatial ion buffering (spatial ion buffering), visual cycle (visual cycle), phagocytosis, secretion and immunomodulation (Strauss, 2005, The retinal pigment epithelium in visual function. Physiol Rev 85:845-81).Therefore, the dysfunction of RPE and degeneration cause the photoreceptor dysfunction and degeneration that cause vision loss.Considering that BCD is relevant to the progressive degeneration and degeneration of RPE, RPE is most important for research and treatment BCD.

[0008] BCD is a rare disease. One source estimates the incidence of BCD to be 1:67,000 (ghr.nlm.nih.gov / condition / bietti-crystalline-dystrophy#statistics on the World Wide Web). Another source estimates the prevalence of BCD to be 2.5% of all RP patients (3 of 121 RP index patients had BCD, see Mataftsi et al., Retina. 24:416-426, 2004). Based on this estimate and considering that the incidence of RP is estimated to be 1:4000 (Hartong et al., Lancet. 368:1795-1809, 2006), the incidence of BCD is estimated to be 1:160,000. Because BCD symptoms are similar to those of other eye conditions that progressively damage the retina, it is sometimes generally diagnosed as retinitis pigmentosa (RP) (Mataftsi A et al. Bietti's crystalline corneoretinal dystrophy: a cross-sectional study. Retina. 2004; 24: 416-426). Although BCD has been reported in various regions of the world, including Asia, Africa, Europe, the Middle East, North America, and South America, it has been reported to be more common in people of East Asian descent, particularly in Chinese and Japanese populations (Hu 1983, Ophthalmic genetics in China. Ophthal Paed Genet 2: 39-45; Li et al. Am J Hum Genet. 2004 May; 74(5): 817-826).

[0009] There are currently no approved treatments for BCD, and patients eventually become blind. There is a high unmet medical need to develop life-changing treatment options for patients with this rare disease.

[0010] CYP4V2

[0011] CYP4V2 (cytochrome P450, family 4, subfamily V, polypeptide 2, (OMIM 608614), synonym: CYP4AH1) is a protein in the cytochrome P450 superfamily and a member of the heme-thiolate cytochrome P450 subfamily 4 (CYP4). Cytochrome P450s (CYPs) are important heme-containing proteins known for their monooxygenase reactions. They participate in the metabolism of xenobiotics and endogenous compounds such as steroids and fatty acids. Human CYPs are primarily membrane-associated proteins located in the inner membrane of mitochondria or in the endoplasmic reticulum of cells. P450 proteins can be identified by their signature sequence element, FxxGxxxCxG (SEQ ID NO:30), in which the underlined cysteine ​​serves as the axial ligand for the heme iron. Another signature sequence element of P450 proteins is ExxR (SEQ ID NO:31). The Human Genome Project has estimated the number of human P450 genes to be 57. For reference, there are 103 mouse P450 genes and 89 rat P450 genes (Guengerich & Cheng, Pharmacological Reviews, September 2011, 63(3): 684-699).

[0012] The human CYP4 family consists of 12 genes and 10 pseudogenes. The human CYP4V2 gene (HGNC:23198) is located at 4q35 and has 11 exons. Mutations in the CYP4V2 gene cause BCD (Li et al., Am J Hum Genet. 74:817-826, 2004). Although CYP4V2 is expressed in almost all tissues, it is expressed at high levels in the retina and RPE and at slightly lower levels in the cornea, tissues that display the main clinical findings of BCD (Li et al., Am J Hum Genet. 74:817-826, 2004; Nakano M, Kelly EJ, Rettie AE: Expression and Characterization of CYP4V2 as a Fatty Acid omega-Hydroxylase. Drug Metab Dispos 2009; Nakano M, Kelly EJ, Wiek C, Hanenberg H, Rettie AE: CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of omega-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant. Mol Pharmacol 2012;82:679-686).

[0013] Because CYP4V2 is a relatively new member of the P450 family and BCD is a rare disease, the function of CYP4V2 has not been extensively studied. Previous studies have shown that the CYP4V2 protein is primarily active in fatty acid metabolism. Abnormalities in fatty acids and their metabolism have been demonstrated in the serum, lymphocytes, and skin fibroblasts of BCD patients (Lee J, Jiao X, Hejtmancik JF, et al.: The metabolism of fatty acids in human Bietti crystalline dystrophy. Invest Ophthalmol Vis Sci 2001; 42: 1707-1714; Lai T, Chu KO, Chan KP, et al.: Alterations in serum fatty acid concentrations and desaturase activities in Bietti crystalline dystrophy unaffected by CYP4V2 genotypes. Invest Ophthalmol Vis Sci 2010; 51: 1092-1097). Another study showed that CYP4V2 is an ω-3-polyunsaturated fatty acid (PUFA) hydroxylase and a P450 highly expressed in the transformed human RPE cell line ARPE-19 (Nakano M, Kelly EJ, Wiek C, Hanenberg H, Rettie AE: CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of omega-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant. Molecular pharmacology 2012; 82: 679-686).

[0014] Numerous mutations have been identified in the CYP4V2 gene and cause BCD, with at least one mutation in each of the gene's 11 exons. The most common CYP4V2 mutation in BCD patients is c.802-8_810del17insGC (referring to a 17-base deletion and a two-base (GC) insertion eight bases from the end of intron 6 of the CYP4V2 gene, also known as IVS6-8del / insGC; see SEQ ID NO:46, which shows the sequence of the human CYP4V2 genomic DNA region containing the c.802-8_810del17insGC mutation; and SEQ ID NO:47, which shows the corresponding wild-type sequence). The c.802-8_810del17insGC mutation is shown in the sequence below showing the human CYP4V2 intron 6-exon 7 junction. The intron 6 sequence is shown in lowercase letters, and the exon 7 sequence is shown in uppercase letters. The 17 bp deletion and GC insertion are in brackets: caa aca gaa gca tgt gat tat cat tca aa(tca tac agG TCA TCG CT)(GC)GAA CGGGCC AAT GAA ATG AAC GCC AAT GA (SEQ ID NO: 46), resulting in skipping of exon 7. (Xiao et al., Biochem Biophys Res Commun. 409: 181-6, 2011; Meng et al., 2014, Mol. Vis., 20: 1806-14; Wada et al., Am J Ophthalmol. 139: 894-9, 2005; Jiao et al., European Journal of Human Genetics (2017) 25, 461-471). Recent studies estimate that the c.802-8_810del17insGC mutation is present for 1,040 to 8,200 generations in Chinese populations and 300 to 1,100 generations in Japanese populations. See Jiao et al., European Journal of Human Genetics (2017) 25, 461-471.

[0015] A variety of CYP4V2 mutations have been found to be associated with BCD, including but not limited to missense, duplication, splice site, frameshift, deletion, insertion, insertion or deletion (indel), nonsense, polymorphism (e.g., single nucleotide polymorphism), and premature termination, as well as deletion of the entire CYP4V2 gene. An overview of selected CYP4V2 mutations in human BCD patients is provided in Table 1 herein and can be found in various publications and online databases, such as LOVD (databases.lovd.nl / shared / genes / CYP4V2 on the World Wide Web), OMIM (omim.org / allelicVariant / 608614 on the World Wide Web), and ClinVar (ncbi.nlm.nih.gov / clinvar?term=608614[MIM] on the World Wide Web).

[0016] Table 1: Selected CYP4V2 mutations in BCD patients

[0017]

[0018]

[0019]

[0020] This is only a selection list and may not include all pathological CYP4V2 mutations / variants identified and reported to date in patients with BCD. Mutations are relative to the reference sequences (NM_207352.3) and (NP_997235.3). New pathological CYP4V2 mutations are continually being identified in patients with BCD. All identified and future pathological CYP4V2 mutations / variants associated with BCD are incorporated herein by reference.

[0021] Inherited Retinal Degeneration (IRD)

[0022] Inherited retinal degenerations (IRDs) are a leading cause of blindness. Over 200 genes are known to be involved in IRDs and related conditions. In humans, retinitis pigmentosa (RP) is the predominant form of IRD. RP has three general inheritance patterns (autosomal dominant, autosomal recessive, and X-linked). The estimated worldwide incidence of RP is 1 in 4,000, with autosomal recessive RP accounting for 50%-60% of RP cases (Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. 2006;368:1795-809). A European study estimated the prevalence of BCD to be 2.5% of all RP patients and approximately 10% of those with nonsyndromic autosomal recessive RP (Mataftsi A, Zografos L, Millá E, Secrétan M, Munier FL. Bietti's crystalline corneoretinal dystrophy: a cross-sectional study. Retina. 2004;24:416-26). The same study also noted that BCD is often commonly diagnosed as RP. Therefore, BCD may have been under-diagnosed. BCD is a worldwide disease, but it is most common in East Asia, particularly in Chinese and Japanese populations (Li et al., Am J Hum Genet. 2004 May;74(5):817-826).

[0023] References for mutations in Table 1:

[0024] Li A,Jiao

[0025] Xiao X,Mai G,Li S,Guo X,Zhang Q(2011)Identification of CYP4V2mutations in 21families and overview of mutation spectrum in Bietti crystallinecorneoretinal dystrophy.Biochem Biophys Res Commun 409:181-186.

[0026] Shan M, Dong B, Zhao X, Wang J, Li G, et al.(2005)Novel mutations in theCYP4V2 gene associated with Bietti crystalline corneoretinal dystrophy.MolVis11:738-743.

[0027] Rossi S, Testa F, Li A, Yaylacioglu F, Gesualdo C, et al.(2013)Clinical and genetic features in Italian Bietti crystalline dystrophy patients.Br JOphthalmol 97:174-179.

[0028] Lin J, Nishiguchi KM, Nakamura M, Dryja TP, Berson EL, et al.(2005)Recessive mutations in the CYP4V2 gene in East Asian and Middle Eastern patients with Bietti crystalline corneoretinal dystrophy.J Med Genet 42:e38.

[0029] Manzouri B,Sergouniotis PI,Robson AG,Webster AR,Moore A(2012)Bietticrystalline retinopathy:report of retinal crystal deposition in maleadolescent siblings.ARCH OPTHALMOL 130:1470-1473.

[0030] Lai TY, Ng TK, Tam PO, Yam GH, Ngai JW, et al.(2007)Genotype phenotype analysis of Bietti's crystalline dystrophy in patients with CYP4V2 mutations.Invest Ophthalmol Vis Sci 48:5212-5220.

[0031] Parravano M,Sciamanna M,Giorno P,Boninfante A,Varano M(2012)Bietticrystalline dystrophy:a morpho-functional evaluation.Doc Ophthalmol 124:73-77.

[0032] Wada Y, Itabashi T, Sato H, Kawamura M, Tada A, et al.(2005)Screening for mutations in the CYP4V2 gene in Japanese patients with Bietti's crystalline corneoretinal dystrophy.Am J Ophthalmol 139:894-899.

[0033] Zenteno JC, Ayala-Ramirez R, Graue-Wiechers F(2008)Novel CYP4V2gene mutation in a Mexican patient with Bietti's crystalline corneoretinal dystrophy.Curr Eye Res 33:313-318.

[0034] Lee KY,Koh AH,Aung T,Yong VH,Yeung K,et al.(2005)Characterization ofBietti crystalline dystrophy patients with CYP4V2 mutations.Invest OphthalmolVis Sci 46:3812-3816.

[0035] Yokoi Y,Sato K,Aoyagi H,Takahashi Y,Yamagami M,et al.(2011)A NovelCompound Heterozygous Mutation in the CYP4V2 Gene in a Japanese Patient 3ithBietti's Crystalline Corneoretinal Dystrophy.Case Rep Ophthalmol 2:296-301.

[0036] Haddad NM,Waked N,Bejjani R,Khoueir Z,Chouery E,et al.(2012)Clinicaland molecular findings in three Lebanese families with Bietti crystallinedystrophy:report on a novel mutation.Mol Vis 18:1182-1188.

[0037] Fu Q,Wang F,Wang H,Xu F,Zaneveld JE,et al.(2013)Next-generationsequencing-based molecular diagnosis of a Chinese patient cohort withautosomal recessive retinitis pigmentosa.Invest Ophthalmol Vis Sci 54:4158-4166.

[0038] Song Y,Mo G,Yin G(2013)A novel mutation in the CYP4V2 gene in a Chinese patient with Bietti's crystalline dystrophy.Int Ophthalmol 33:269-276.

[0039] Jin ZB, Ito S, Saito Y, Inoue Y, Yanagi Y, et al.(2006)Clinical and molecular findings in three Japanese patients with crystalline retinopathy.Jpn J Ophthalmol50:426-431.

[0040] Halford S,Liew G,Mackay DS,Sergouniotis PI,Holt R,Broadgate S,VolpiEV,Ocaka L,Robson AG,Holder GE,Moore AT,Michaelides M,Webster AR crystalline dystrophy.Ophthalmology.2014:121:1174-84

[0041] Molecular Analysis and Phenotypic Study in 14 Chinese Families With Bietti Crystalline Dystrophy.PLoSOne 9(4),e94960.2014 Apr

[0042] Xiao Hong Meng,Hong Guo,Hai Wei Xu,Qi You Li,Xin Jin,Yun Bai,Shi YingLi,Zheng Qin Yin,Identification of novel CYP4V2 gene mutations in 92Chinesefamilies with Bietti's crystalline corneoretinal dystrophy,Molecular Vision(2014):20:1806-1814

[0043] Galuh DN Astuti,Vincent Sun,Miriam Bauwens,Ditta Zobor,Bart P Leroy,Amer Omar,Bernhard Jurklies,Irma Lopez,Huanan Ren,Volkan Yazar,ChristianHamel,Ulrich Kellner,Bernd Wissinger,Susanne Kohl,Elfride De Baere,Rob Colinkoop,Robert WJNovel,Robert Kolene,Kolene,Irma Lopez insights into the molecular pathogenesisof CYP4V2-associated Bietti's retinal dystrophy,Mol Genet GenomicMed.2015January;3(1):14-29.

[0044] Xiaodong Jiao,Anren Li,Zi-Bing Jin,Xinjing Wang,AlessandroIannaccone,Elias I Traboulsi,Michael B Gorin,Francesca Simonelli and JFielding Hejtmancik,Identification and Population History of CYP4V2 mutationsin Patients with Cryp4V2 Crystalline,Europeanneurope, Journalof Human Genetics(2017)25,461-471. [Summary of the invention]

[0045] Summary of the Invention

[0046] Cell line claims

[0047] Cell line and disease model claims

[0048] Cell line composition

[0049] In one aspect, a cellular disease model is provided, comprising a cell line. Such a disease model comprises (a) a stem cell provided by a subject or a stem cell reprogrammed from a cell provided by a subject, or (2) a cell derived from a stem cell provided by a subject or a stem cell reprogrammed from a cell provided by a subject, comprising one or more mutations in a target gene.

[0050] In some embodiments, stem cells are induced pluripotent stem (induced pluripotent stem, iPS) cells. In some embodiments, stem cells are embryonic stem (ES) cells, somatic (or adult) stem cells or mesenchymal stem cells (MSC). In some embodiments, the cells provided by the subject belong to any cell type and / or any tissue from the subject. In some embodiments, the cells provided by the subject are skin cells, fibroblasts or blood cells. In some embodiments, the cells wherein provided by the subject are skin fibroblasts or peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC). In some embodiments, the cells provided by the subject are urinary cells (urinary cell), renal epithelial cells, hair follicles or dermal papilla cells (dermal papilla cell).

[0051] In some embodiments, the cell derived from stem cells is an ocular cell. In some embodiments, the ocular cell is a retinal pigment epithelium (RPE) cell, a photoreceptor cell (PRC, including rods, cones, and photoreceptor progenitor cells), a retinal cell, a keratocyte, a corneal epithelial cell (CEC), a retinal nerve cell, a lens cell, a choroidal endothelial (CE) cell, a retinal nerve cell, or a choroidal cell. In some embodiments, the cell derived from stem cells is a neuronal cell.

[0052] In some embodiments, the mutation is endogenous to the subject. In some embodiments, the mutation is exogenous to the subject. In some embodiments, the mutation is artificially introduced via gene editing or genetic manipulation. In some embodiments, the cell line comprises a plurality of mutations that are endogenous and / or exogenous to the subject.

[0053] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0054] In some embodiments, the target gene comprises a gene set forth in Table 4. In some embodiments, the target gene comprises a mutated or defective CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNR D2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SY NJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A genes, or CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNR, or proteins encoding proteins with defective or partial function or activity In some embodiments, the target gene is CYP4V2.

[0055] In some embodiments, cell line comprises iPS cell.In some embodiments, cell line comprises iPS-RPE cell.In some embodiments, cell line comprises iPS-photoreceptor (iPS-PRC) cell, iPS-corneal epithelial cell (iPS-CEC), iPS-choroidal endothelial (CE) cell, iPS-keratocyte, iPS-choroidal cell, iPS-optic nerve cell, iPS-eye cell or iPS-neuron cell.In some embodiments, the CYP4V2 mutation in cell line is endogenous to experimenter.In some embodiments, experimenter has pathological mutation in CYP4V2 gene or in the ortholog (ortholog) of CYP4V2 gene.

[0056] In some embodiments, the subject has at least one mutation set forth in Table 1. In some embodiments, the subject has an inherited retinal degeneration (IRD) or retinitis pigmentosa (RP). In some embodiments, the subject has or is at risk for developing crystalline retinitis pigmentosa (BCD, also known as crystalline keratoretinal degeneration, crystalline retinopathy, Bietti retinal degeneration).

[0057] In some embodiments, the cell line comprises a CYP4V2 mutation that is exogenous to the subject and was artificially introduced via gene editing or genetic manipulation.

[0058] In some embodiments, cell line comprises iPS cell, ES cell, MSC or adult stem cell, or is RPE cell, photoreceptor cell, corneal epithelial cell, choroidal endothelial (CE) cell or choroid cell derived from iPS cell, ES cell, MSC or adult stem cell.In some embodiments, the stem cell of iPS cell or other types is characterized by following one or more:A.iPS, ES or MSC unique form;B. one or more pluripotency markers, such as Oct-4, Sox-2, SSEA4, TRA-1-60, TRA-1-81, NANOG and AP;C. be divided into the ability of desired cell type (for example, RPE);And / or d. teratoma analysis (terotoma assay).

[0059] In some embodiments, iPS-RPE cells or RPE cells derived from other types of stem cells are characterized by: a. morphology: pigment and hexagonal shape, and / or b. one or more of the following biomarkers: retinaldehyde-binding protein 1 (RLBP1, also known as CRALBP), RPE65, BESTROPHIN-1, MITF, LRAT, RDH5, PAX6, MERTK, TYR, ZO-1 and / or VINCULIN.

[0060] In another aspect, a BCD human cell model or a CYP4V2 functional cell model is provided. Such models include iPS cells or iPS cell lines comprising cells or cell lines derived from BCD patients or cells or cell lines derived from cells or cell lines with artificially produced CYP4V2 mutations, or iPS-RPE cells or iPS-RPE cell lines.

[0061] In some embodiments, the cell line has an abnormal biochemical profile compared to a corresponding cell line of a healthy control group for one or more compounds in the following group of compounds: (i) fatty acids, (ii) ceramides, (iii) sphingomyelins, (iv) sphingosines, (v) dihydrosphingosine, or (vi) hydroxy-fatty acids. In some embodiments, the cell line has an abnormal biochemical profile compared to a corresponding cell line of a healthy control group for one or more compounds set forth in Table 2.

[0062] Methods for making cell disease models:

[0063] In another aspect, methods for producing an iPS-derived BCD disease model are provided. Such methods include: obtaining cells from a subject having an endogenous mutation in the CYP4V2 gene, or obtaining cells that do not have an endogenous mutation in the CYP4V2 gene but have an exogenous CYP4V2 mutation artificially introduced through gene editing or genetic manipulation at this or any subsequent stage; inducing pluripotency in these cells or reprogramming these cells to generate iPSCs; and culturing the iPSCs under conditions that cause the iPSCs to differentiate into desired ocular cells, thereby generating an iPS-derived ocular cell line.

[0064] In some embodiments, the cell obtained from the subject is a somatic cell. In some embodiments, the cell obtained from the subject is a skin cell, a fibroblast, a blood cell, a peripheral blood mononuclear cell (PBMC) or an eye cell. In some embodiments, the cell obtained from the subject is a urinary tract cell, a renal epithelial cell, a hair follicle or a dermal papilla cell. In some embodiments, the eye cell is a retinal pigment epithelium (RPE) cell, a corneal epithelial cell (CEC), a photoreceptor cell (PRC), a choroidal endothelial (CE) cell, a retinal cell, a keratocyte or a choroid cell. In some embodiments, one or more of OCT4, SOX2, KLF4 and c-MYC transcription factors are used to induce pluripotency or reprogrammed cells.

[0065] In some embodiments, the mutation is pathological. In some embodiments, the cell line comprises one or more of the mutations set forth in Table 1. In some embodiments, the cell line is heterozygous for the mutation. In some embodiments, the cell line is homozygous for the mutation.

[0066] In some embodiments, the cellular disease model shows abnormal levels of one or more compounds from the following groups of compounds: (i) fatty acids, (ii) ceramides, (iii) sphingomyelins, (iv) sphingosines, (v) dihydrosphingosines, or (vi) hydroxy-fatty acids, compared to the levels in the relevant cell line of the healthy control group. In some embodiments, the cellular disease model shows abnormal levels of one or more compounds set forth in Table 2 in the relevant cell line compared to the levels in the healthy control group.

[0067] Biochemical analysis methods:

[0068] In one aspect, a method for discovering an abnormality or phenotype in a cell model of a disease is provided. Such methods generally comprise assessing and comparing the levels of one or more compounds between a patient cell line (or a cell line that has been genetically edited or manipulated to contain an exogenous mutation in the gene causing the disease) and a cell line of a healthy control group, wherein the one or more compounds are selected from the group consisting of: (i) fatty acids, (ii) ceramides, (iii) sphingomyelins, (iv) sphingosines, (v) dihydrosphingosines, and / or (vi) hydroxy-fatty acids.

[0069] In some embodiments, one or more of the compounds evaluated are described in Table 2. In some embodiments, identification and / or evaluation of compound levels are performed using LC-MS, LC-MS / MS, GC-MS, GC-MS / MS, and / or FIA-MS / MS. In some embodiments, the disease cell model comprises a mutated or defective gene as described in Table 4. In some embodiments, the disease cell model comprises a mutated or defective gene in the following genes: CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TE K, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA , SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A genes.

[0070] Methods using the BCD cell model (drug, dose, and device screening)

[0071] In another aspect, a method for screening a test agent for the therapeutic efficacy for BCD is provided.Such methods generally include: making the cell from the iPS-RPE cell line derived from BCD patient or from the cell of the iPS-RPE cell line comprising mutation or defective CYP4V2 gene because of artificial gene editing or operation contact with test agent;And these cells are assessed for the following: compared to before contacting the test agent, the standardization of the level of one or more compounds set forth in table 2;The increase of the non-defective CYP4V2 nucleotide sequence in these cells;The increase of the amount of CYP4V2 polypeptide in these cells;And / or improved cell structure, form or function;Wherein compared to before being processed with the test agent, the standardization of the level of one or more compounds set forth in table 2;The increase of the non-defective CYP4V2 nucleotide sequence in these cells;The increase of the amount of CYP4V2 polypeptide in these cells;And / or improved cell structure, form or function, show that test agent presents the therapeutic efficacy for BCD.

[0072] In some embodiments, the test agent is selected from the group consisting of: a nucleic acid or its analogue, a vector containing a nucleic acid sequence or encoding a polypeptide, a polypeptide or its analogue, an antibody, a chemical substance, a small molecule and / or any combination thereof. In some embodiments, the cells are evaluated using PCR technology, immunoassays, sequencing, biochemical assays, functional assays, microscopy or a combination thereof.

[0073] In another aspect, a method of screening a formulation, vector, or construct comprising a test agent for BCD for efficacy or efficiency is provided. Such methods generally include: contacting multiple cell samples from iPS-RPE cell lines derived from BCD patients or multiple cell samples from iPS-RPE cell lines containing mutated or defective CYP4V2 genes due to artificial gene editing or manipulation with test agents formulated or packaged in various preparations, carriers or constructs; and evaluating these cell samples for the following: standardization of the levels of one or more compounds described in Table 2 compared to cell samples before treatment with the test agent and / or compared to cell samples treated with the same but formulated or packaged in different preparations, carriers or constructs; an increase in non-defective CYP4V2 nucleic acid sequences in these cells; an increase in the amount of CYP4V2 polypeptides in these cells; improved cell structure, morphology or function; and / or cell tolerance or death to measure and compare the efficiency or efficacy of the preparation, carrier or construct; wherein these cells are evaluated using PCR technology, immunoassays, sequencing, biochemical assays, cell viability assays, microscopy or a combination thereof.

[0074] In one aspect, a method for the effective and safe dose range of the test agent for screening BCD is provided.Such methods generally include: making multiple cell samples from the iPS-RPE cell line derived from BCD patients or from multiple cell samples of the iPS-RPE cell line comprising mutation or defective CYP4V2 gene because of artificial gene editing or operation and test agent with different doses for each cell sample contact;Assess these cell samples for the following: compared to before being treated with the test agent and / or compared to using the same test agent but with different doses of the cell sample, the standardization of the level of one or more compounds set forth in Table 2;The increase of the non-defective CYP4V2 nucleotide sequence in these cells;The increase of the amount of the CYP4V2 polypeptide in these cells;Improved cell structure, morphology or function and / or cell tolerance or death, in order to measure and compare the effectiveness and safety of different doses, so as to determine appropriate dosage range;Wherein these cells use PCR technology, immunoassay, sequencing, biochemical analysis, cell viability analysis, functional analysis, microscopy or its combination to assess.

[0075] In another aspect, a method for screening a delivery device or method for delivering a therapeutic agent to the retina or retinal cells or evaluating its efficacy or efficiency is provided. Such methods generally include: (i) contacting a cell sample from an iPS-RPE cell line derived from a BCD patient or a cell sample from an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene due to artificial gene editing or manipulation with a test agent without using the delivery device or method; (ii) contacting another cell sample from an iPS-RPE cell line derived from a BCD patient or another cell sample from an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene due to artificial gene editing or manipulation with the test agent at the same dose as in (i) using the delivery device or method; (iii) contacting the test agent with ... delivery device or method; (iv) contacting the test agent with the test agent with the test agent with the Each evaluates and compares cell samples from (i) and (ii) for: normalization of the levels of one or more compounds described in Table 2 compared to before treatment with the test agent and / or with the same test agent at the same dose but without the delivery device or method; an increase in non-defective CYP4V2 nucleic acid sequences in the cells; an increase in the amount of CYP4V2 polypeptide in the cells; improved cell structure, morphology or function; cell tolerance or death; and / or the levels of the test agent in the cells, thereby determining the efficacy or efficiency of the delivery device or technology; wherein the cells are evaluated using PCR technology, immunoassays, sequencing, biochemical assays, functional assays, microscopy, or a combination thereof.

[0076] In some embodiments, the retinal cells are RPE cells.

[0077] CRISPR gene editing therapy

[0078] In one aspect, a composition is provided, comprising: (a) a CRISPR guide RNA that targets a nucleic acid sequence within 100 bp of a CYP4V2 gene ("target sequence"), and (b) a functional CRISPR-associated protein (Cas). In some embodiments, such a composition may further comprise (c) a donor nucleic acid sequence comprising all or a portion of a wild-type sequence or a functional sequence of the CYP4V2 gene, which is used to correct, disrupt, or replace the CYP4V2 gene or a portion thereof.

[0079] In some embodiments, one or more components thereof are provided in the following form: a DNA molecule encoding the component, an mRNA molecule encoding the component, an RNA molecule, a polypeptide and / or a ribonucleoprotein (RNP) or a protein-RNA complex. In some embodiments, two or more components thereof are in separate molecules or combined in one molecule or a complex, in a separate vector or combined in one vector, in one or more nucleic acid complexes, in one or more RNP complexes. In some embodiments, the donor nucleic acid sequence is provided in the form of a single-stranded donor oligonucleotide (ssODN) or a vector. In some embodiments, the vector is a plasmid, a recombinant AAV vector, a recombinant lentiviral vector and / or a combination thereof.

[0080] In some aspects, a composition comprising a cell having a pathological CYP4V2 mutation is provided, comprising any one of the compositions described herein. In some embodiments, (a) the CRISPR guide RNA comprises (i) CRISPR RNA (crRNA) comprising a protospacer element sequence complementary to a target sequence within 100 bp or less of a target gene ("target gene") and a sequence corresponding to a complementary region of a transactivating crRNA (tracrRNA), and (ii) tracrRNA comprising a region complementary to a corresponding region of the crRNA and a sequence that interacts with CRISPR-associated protein 9 (Cas9), and (b) the functional CRISPR-associated protein comprises Cas9.

[0081] In some embodiments, the protospacer element is about 20 bases, about 19 bases, about 21 bases, about 19-21 bases, about 18-22 bases, or about 16-24 bases. In some embodiments, crRNA and tracrRNA are in separate molecules. In some embodiments, crRNA and tracrRNA are combined into a single guide RNA (single guide RNA, sgRNA). In some embodiments, sgRNA is about 88-150bp.

[0082] In some embodiments, the Cas9 comprises a Cas9 ortholog or mutant Cas9 selected from the group consisting of: Streptococcus pyogenes (SpCas9), SpCas9 nickase (Cas9nD10A), SpCas9 (D1135E), eSpCas9, SpCas9-HF1, SpCas9 VRER, SpCas9VQR, SpCas9EQR, Staphylococcus aureus (SaCas9), Neisseria Meningitidis, Streptococcus thermophilus, Streptococcus pneumoniae, Campylobacter coli, Campylobacter jejuni, Streptococcus mutans, Pasteurella multocida, Bifidobacterium longum longum), Bacillus smithii, Treponema denticola, Mycoplasma canis, and Enterococcus faecalis. In some embodiments, the CRISPR-associated protein Cas9 or Cpf1 further comprises one, two, three, or more nuclear localization sequences (NLS) located at the N-terminus and / or C-terminus, and / or a selection marker, including but not limited to GFP or EGFP.

[0083] In some embodiments, (a) the CRISPR guide RNA comprises a crRNA comprising a protospacer element sequence that is complementary to a target sequence at or within 100 bp of a target gene, and (b) the functional CRISPR-associated protein comprises Cpf 1. In some embodiments, the protospacer element is about 20 bases, about 21 bases, about 22 bases, about 23 bases, about 24 bases, about 19-25 bases, about 18-26 bases, or about 16-28 bases.

[0084] In some embodiments, the protospacer element sequence is selected from the group consisting of SEQ ID NOs: 48 to 52, or shares at least 85% sequence identity with one of SEQ ID NOs: 48 to 52, and is used with a Cas protein having NGG as a protospacer adjacent motif (PAM) to target the c.802-8_810del17insGC mutation of the CYP4V2 gene. In some embodiments, the donor nucleic acid sequence is selected from SEQ ID NOs: 56 and 57 (which are two donor template sequences), or shares at least 90% sequence identity with one of SEQ ID NOs: 56 and 57, or their complements, and is used to correct, disrupt, or replace the c.802-8_810del17insGC mutation of the CYP4V2 gene.

[0085] CRISPR gene therapy method claims

[0086] In another aspect, a method for treating or preventing BCD in a subject or cell having a mutated CYP4V2 gene is provided. Such a method comprises (i) identifying a pathological mutation in the subject or cell by sequencing; (ii) finding a Cas-associated PAM site within a region spanning from about 100 bp upstream of the first nucleotide involved in the mutation to about 100 bp downstream of the last nucleotide involved in the mutation; (iii) identifying various protospacer element sequences targeting the CYP4V2 sequence associated with each PAM site identified in (ii); (iv) assessing the activity level and off-target editing profile of each CRISPR guide RNA comprising the protospacer element sequence identified in (iii) based on the protospacer element sequence and PAM; (v) selecting one or more CRISPR guide RNA designs based on (iv); (vi) determining the activity level and off-target editing profile of each CRISPR guide RNA based on (iv); and (vii) determining the activity level and off-target editing profile of each CRISPR guide RNA based on (iv). (vii) constructing the CRISPR guide RNA, Cas and donor nucleic acid sequence as provided in claims 1 to 18 of the composition; (viii) optionally validating and further selecting the components of (vii) in cells isolated from the subject, or iPS cells derived from the subject, or cells differentiated from stem cells derived from the subject, or genomic DNA isolated from the subject, or cells isolated or derived therefrom, to assess the activity level and / or off-target editing spectrum; and ( ix) administering the components in (viii) to the subject or the cell via a delivery system selected from the group consisting of: ribonucleoprotein or protein-RNA complexes, vectors, proteins, nucleic acid molecules, nanoparticles, liposomes, micelles, virions, nucleic acid complexes and / or combinations thereof, wherein the delivery is by electroporation or via lipid-mediated transfection or nucleofection or viral transduction or injection or a combination thereof; (x) wherein for treatment in cells in vitro, a selection marker is optionally added or added to the components in (viii), including but not limited to GFP, EGFP or puromycin resistance.

[0087] In one aspect, a gene editing composition is provided for correcting or replacing the c.802-8_810del17insGC mutation in the CYP4V2 gene in vivo or in cells in vitro. Such compositions generally include: (i) a CRISPR guide RNA comprising a protospacer element sequence selected from one of SEQ ID NOs: 48 to 52 or sharing at least 80% sequence identity with one of the sequences in SEQ ID NOs 48 to 52; (ii) a donor nucleic acid sequence selected from one of SEQ ID NOs: 56 and 57 or sharing at least 90% sequence identity with one of SEQ ID NOs: 56 and 57 or their complements; and (iii) a Cas9 protein (an exemplary sequence is shown in SEQ ID NO: 58), optionally containing 1, 2, 3 or more NLSs, and / or a selection marker, including but not limited to GFP or EGFP.

[0088] In some embodiments, an optional nucleotide G is added before the protospacer element sequence. In some embodiments, the CRISPR guide RNA comprises a crRNA (exemplary sequence (excluding the 5' protospacer element sequence) is shown in SEQ ID NO: 53) and a tracrRNA (exemplary sequence is shown in SEQ ID NO: 54); and the protospacer element sequence is contained in the crRNA. In some embodiments, the CRISPR guide RNA comprises a single guide RNA (sgRNA) containing a protospacer element sequence (exemplary sgRNA sequence (excluding the 5' protospacer element sequence) is shown in SEQ ID NO: 55).

[0089] In some embodiments, one or more components of (i), (ii) and (iii) are provided in the form of a DNA molecule encoding the component, an mRNA molecule encoding the component, a nucleic acid molecule, a vector, an RNA molecule, a polypeptide, a ribonucleoprotein (RNP) or a protein-RNA complex and / or a combination thereof.

[0090] BCD cell therapy, autologous cell therapy for ocular diseases, and combined therapeutic claims

[0091] BCD Cell Therapy

[0092] Allogeneic or autologous cell therapy for BCD without gene repair

[0093] In some aspects, a method for treating or preventing an eye disease of a subject is provided, wherein the disease is associated with a pathological hereditary or epigenetic change in a CYP4V2 gene. Such methods generally include administering a cell composition to the subject, wherein the cell composition includes: retinal pigment epithelium (RPE) cells, photoreceptors or photoreceptor progenitor cells (PRCs), corneal epithelial cells (CECs), choroidal endothelial (CE) cells and / or other eye cells derived from stem cells.

[0094] In some embodiments, the stem cells are embryonic stem (ES) cells, iPC cells, MSCs, adult stem cells, or tissue-specific stem cells. In some embodiments, the stem cells are derived from or are derived from one or more subjects that do not have BCD or do not have a pathological CYP4V2 gene. In some embodiments, the stem cells are derived from or are derived from one or more subjects that have a pathological mutation in the CYP4V2 gene. In some embodiments, the subject is a human individual.

[0095] Autologous cell therapy for gene repair of BCD

[0096] In another aspect, a cellular composition is provided comprising (a) stem cells reprogrammed from cells isolated from a subject having BCD or a pathological mutation in a CYP4V2 gene or stem cells isolated from the subject, or (b) cells differentiated from stem cells isolated from a subject having BCD or a pathological mutation in a CYP4V2 gene or reprogrammed from cells isolated from the subject.

[0097] In some embodiments, the stem cell from the cell reprogramming separated from the experimenter is iPC cell.In some embodiments, iPS cell is reprogrammed from any cell of any tissue of the experimenter.In some embodiments, iPS cell is reprogrammed from skin cell, blood cell, urinary tract cell, hair cell, fibroblast, peripheral blood mononuclear cell (PBMC), renal epithelial cell, hair follicle or dermal papilla cell.In some embodiments, the stem cell separated from the experimenter is MSC, adult stem cell or tissue-specific stem cell.In some embodiments, the cell from stem cell differentiation is eye cell.In some embodiments, the cell from stem cell differentiation is RPE cell, PRC, retinal cell, keratocyte, choroid cell, CEC or CE cell.In some embodiments, the cell from stem cell differentiation is iPS-RPE, iPS-PRC, iPS-CEC or iPS-CE cell.

[0098] In some embodiments, gene repair is performed on the following cells to improve the effects of a mutated CYP4V2 gene: (i) cells isolated from a subject with BCD or a pathological mutation in the CYP4V2 gene for reprogramming into iPSCs; (ii) stem cells isolated from a subject with BCD or a pathological mutation in the CYP4V2 gene or iPS cells reprogrammed from cells isolated from the subject; or (iii) cells differentiated from stem cells isolated from a subject with BCD or a pathological mutation in the CYP4V2 gene or cells differentiated from iPS cells reprogrammed from cells isolated from the subject. In some embodiments, gene repair is performed before reprogramming into iPS cells. In some embodiments, gene repair is performed after reprogramming into iPS cells. In some embodiments, gene repair is performed before differentiation of stem cells or iPS cells. In some embodiments, gene repair is performed after differentiation of stem cells or iPS cells. In some embodiments, gene repair is performed via gene transfer therapy. In some embodiments, gene repair is performed via gene transfer therapy, by using any composition or method of any one of the gene therapy claims. In some embodiments, gene repair is via gene editing. In some embodiments, gene repair is via gene editing, by using any composition or method of any one of the CRiSPR gene therapy claims.

[0099] In another aspect, a method for treating or preventing an eye disease in a subject with BCD or a pathological genetic or epigenetic change in the CYP4V2 gene is provided. Such methods generally include administering to the subject any one of the CYP4V2 autologous cell compositions described herein, wherein the cell composition includes: retinal pigment epithelium (RPE) cells, photoreceptors or photoreceptor progenitor cells (PRCs), corneal epithelial cells (CECs), choroidal endothelial (CE) cells, and / or other eye cells derived from stem cells of the subject.

[0100] In some embodiments, the stem cells are iPC cells, MSCs, adult stem cells, or tissue-specific stem cells. In some embodiments, iPS cells are reprogrammed using one or more of OCT4, SOX2, KLF4, and c-MYC transcription factors. In some embodiments, the gene-repaired cells exhibit one or more of the following compared to before gene repair: normalization of the levels of one or more compounds described in Table 2; an increase in non-defective CYP4V2 nucleic acid sequences in these cells; an increase in the amount of functional CYP4V2 polypeptides in these cells; and / or improved cell structure, morphology, or function.

[0101] In some embodiments, the amount of cells administered in a single dose is about 1,000 to about 100 million cells. In some embodiments, administration is via injection. In some embodiments, administration is via subretinal injection. In some embodiments, administration is via intravitreal injection. In some embodiments, administration is via direct retinal injection. In some embodiments, administration is via corneal injection. In some embodiments, administration is by any other method of administration that effectively delivers cells to the subretinal position, posterior segment, or cornea of ​​the subject's eye. In some embodiments, cells are administered via injection of a cell suspension. In some embodiments, cells are administered as part of a sheet, matrix, scaffold, or tissue.

[0102] In some embodiments, RPE cells are administered using natural and / or synthetic scaffolds to produce a functional RPE monolayer.In some embodiments, the subject is a human subject.

[0103] Autologous cell therapy for gene repair of ocular diseases

[0104] In another aspect, a cellular composition is provided comprising (a) stem cells reprogrammed from cells isolated from a subject affected by a disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity, or (b) cells differentiated from stem cells isolated from a subject affected by a disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity, or reprogrammed from cells isolated from a subject affected by a disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity.

[0105] In some embodiments, the stem cells reprogrammed from cells isolated from the subject are iPS cells. In some embodiments, iPS cells are reprogrammed from any cells of any tissue of the subject. In some embodiments, iPS cells are reprogrammed from skin cells, blood cells, urinary tract cells, hair cells, fibroblasts, peripheral blood mononuclear cells (PBMC), renal epithelial cells, hair follicles, or dermal papilla cells. In some embodiments, the stem cells isolated from the subject are MSCs, adult stem cells, or tissue-specific stem cells.

[0106] In some embodiments, the gene is involved in eye development or function and / or mutation thereof that causes an eye disease or is a risk factor for causing an eye disease. In some embodiments, the gene is involved in neuronal development or function and / or mutation thereof that causes a neurodegenerative disease or is a risk factor for causing a neurodegenerative disease. In some embodiments, the gene is a cytochrome P450 gene. In some embodiments, the gene is a gene set forth in Table 4.

[0107] In some embodiments, the genes include mutated or defective CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNR D2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SY NJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A genes, or CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNR, or proteins encoding proteins with defective or partial function or activity D2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SY NJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A genes.

[0108] In some embodiments, the cell differentiated from stem cells is any type of cell. In some embodiments, the cell differentiated from stem cells is an eye cell. In some embodiments, the cell differentiated from stem cells is an RPE cell, PRC, retinal cell, corneal cell, choroid cell, CEC, CE cell or optic nerve cell. In some embodiments, the cell differentiated from stem cells is iPS-RPE, iPS-PRC, iPS-CEC or iPS-CE cell. In some embodiments, the cell differentiated from stem cells is a neuron.

[0109] In some embodiments, gene repair is performed on the following cells to ameliorate the effects of a mutated or defective gene: (i) cells isolated from a subject affected by a disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity for reprogramming into iPSCs; (ii) stem cells isolated from a subject affected by a disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity, or iPS cells reprogrammed from cells isolated from the subject; or (iii) cells differentiated from stem cells isolated from a subject affected by a disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity, or iPS cells reprogrammed from cells isolated from the subject.

[0110] In some embodiments, gene repair is performed before reprogramming into iPS cells. In some embodiments, gene repair is performed after reprogramming into iPS cells. In some embodiments, gene repair is performed before differentiation of stem cells or iPS cells. In some embodiments, gene repair is performed after differentiation of stem cells or iPS cells. In some embodiments, gene repair is performed via gene transfer therapy. In some embodiments, gene repair is performed via gene transfer therapy, by using any composition or method of any one of the claims related to gene therapy. In some embodiments, gene repair is performed via gene editing therapy. In some embodiments, gene repair is performed via gene editing therapy, by using any composition or method of any one of the claims related to CRiSPR gene therapy.

[0111] In another aspect, a method of treating or preventing a disease in a subject affected by a disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity as set forth in Table 4 is provided. Such methods generally comprise administering to the subject an autologous cell composition as described herein, wherein the cell composition comprises: retinal pigment epithelium (RPE) cells, photoreceptors or photoreceptor progenitor cells (PRCs), corneal epithelial cells (CECs), neurons, choroidal endothelial (CE) cells, and / or other ocular cells derived from stem cells of the subject, and wherein the mutated or defective gene in the cell composition has been genetically repaired.

[0112] In another aspect, a method of autologously treating a subject is provided. Such methods generally include (i) providing cells from a subject having an ocular disease; (ii) inducing pluripotency in the cells from the subject to generate iPSCs; (iii) genetically repairing one or more mutations in the mutated or defective genes described in Table 4 in the iPSCs derived from the subject; (iv) differentiating the iPSCs into ocular cells; (v) as an alternative to step (iii), genetically repairing the iPS-ocular cells via gene transfer therapy; and (vi) introducing the iPS-ocular cells into the subject, thereby autologously treating the subject having the ocular disease.

[0113] In some embodiments, the stem cells are iPC cells, MSCs, adult stem cells, or tissue-specific stem cells. In some embodiments, iPS cells are reprogrammed using one or more of OCT4, SOX2, KLF4, and c-MYC transcription factors. In some embodiments, the gene-repaired cells exhibit one or more of the following compared to before gene repair: an increase in the number of non-defective target gene nucleic acid sequences in these cells; an increase in the amount of functional polypeptides encoded by the target gene in these cells; improved cell structure, morphology, or function; and / or improved or standardized biochemical functions in these cells. In some embodiments, the amount of cells administered in a single dose is about 1,000 to about 100 million cells.

[0114] In some embodiments, administration is via injection. In some embodiments, administration is by subretinal injection. In some embodiments, administration is by intravitreal injection. In some embodiments, administration is by direct retinal injection. In some embodiments, administration is by corneal injection. In some embodiments, administration is by any other method of administration that effectively delivers cells to the subretinal position, posterior segment or cornea of ​​experimenter's eyes. In some embodiments, cells are administered via injection cell suspension. In some embodiments, cells are administered as a part for lamella, matrix, support or tissue. In some embodiments, natural and / or synthetic support is used to administer RPE cells to produce functional RPE monolayer. In some embodiments, experimenter is a human experimenter.

[0115] In some embodiments, the disease is associated with a genetic or epigenetic change or risk factor in the subject. In some embodiments, the disease is photoreceptor degeneration, retinal pigment epithelial degeneration, retinal degeneration, corneal degeneration and / or choroidal disorder. In some embodiments, the disease is hereditary retinal degeneration (IRD). In some embodiments, the disease is retinitis pigmentosa (RP). In some embodiments, the disease is crystalline retinitis pigmentosa (also known as crystalline corneal retinal degeneration; BCD). In some embodiments, the disease is associated with neurodegeneration. In some embodiments, the disease is corneal degeneration. In some embodiments, the subject has BCD or is at risk of developing BCD.

[0116] In some embodiments, these cells are fibroblasts, hemocytes or eye cells. In some embodiments, these cells are obtained from urine or from hair or hair follicles. In some embodiments, eye cells are retinal pigment epithelial (RPE) cells, corneal epithelial cells (CEC), choroidal endothelial (CE) cells or photoreceptor cells (PRC).

[0117] In some embodiments, the genetic or epigenetic changes are selected from the group consisting of: mutations, insertions, single nucleotide polymorphisms, improper methylation, improper demethylation, and combinations thereof. In some embodiments, the genetic or epigenetic changes are mutations. In some embodiments, the genetic or epigenetic changes in the subject's iPS- eye cells have been genetically repaired using gene editing. In some embodiments, the gene editing method utilizes zinc finger nucleases, TALEN technology, or CRISPR technology. In some embodiments, the genetic or epigenetic changes in the subject's iPSC- eye cells have been genetically repaired using gene transfer. In some embodiments, the gene transfer method utilizes a recombinant AAV vector or another viral vector or non-viral vector to deliver a healthy copy of the target gene (e.g., cDNA) to the cells to be transplanted.

[0118] In some embodiments, the administering step occurs before the onset of disease symptoms or occurs after the onset of disease symptoms. In some embodiments, administration is performed into the eye or into another organ or tissue containing neurons. In some embodiments, administration is performed by injection. In some embodiments, administration is performed by subretinal or intravitreal injection. In some embodiments, administration is performed by direct retinal injection. In some embodiments, administration is performed by corneal injection. In some embodiments, administration is performed by any other method of administration that effectively delivers cells to a subretinal location, posterior segment, or cornea of ​​the subject's eye.

[0119] In some embodiments, the method further comprises: prior to administration or transplantation, genotyping the cells to identify the presence or absence of a genetic or epigenetic alteration in one or more genes listed in Table 4. In some embodiments, the genetic or epigenetic alteration is a mutation. In some embodiments, the mutation is in a CYP4V2 nucleic acid molecule. In some embodiments, the method further comprises: prior to administration, assessing the subject's eye to identify the area and extent of damaged or remaining photoreceptors, retinal cells, or corneal cells.

[0120] In some embodiments, the method further comprises monitoring the subject after administration. In some embodiments, monitoring comprises performing non-invasive retinal imaging, corneal testing, perimetry, ERG, OCT, visual acuity testing, and / or functional studies. In some embodiments, monitoring comprises assessing the subject's immune response. In some embodiments, the method further comprises: after administration, assessing the subject's eye to identify the area and extent of damaged or residual photoreceptors, retinal cells, or corneal cells.

[0121] Cell Therapy-RNP Claims

[0122] RNP claim

[0123] In another aspect, a composition is provided comprising: (a) a CRISPR guide RNA that targets a nucleic acid sequence ("target sequence") that is 100 bp or within 100 bp of a target gene ("target gene"), and (b) a functional CRISPR-associated protein in a ribonucleoprotein (RNP) or protein-RNA complex.

[0124] In some embodiments, the composition further comprises (c) a donor nucleic acid sequence comprising all or part of a wild-type sequence or functional sequence of a target gene, which is used to correct or replace the target gene or a part thereof. In some embodiments, the target gene is involved in eye development or function and / or its mutation causes an eye disease or is a risk factor for causing an eye disease. In some embodiments, the target gene is involved in neuronal development or function and / or its mutation causes a neurodegenerative disease or is a risk factor for causing a neurodegenerative disease.

[0125] In some embodiments, the target gene is a cytochrome P450 gene. In some embodiments, the target gene includes a gene set forth in Table 4, which is mutated or defective, or encodes a protein with defective or partial function or activity. In some embodiments, the donor nucleic acid sequence is provided in the form of a single-stranded donor oligonucleotide (ssODN) or a vector.

[0126] In some embodiments, (a) the CRISPR guide RNA comprises (i) a CRISPR RNA (crRNA) comprising a protospacer element sequence complementary to a target sequence at or within 100 bp of a target gene and a sequence corresponding to a complementary region of a trans-activating crRNA (tracrRNA), and (ii) a tracrRNA comprising a region complementary to a corresponding region of the crRNA and a sequence that interacts with CRISPR-associated protein 9 (Cas9), and (b) the functional CRISPR-associated protein comprises Cas9.

[0127] In some embodiments, the protospacer element is about 20 bases, about 19 bases, about 21 bases, about 19-21 bases, about 18-22 bases, or about 16-24 bases. In some embodiments, crRNA and tracrRNA are in different nucleic acid molecules. In some embodiments, crRNA and tracrRNA are combined into a single guide RNA (sgRNA). In some embodiments, sgRNA is about 88-150bp.

[0128] In some embodiments, Cas9 comprises a Cas9 ortholog or mutant Cas9 selected from the group consisting of: Streptococcus pyogenes (SpCas9), SpCas9 nickase (Cas9n D10A), SpCas9 (D1135E), eSpCas9, SpCas9-HF1, SpCas9VRER, SpCas9 VQR, SpCas9EQR, Staphylococcus aureus (SaCas9), Neisseria meningitidis, Streptococcus thermophilus, Streptococcus pneumoniae, Campylobacter coli, Campylobacter jejuni, Streptococcus mutans, Pasteurella multocida, Bifidobacterium longum, Bacillus smithii, Treponema denticola, Mycoplasma canis, and Enterococcus faecalis.

[0129] In some embodiments, (a) CRISPR guide RNA comprises crRNA, which comprises a protospacer element sequence complementary to a target sequence within 100bp or 100bp of the target gene, and (b) the functional CRISPR-associated protein comprises Cpf1. In some embodiments, the protospacer element is about 20 bases, about 21 bases, about 22 bases, about 23 bases, about 24 bases, about 19-25 bases, about 18-26 bases, or about 16-28 bases. In some embodiments, the CRISPR-associated protein Cas9 or Cpf1 further comprises one, two, three or more nuclear localization sequences (NLS) at the N-terminus and / or C-terminus, and / or a selection marker, including but not limited to GFP or EGFP.

[0130] In some embodiments, the protospacer element is 100% complementary to the target sequence or contains 1, 2, 3, 4, or 5 nucleotide mismatches corresponding to the target sequence. In some embodiments, the crRNA sequence further comprises a G nucleotide, which is optionally added to the crRNA sequence immediately before the protospacer element. In some embodiments, the CRISPR guide RNA, crRNA, and / or tracrRNA or sgRNA is chemically modified.

[0131] In some embodiments, for donor nucleic acid sequences provided as ssODN, the donor nucleic acid sequence is no longer than about 1 kb, 800 bp, 600 bp, 500 bp, 400 bp, 300 bp, 280 bp, 260 bp, 240 bp, 220 bp, or 200 bp; and for donor nucleic acid sequences provided as a vector, the donor nucleic acid sequence is no longer than about 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 0.5 kb, 0.2 kb, or 0.1 kb. In some embodiments, the wild-type form of the target gene encodes an enzyme.

[0132] In some embodiments, target genes include mutated or defective CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNR D2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SY NJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A genes, or CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNR, or proteins encoding proteins with defective or partial function or activity D2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SY NJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A genes.

[0133] In some embodiments, any one or more components thereof, including CRISPR guide RNA, CRISPR-associated protein and / or donor nucleic acid sequence, are provided alone and / or additionally as a vector, DNA and / or mRNA capable of being transcribed and / or translated into the component. In one aspect, a pharmaceutical formulation comprising any one of the compositions described herein is provided.

[0134] In another aspect, a method of treating a disease in a subject caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity is provided. Such methods include disrupting, correcting, or replacing the gene by administering to the subject any of the compositions described herein.

[0135] In another aspect, a method of treating an eye disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity, or ameliorating risk factors associated therewith, in a subject is provided. Such methods include disrupting, correcting, or replacing the gene by administering to the subject any of the compositions described herein.

[0136] In another aspect, a method for treating a neurodegenerative disease caused by a mutated or defective gene or a gene encoding a protein with defective or partial function or activity, or ameliorating risk factors associated therewith, in a subject is provided. Such methods include disrupting, correcting, or replacing the gene by administering to the subject any of the compositions described herein.

[0137] In another aspect, a method for treating a disease caused by a mutated or defective cytochrome P450 gene or a cytochrome P450 gene encoding a protein with defective or partial function or activity, or ameliorating risk factors associated therewith, in a subject is provided. Such methods include disrupting, correcting, or replacing the gene by administering to the subject any of the compositions described herein.

[0138] In some embodiments, the gene of the sudden change or defective gene or coding that is destroyed, corrected or substituted has defective or partial function or the gene of active protein is a sudden change or defective form of the gene set forth in Table 4, or the coding of the gene set forth in Table 4 has defective or partial function or the form of active protein.In some embodiments, the gene of sudden change or defective gene or coding that is destroyed, corrected or substituted has defective or partial function or the gene of active protein is present in fibroblast, hemocyte, RPE cell, photoreceptor cell, retinal cell, keratocyte, choroid cell, eye cell, optic nerve cell, neuronal cell or stem cell or any type of cell derived from stem cell.

[0139] In some embodiments, the composition is delivered to fibroblasts, hemocytes, RPE cells, photoreceptor cells, retinal cells, corneal cells, choroidal cells, eye cells, optic nerve cells, neuronal cells or stem cells or any type of cells derived from stem cells. In some embodiments, delivery is carried out by electroporation or via lipid-mediated transfection or nuclear transfection or viral transduction or injection or its combination. In some embodiments, any one or more components, including CRISPR guide RNA, CRISPR associated proteins and / or donor nucleic acid sequences, are administered to the subject or to these cells via the delivery system selected from the group consisting of: ribonucleoprotein or protein-RNA complex, nanoparticle, liposome, micelle, virion, nucleic acid complex and / or its combination.

[0140] In some embodiments, this process is carried out in the subject's body.In some embodiments, this process is carried out in vitro in the following cell: fibroblast, hemocyte, RPE cell, photoreceptor cell, retinal cell, corneal cell, choroid cell, eye cell, optic nerve cell, neuronal cell or stem cell or any type of cell derived from stem cell.In some embodiments, treated cell body is transplanted to the subject, or if treated cell is stem cell, then this stem cell differentiation is made into the cell of the desired type for transplanting, then is transplanted in the subject through differentiated cell body.

[0141] In some embodiments, a mutated or defective gene or a gene encoding a protein with defective or partial function or activity is replaced. In some embodiments, one or more mutations in a mutated or defective gene or a gene encoding a protein with defective or partial function or activity are corrected or replaced. In some embodiments, a mutated or defective gene or a gene encoding a protein with defective or partial function or activity is destroyed.

[0142] In some embodiments, 1-20, 21-40, 41-60, 61-80, 81-100, 101-1000, 1001-10000 base pairs of nucleotides or mutations are destroyed, corrected or replaced in a mutated or defective gene or a gene encoding a protein with a defective or partial function or activity. In some embodiments, a region of a mutated or defective gene or a gene encoding a protein with a defective or partial function or activity is destroyed, corrected or replaced. In some embodiments, a region of less than about 10, 8, 6, 4, 2 or 1 kb of a mutated or defective gene or a gene encoding a protein with a defective or partial function or activity is destroyed, corrected or replaced.

[0143] In some embodiments, a mutated or defective gene or a gene encoding a protein with defective or partial function or activity is disrupted, corrected, or replaced by inserting and / or deleting nucleotides. In some embodiments, a mutated or defective gene or a gene encoding a protein with defective or partial function or activity is disrupted, corrected, or replaced in one or both alleles. In some embodiments, two or more different CRISPR guide RNAs, CRISPR-associated proteins, and / or donor nucleic acid sequences are used to disrupt, correct, or replace one or more mutations or defects in a mutated or defective gene or a gene encoding a protein with defective or partial function.

[0144] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the method improves ocular development or function, or prevents degeneration of the eye, retina, or cornea. In some embodiments, the method improves neural development or function, or prevents neural degeneration. In some embodiments, the method improves the expression or function of a P450 enzyme.

[0145] In some embodiments, homology-directed repair based on a donor nucleic acid sequence obtains introns and / or exons of a target gene. In some embodiments, homology-directed repair based on a donor nucleic acid sequence obtains a splice acceptor of a target gene. Such methods may further include (c) a donor nucleic acid sequence comprising all or part of a target gene as described in Table 4, having a mutation or change in a target gene or a part thereof for generating a mutation or change.

[0146] In some aspects, a method is provided for producing a cell disease model for a disease caused by a gene of a mutated or defective gene or coding with a defective or partial function or active protein, the method being by producing a mutation in the gene. Such methods include delivering such genes to the cell of healthy form via any one of the compositions described herein. In some embodiments, delivery is carried out by electroporation or via lipid-mediated transfection or nuclear transfection or viral transduction or microinjection or its combination. In some embodiments, these cells are fibroblasts, hemocytes, RPE cells, photoreceptor cells, retinal cells, keratocytes, choroidal cells, eye cells, optic nerve cells, neuronal cells or stem cells or any type of cell derived from stem cells.

[0147] In another aspect, a composition comprising cells containing a mutated or defective gene set forth in Table 4 is provided.

[0148] In another aspect, a composition is provided that includes cells containing mutated or defective genes: CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WF S1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A gene, the composition comprising any one of the claims herein.

[0149] In some embodiments, the vector is an AAV vector. In some embodiments, the protospacer element sequence is selected from the group consisting of SEQ ID NOs: 48 to 52, or shares at least 80% sequence identity with one of SEQ ID NOs: 48 to 52, which is used with a Cas protein having NGG as a protospacer adjacent motif (PAM) to target the c.802-8_810del17insGC mutation of the CYP4V2 gene. In some embodiments, the donor nucleic acid sequence is selected from SEQ ID NOs: 56 and 57, or shares at least 90% sequence identity with one of SEQ ID NOs: 56 and 57 or their complements, which is used to correct, disrupt, or replace the c.802-8_810del17insGC mutation of the CYP4V2 gene.

[0150] Gene therapy claims

[0151] Codon-optimized sequence-related claims:

[0152] In one aspect, a nucleic acid molecule is provided, which includes the nucleic acid sequence of SEQ ID NO: 2 encoding a human CYP4V2 protein or a nucleic acid sequence sharing at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 2.

[0153] In another aspect, an expression cassette is provided, comprising a nucleic acid molecule as described herein and one or more regulatory sequences operably linked to the nucleic acid sequence. In another aspect, a vector is provided, comprising a nucleic acid molecule as described herein or an expression cassette as described herein.

[0154] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of: a recombinant adenoviral vector, a recombinant lentiviral vector, a recombinant herpes simplex virus vector, a recombinant Sendai virus vector, and a recombinant retroviral vector. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector or plasmid. In some embodiments, the vector is a plasmid or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of naked nucleic acid, liposomes, dendrimers, and nanoparticles.

[0155] In some embodiments, the host cell comprises any one of the nucleic acid molecules described herein and / or any one of the compositions described herein. In some embodiments, the host cell is a bacterial cell, an E. coli cell (E. Coli cell), a plant cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a HEK293, HeLa cell (HeLa), Vero, V27, A549, K562, B50, WI38, Hep G2, or BHK cell.

[0156] In another aspect, the product encoded by any of the nucleic acid molecules described herein, any of the expression cassettes described herein, or any of the vectors described herein is expressed in bacterial cells, insect cells, plant cells, mammalian cells, RPE cells, photoreceptors or photoreceptor progenitor cells (PRCs), retinal cells, corneal cells, eye cells, neurons, neuronal cells, blood cells, epithelial cells, somatic cells, iPS cells, ES cells, MSCs, adult stem cells, stem cells, or any cell derived from stem cells.

[0157] EFS and / or SPA related claims

[0158] In another aspect, a self-complementary adeno-associated virus (scAAV) vector is provided, comprising an elongation factor 1α short (EFS) promoter and / or a small polyadenylation (polyA) signal (SPA) operably linked to a nucleic acid molecule, interfering RNA molecule, or oligonucleotide encoding a polypeptide. In some embodiments, the EFS promoter consists of a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 35 and the SPA consists of a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 36.

[0159] In some embodiments, the scAAV vector is delivered to a cell such that the product encoded by the nucleic acid molecule is expressed in the cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a retinal cell, a corneal cell, a choroidal cell, an eye cell, a brain cell, a neuron, a neuronal cell, an iPS cell, an ES cell, a MSC, a stem cell, or any cell derived from a stem cell.

[0160] In one aspect, a method is provided for reducing the immune response to viral vectors and maintaining the transduction efficiency in gene therapy and / or maximizing the therapeutic effect on different patients with the same genetic disease. Such methods include (a) establishing a library of more than one recombinant viral vector (e.g., rAAV) that has sufficient transduction efficiency in the target cell type targeted by the gene therapy. The library of viral vectors can be amplified by generating variants with mutations in the antigenic region or generating other mutations or variants on the capsids of these viral vectors, and these mutations or variants are confirmed to have sufficient transduction efficiency in target cells associated with the disease (e.g., in iPS-RPE cell lines for CYP4V2 gene therapy for BCD); (b) detecting pre-existing neutralizing anti-viral vector antibodies against different viral vector serotypes and / or capsid mutations or variants in subjects who need the gene therapy. antibodies, NAb), and / or testing and comparing different viral vectors in patient-specific cells derived from the subject (e.g., iPS-RPE cells); (c) selecting a viral vector with sufficient transduction efficiency and minimal cross-reactivity with pre-existing NAbs in the subject and / or a viral vector with the best phenotypic rescue results in the subject's patient-specific cells from the library of the viral vectors, the library of the viral vectors comprising viral vectors of different serotypes and capsids modified (e.g., including but not limited to capsid mutant AAV and / or capsid protein variant AAV); (d) administering to the subject using a viral vector selected from (c); and (e) repeating (b) to (d) above (repeating only the portion associated with pre-existing NAbs) each time the subject requires gene therapy, including but not limited to subsequent administration to the same eye or administration to the contralateral eye or to other organs.

[0161] In another aspect, a composition for treating or preventing a disease in a subject is provided, comprising an effective amount of a vector and a pharmaceutically acceptable carrier. The vector generally comprises a nucleic acid molecule encoding a non-mutant or functional CYP4V2 protein or a non-pathogenic variant thereof operably linked to a regulatory sequence.

[0162] In some embodiments, the disease is crystalline retinitis pigmentosa (also known as crystalline keratoretinal degeneration; BCD). In some embodiments, the disease is associated with a genetic or epigenetic change in the subject. In some embodiments, the disease is photoreceptor degeneration, retinal pigment epithelial degeneration, retinal degeneration, corneal degeneration, or choroidal degeneration. In some embodiments, retinal degeneration is retinitis pigmentosa (RP). In some embodiments, retinal degeneration is inherited retinal degeneration (IRD). In some embodiments, the disease is BCD. In some embodiments, the disease is corneal degeneration. In some embodiments, the subject has BCD or is at risk of developing BCD.

[0163] In one aspect, a vector is provided that includes a nucleic acid molecule encoding a non-mutant or functional CYP4V2 protein or a non-pathogenic variant thereof operably linked to a regulatory sequence.

[0164] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of: adeno-associated virus (AAV) vector, adenoviral vector, lentiviral vector, herpes simplex virus vector, Sendai virus vector and retroviral vector. In some embodiments, AAV is recombinant AAV (rAAV). In some embodiments, rAAV comprises an AAV genome or a derivative thereof, and / or an AAV capsid protein or a derivative thereof. In some embodiments, rAAV is a chimeric AAV, a rearranged AAV (shuffled AAV) or an AAV with a modified capsid.

[0165] In some embodiments, the AAV genome or AAV capsid protein is from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or clade of AAV, or any derivative or hybrid thereof. In some embodiments, the rAAV is a pseudotyped AAV (e.g., AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 4, AAV2 / 6, AAV2 / 7, AAV2 / 12, AAV2 / 10, and AAV2 / 9). In some embodiments, the rAAV is a hybrid AAV (e.g., AAV-DJ, AAV-DJ / 8, or AAV-DJ / 9). In some embodiments, rAAV is developed through directed evolution and / or rational design (eg, AAV 7m8 or AAV-PHP.B).

[0166] In some embodiments, the rAAV comprises one or more capsid mutations (e.g., YF, KR, TA, SA, and / or TV mutations (e.g., AAV2 has one or more capsid mutations of Y444F, Y500F, Y730F, Y252F, Y272F, Y700F, Y704F, and T491V, or corresponding mutations for a different AAV serotype (e.g., AAV2 / 8 (Y733F), AAV2 (Y444F+Y500F+Y730F), and AAV2 (quadY-F+TV)))). In some embodiments, the serotype of the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Anc80, rh10, and ShH10. In some embodiments, the rAAV vector is selected from the group consisting of: AAV2 / 5, AAV2 / 8, AAV2 / 8(Y733F), AAV2(Y444F+Y500F+Y730F), AAV2 / 1, AAV2 / 4, AAV2 / 9, AAV2 / 6, AAV2 / 7, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV-PHP.B, or hybrids, derivatives or variants thereof.

[0167] In some embodiments, the rAAV vector is a single-stranded AAV vector or a self-complementary AAV (scAAV) vector. In some embodiments, the vector is a plasmid or a non-viral vector (e.g., naked nucleic acid, liposomes, dendrimers, and nanoparticles).

[0168] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises: (i) human CYP4V2 protein (SEQ ID NO:4); (ii) a variant (e.g., altered amino acid and / or splice variant) of a human CYP4V2 protein or a functional CYP4V2 protein (e.g., SEQ ID NO:5); (iii) one or more fragments of a functional CYP4V2 protein (e.g., SEQ ID NO:6); (iv) all or a portion of the sequence of one or more CYP4V2 orthologs from other species; (v) all or a portion of the sequence of one or more other P450 proteins, including but not limited to other CYP4 proteins and CYP46A1; (vi) a polypeptide that can improve, treat, or suppress one or more biochemical abnormalities of one or more of the genes listed in Table 4 in patient cells (e.g., iPS-RPE cells from a BCD patient); and / or (vii) a combination of the above.

[0169] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or a portion of the amino acid sequence set forth in SEQ ID NO: 4, 5, or 6. In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or a portion of an amino acid sequence selected from the group consisting of CYP4V2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1, and CYP46A1 (SEQ ID NOs: 4-18), and derivatives, hybrids, variants, and / or fragments thereof. In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or part of an amino acid sequence selected from the group consisting of chimpanzee, rhesus monkey, dog, cow, mouse, rat, chicken, frog, horse, rabbit and fruit fly CYP4V2 (or an ortholog of CYP4V2) (SEQ ID NOs: 19-29), and derivatives, hybrids, variants and / or fragments thereof.

[0170] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises a polypeptide having at least 80% amino acid sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) to any of the sequences selected from the group consisting of SEQ ID NO: 4-29. In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises sequence elements of FxxGxxxCxG and ExxR (SEQ ID NO: 30 and 31). In some embodiments, the non-mutant or functional CYP4V2 protein is a compound or agent that can improve, treat or suppress one or more biochemical abnormalities of one or more of the genes listed in Table 4 in patient cells (e.g., iPS-RPE cells of BCD patients).

[0171] In some embodiments, the nucleic acid molecule encodes a non-mutated or functional CYP4V2 protein according to any one of claims 43 to 50. In some embodiments, the nucleic acid molecule encodes a non-mutated or functional CYP4V2 protein comprising the amino acid sequence shown in SEQ ID NO: 4, 5, or 6, or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 4, 5, or 6. In some embodiments, the nucleic acid molecule has at least 60% sequence identity to any of the sequences in SEQ ID NO: 1, 2, or 3. In some embodiments, the nucleic acid molecule has at least 70% sequence identity to any of the sequences in SEQ ID NO: 1, 2, or 3. In some embodiments, the nucleic acid molecule has at least 75% sequence identity to any of the sequences in SEQ ID NO: 1, 2, or 3. In some embodiments, the nucleic acid molecule has at least 76% sequence identity to any of the sequences in SEQ ID NO: 1, 2, or 3. In some embodiments, the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 1, 2, or 3.

[0172] In some embodiments, the regulatory sequence comprises a promoter. In some embodiments, the promoter is a RPE cell-specific promoter, a retinal cell-specific promoter, a keratinocyte-specific promoter, an eye cell-specific promoter, or a constitutive promoter. In some embodiments, the promoter is a mammalian beta-actin promoter or a viral promoter.

[0173] In some embodiments, the promoter is selected from the group consisting of: CAG promoter (hybrid CMV early enhancer / chicken beta actin promoter, also known as CAGGS promoter, CB promoter or CBA promoter), chicken beta actin promoter, small CBA (smCBA) promoter, CB promoter, SB promoter or CBh promoter, another β-actin promoter such as the human β-actin promoter, elongation factor 1 alpha short form (EFS) promoter, elongation factor 1 alpha short form (EF-1α) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (vitelliform macular dystrophy 2; also known as BEST1) promoter, RPE65 promoter, or a hybrid or derivative thereof.

[0174] In some embodiments, the promoter is a CAG promoter (hybrid CMV early enhancer / chicken beta actin promoter, also known as CAGGS promoter, CB promoter, or CBA promoter), elongation factor 1 alpha short (EFS) promoter, elongation factor 1 alpha short (EF-1 alpha) promoter, or CMV promoter, or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises an enhancer.

[0175] In some embodiments, the enhancer is a viral enhancer, including but not limited to the WPRE enhancer, the HPRE enhancer, the CTE enhancer, or derivatives or hybrids thereof. In some embodiments, the regulatory sequence comprises a polyadenylation (polyA) signal. In some embodiments, the polyA signal is a bovine growth hormone polyadenylation signal (bGH polyA), a small polyA signal (SPA), a human growth hormone polyadenylation signal (hGH polyA), an SV40 polyA signal, an SV40 late polyA signal, or derivatives or hybrids thereof. In some embodiments, the regulatory sequence comprises a Kozak sequence (SEQ ID NO: 37 or 38).

[0176] In some embodiments, the compositions are formulated with carriers and additional components appropriate for a particular route of administration.

[0177] In another aspect, a host cell is provided, comprising any one of the vectors described herein.

[0178] In another aspect, a method of treating or preventing an eye disease in a subject is provided, comprising administering to the subject a vector comprising a nucleic acid molecule encoding a human CYP4V2 protein or a functional CYP4V2 protein or a non-pathogenic variant thereof operably linked to a regulatory sequence.

[0179] In one aspect, a method for preventing, arresting or slowing the progression or ameliorating dysfunction, degeneration, disorder, degeneration and / or death of ocular cells is provided, the method comprising delivering a vector to the ocular cells, wherein the vector comprises a nucleic acid molecule encoding a human CYP4V2 protein or a functional CYP4V2 protein or a non-pathogenic variant thereof operably linked to a regulatory sequence.

[0180] In some embodiments, the disease is crystalline retinitis pigmentosa (also known as crystalline keratoretinal degeneration; crystalline retinopathy; Bietti retinal degeneration; BCD). In some embodiments, the subject suffers from other clinically defined ophthalmic conditions caused by CYP4V2 gene mutations (e.g., inherited retinal degeneration (IRD), retinitis pigmentosa (RP), or corneal degeneration). In some embodiments, the eye disease is photoreceptor degeneration, retinal pigment epithelial degeneration, retinal degeneration, corneal degeneration, or BCD.

[0181] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of: a recombinant adeno-associated virus (rAAV) vector, a recombinant adenoviral vector, a recombinant lentiviral vector, a recombinant herpes simplex virus vector, a recombinant Sendai virus vector, and a recombinant retroviral vector. In some embodiments, the viral vector is a rAAV vector. In some embodiments, the rAAV vector comprises VP1, VP2, or VP3 capsid proteins selected from any of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or branch of AAV, or a hybrid, variant, or derivative thereof.

[0182] In some embodiments, the rAAV vector 5' AAV ITR is selected from any one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or branch of AAV, or a mutation, chimera, variant, or fusion thereof. In some embodiments, the rAAV vector 3' AAV ITR is selected from any one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or branch of AAV, or a mutation, chimera, variant, or fusion thereof. In some embodiments, the rAAV is a chimeric AAV, a rearranged AAV, or an AAV with a modified capsid. In some embodiments, the rAAV is a pseudotyped AAV (e.g., AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 4, AAV2 / 6, AAV2 / 7, AAV2 / 12, AAV2 / 10, and AAV2 / 9). In some embodiments, the rAAV is a hybrid AAV (e.g., AAV-DJ, AAV-DJ / 8, or AAV-DJ / 9). In some embodiments, the rAAV is developed through directed evolution and / or rational design (e.g., AAV 7m8 or AAV-PHP.B).

[0183] In some embodiments, the rAAV comprises one or more capsid mutations (e.g., YF, KR, TA, SA, and / or TV mutations (e.g., AAV2 has one or more capsid mutations of Y444F, Y500F, Y730F, Y252F, Y272F, Y700F, Y704F, and T491V, or corresponding mutations for different AAV serotypes (e.g., AAV2 / 8 (Y733F), AAV2 (Y444F+Y500F+Y730F), and AAV2 (two-Y-F+TV))). In some embodiments, the serotype of rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Anc80, rh10, and ShH10. In some embodiments, the rAAV vector is selected from the group consisting of: AAV2 / 5, AAV2 / 8, AAV2 / 8(Y733F), AAV2(Y444F+Y500F+Y730F), AAV2 / 1, AAV2 / 4, AAV2 / 9, AAV2 / 6, AAV2 / 7, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV-PHP.B, or hybrids, derivatives or variants thereof.

[0184] In some embodiments, the rAAV vector is a single-stranded AAV vector or a self-complementary AAV (scAAV) vector. In some embodiments, the vector is a plasmid or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of naked nucleic acid, liposomes, dendrimers, and nanoparticles.

[0185] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises: (i) human CYP4V2 protein (SEQ ID NO: 4); (ii) a variant (e.g., altered amino acid and / or splice variant) of a human CYP4V2 protein or a functional CYP4V2 protein (e.g., SEQ ID NO: 5); (iii) one or more fragments of a functional CYP4V2 protein (e.g., SEQ ID NO: 6); (iv) all or a portion of the sequence of one or more CYP4V2 orthologs from other species; (v) all or a portion of the sequence of one or more other P450 proteins, including but not limited to other CYP4 proteins and CYP46A1; (vi) a polypeptide that can improve, treat, or suppress one or more biochemical abnormalities in one or more genes listed in Table 4 in patient cells (e.g., iPS-RPE cells from a BCD patient); and / or (vii) a combination of the above.

[0186] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or a portion of the amino acid sequence set forth in SEQ ID NO: 4, 5, or 6. In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or a portion of an amino acid sequence selected from the group consisting of CYP4V2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1, and CYP46A1 (SEQ ID NOs: 4-18), and derivatives, hybrids, variants, and / or fragments thereof.

[0187] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all of a portion of an amino acid sequence selected from the group consisting of chimpanzee, rhesus monkey, dog, cow, mouse, rat, chicken, frog, horse, rabbit, and fruit fly CYP4V2 (or an ortholog of CYP4V2) (SEQ ID NOs: 19-29), and derivatives, hybrids, variants, and / or fragments thereof. In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises a polypeptide having at least 80% amino acid sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any of the sequences selected from the group consisting of SEQ ID NOs: 4-29.

[0188] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises sequence elements of FxxGxxxCxG and ExxR (SEQ ID NO:30 and 31). In some embodiments, the non-mutant or functional CYP4V2 protein is a compound or agent that can improve, treat or curb one or more biochemical abnormalities of one or more of the genes listed in Table 4 in patient cells (e.g., iPS-RPE cells of BCD patients). In some embodiments, the nucleic acid molecule encodes a non-mutant or functional CYP4V2 protein according to any one of claims 91 to 97. In some embodiments, the nucleic acid molecule encodes a non-mutant or functional CYP4V2 protein comprising the amino acid sequence shown in SEQ ID NO:4, 5 or 6 or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO:4, 5 or 6. In some embodiments, the nucleic acid molecule encoding a functional CYP4V2 protein has a nucleic acid sequence shown in SEQ ID NO:1, 2 or 3. In some embodiments, the nucleic acid molecule encoding the functional CYP4V2 protein has at least 60% sequence identity to any one of SEQ ID NOs 1, 2, or 3.

[0189] In some embodiments, the regulatory sequence comprises a promoter. In some embodiments, the promoter is an RPE cell-specific promoter, a retinal cell-specific promoter, a keratinocyte-specific promoter, or an eye cell-specific promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a mammalian beta-actin promoter or a viral promoter.

[0190] In some embodiments, the promoter is selected from the group consisting of: CAG promoter (hybrid CMV early enhancer / chicken beta actin promoter, also known as CAGGS promoter, CB promoter or CBA promoter), chicken beta actin promoter, small CBA (smCBA) promoter, CB promoter, SBIn some embodiments, the promoter is a CAG promoter (hybrid CMV early enhancer / chicken β-actin promoter, also known as CAGGS promoter, CB promoter, or CBA promoter), an elongation factor 1 alpha short form (EFS) promoter, an elongation factor 1 alpha short form (EF-1 alpha) promoter, or a CMV promoter, or a derivative or hybrid thereof.

[0191] In some embodiments, the regulatory sequence comprises an enhancer. In some embodiments, the enhancer is a viral enhancer, including but not limited to a WPRE enhancer, an HPRE enhancer, a CTE enhancer, or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises a polyadenylation (polyA) signal. In some embodiments, the polyA signal is a bovine growth hormone polyadenylation signal (bGH polyA), a small polyA signal (SPA), an SV40 polyA signal, a human growth hormone polyadenylation signal (hGH polyA), an SV40 late polyA signal, or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises a Kozak sequence (SEQ ID NO: 37 or 38).

[0192] In some embodiments, for in vitro treatment, about 1×10 3 GC / cell is about 1×10 6 The dose of GC / cell (MOI) to infect target cells (GC: genome copies, a measure of AAV particles containing the genome (also known as vector genome (vg) or genome particle (gp)). In some embodiments, for in vivo administration to the eye of a subject, a single dose can be approximately from about 1×10 6 to 2×10 13 The magnitude of GC (e.g., about 1×10 11 GC to about 1×10 12 The high dose range of GC, about 1×10 10 GC to about 1×10 11 The medium dose range of GC is about 1×10 9 GC to about 1×10 10 The low dose range of GC is about 1×10 6 GC to about 1×10 9The extremely low dose range of GC and about 1×10 12 GC to about 2×10 13 The invention relates to a very high dose range of GC), or any dose within these ranges sufficient to provide the desired effect.

[0193] In some embodiments, the administering step occurs before or after the onset of disease symptoms. In some embodiments, administration is performed to the eyes. In some embodiments, administration is performed by subretinal injection. In some embodiments, administration is performed by intravitreal injection. In some embodiments, administration is performed by direct retinal injection. In some embodiments, administration is performed by any other method of administration that effectively delivers the vector to the subretinal position, posterior segment of the eye, cornea or RPE cells, photoreceptor cells or corneal epithelial cells of the experimenter.

[0194] In some embodiments, administration is delivered by cornea. In some embodiments, administration is achieved by delivering via bloodstream. In some embodiments, administration is via eye drops. In some embodiments, administration is carried out by being delivered to the crystalline lens. In some embodiments, administration is carried out in the subretinal space, cornea, crystalline lens or in the vitreous body. In some embodiments, eye cells are selected from the group consisting of: retinal pigment epithelium (RPE) cells, photoreceptor cells (PRC), corneal epithelial cells (CEC), choroidal endothelial (CE) cells, retinal cells, keratocytes, lens cells, ganglion cells, optic nerve cells and / or choroidal cells, and cells derived from these types of stem cells (including but not limited to iPSC, ES cells, MSC, adult stem cells and / or tissue-specific stem cells).

[0195] In some embodiments, the methods described herein can further comprise identifying a subject having or at risk of developing BCD.

[0196] Related claims for the use of EFS and / or SPA in rAAV vectors comprising nucleic acid sequences encoding Cas

[0197] In one aspect, a composition is provided that includes a recombinant adeno-associated virus (rAAV) vector comprising an elongation factor 1 alpha short form (EFS) promoter and / or a small polyadenylation (polyA) signal (SPA) operably linked to a nucleic acid molecule encoding a CRISPR-associated protein (Cas).

[0198] In some embodiments, the EFS promoter consists of a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 35, and the SPA consists of a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 36. In some embodiments, the Cas encoded by the nucleic acid sequence operably linked to the EFS promoter and / or SPA is Cas9 or Cpf1.

[0199] Provide a host cell comprising rAAV as described herein. In some embodiments, the host cell is a bacterial cell, an E. coli cell, a plant cell, an insect cell, or a mammalian cell. In some embodiments, the cell is a somatic cell or a stem cell. In some embodiments, the host cell is a retinal cell, a corneal cell, a choroidal cell, an eye cell, a brain cell, a neuron, a neuronal cell, an iPS cell, an ES cell, a MSC, an adult stem cell, a tissue-specific cell, a stem cell, or any cell derived from a stem cell. In some embodiments, the rAAV vector is delivered to the host cell so that the Cas encoded by the nucleic acid molecule is expressed in the cell. In some embodiments, the host cell comprises any cell according to any one of claims 131 to 134.

[0200] Other features and advantages of the invention will become apparent from the detailed description, accompanying drawings and examples, and from the claims.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.

Brief Description of the Drawings

[0201] The present invention is further described in the following figures and drawings, which do not limit the scope of the invention described in the claims.

[0202] Cell Line Patents:

[0203] Figure 1: iPS cell lines derived from BCD patients

[0204] (a) iPS cells generated from fibroblasts from skin biopsy samples of BCD patients:

[0205] (i) Patient 1 (P1) iPS cells

[0206] (ii) Patient 2 (P2) iPS cells

[0207] (iii) Characterization of P1 and P2 iPS cell lines using Oct-4, Sox-2, and SSEA-4 markers

[0208] (iv) Characterization of P1 and P2 iPS cell lines using Nanog and Tra-1-60 markers

[0209] (b) iPS cells generated from peripheral blood mononuclear cells (PBMCs) of blood samples from BCD patients and healthy controls:

[0210] (i) Phase contrast image of iPS cell lines

[0211] (ii) AP staining results of iPS cell lines

[0212] (c) Karyotype image of iPS cells derived from a BCD patient, showing an apparently normal human karyotype.

[0213] Figure 2: iPS-RPE cell lines derived from BCD patients:

[0214] (a) Light-field image of an iPS-RPE cell line derived from a BCD patient, showing the unique morphology of RPE—hexagonal shape, pigmentation, and a single layer.

[0215] (i) P1 iPS-RPE cells

[0216] (ii) P2 iPS-RPE cells

[0217] (b) RPE marker results of iPS-RPE cells from BCD patients, showing the presence of RPE-specific markers RPE65, CRALBP, and MITF.

[0218] Figure 3 : qRT-PCR results of CYP4V2 expression in iPS-RPE samples. WT (control group). WT AVG (average value of the control group). P1 (BCD patient 1). P1-AAV8 (P1 sample treated with AAV8.CYP4V2fv at an MOI of 1.5×10e4 GC / cell).

[0219] Figure 4 Figure 2: qRT-PCR results of CYP4V2op expression in iPS-RPE samples. WT (control group). WT AVG (average value of control group). P1 and P2 (BCD patients 1 and 2). P1-AAV2 (P1 sample treated with AAV2.CYP4V2op at an MOI of 2×10e4 GC / cell). P2-AAV2 (P2 sample treated with AAV2.CYP4V2op at an MOI of 2×10e4 GC / cell). P2-scAAV1 (P2 sample treated with scAAV1.CYP4V2op at an MOI of 2×10e4 GC / cell).

[0220] Figure 5: Cell viability images of iPS-RPE samples not exposed to blue light. WT (control group). P1 and P2 (BCD patients 1 and 2). Red (dead / diseased cells); Green (live / healthy cells). Figure 5(a): Red only. Figure 5(b): Red and green.

[0221] Figure 6: Cell viability images of iPS-RPE samples after 1 hour of exposure to blue light. WT (control group). P1 and P2 (BCD patients 1 and 2). Red (dead / diseased cells); Green (live / healthy cells). Figure 6(a): Red only. Figure 6(b): Red and green.

[0222] Gene therapy:

[0223] FIG. 7 : Schematic diagram of an exemplary CYP4V2 expression cassette and recombinant AAV (rAAV) vector and its annotations.

[0224] (a) CYP4V2 expression cassette (including enhancer) packaged in a single-stranded AAV (ssAAV) vector

[0225] (b) CYP4V2 expression cassette (without enhancer) packaged in a single-stranded AAV (ssAAV) vector

[0226] (c) CYP4V2 expression cassette packaged in a self-complementary AAV (scAAV) or ssAAV vector.

[0227] Note: The CYP4V2 expression cassette (flanked by AAV ITRs as shown) can be packaged in an rAAV vector with a capsid from any AAV serotype or hybrid or variant thereof. ITR: Inverted terminal repeats (can be AAV2 ITRs or ITRs from other AAV serotypes). Exemplary AAV2 ITR sequences are shown in SEQ ID NOs:42 and 43. CYP4V2: cDNA encoding human CYP4V2 protein or a functional variant thereof, such as CYP4V2st (SEQ ID NO:1) or CYP4V2op (SEQ ID NO:2), which encode the human CYP4V2 protein (SEQ ID NO:4), or CYP4V2fv (SEQ ID NO:3), which encodes a functional CYP4V2 protein (SEQ ID NO:5). A Kozak sequence (sequence shown in SEQ ID NOs:37 or 38) is inserted immediately before the CYP4V2 cDNA sequence. CAG: hybrid CAG promoter (an exemplary sequence is shown in SEQ ID NO:32). Other promoters discussed herein may also be used, including, but not limited to, the CMV promoter (an exemplary sequence is shown in SEQ ID NO:40) or the EF-1α promoter (an exemplary sequence is shown in SEQ ID NO:41). WPRE: woodchuck hepatitis virus posttranscriptional regulatory element (an exemplary sequence is shown in SEQ ID NO:33). bGH polyA: bovine growth hormone polyadenylation signal (an exemplary sequence is shown in SEQ ID NO:34). Alternative polyA signals, such as the SV40 late polyA signal (an exemplary sequence is shown in SEQ ID NO:39), may be used. EFS: elongation factor 1α short form (EFS) core promoter. An exemplary sequence is shown in SEQ ID NO:35. SPA: small polyA signal. An exemplary sequence is shown in SEQ ID NO:36. Mutated / truncated ITR: One of the two ITRs used in the scAAV vector is a mutated / truncated ITR (shown as ITR*). An exemplary sequence is shown in SEQ ID NO: 44. An enhancer is optional in the CYP4V2 expression cassette.

[0228] Figure 8: Cell viability images of BCD patient-derived iPS-RPE samples after 1 hour of exposure to blue light (no AAV.CYP4V2 treatment vs. treatment with AAV2.CYP4V2op or scAAV1.CYP4V2op at an MOI of 1×10e5 GC / cell). P1 and P2 (BCD patients 1 and 2). Red (dead / diseased cells); Green (live / healthy cells). Figure 8(a): Red only. Figure 8(b): Red and green.

[0229] Figure 9: Cell viability images of BCD patient-derived iPS-RPE samples after 1 hour of exposure to blue light (no AAV.CYP4V2 treatment vs. treatment with AAV5.CYP4V2op, AAV5.CYP4V2st, or AAV8.CYP4V2fv at an MOI of 1×10e5 GC / cell). P1 (BCD patient 1). Red (dead / diseased cells); Green (live / healthy cells). Figure 9(a): Red only. Figure 9(b): Red and green.

[0230] Figure 10: Cell viability images of BCD patient-derived iPS-RPE samples after 1 hour of exposure to blue light (no AAV.CYP4V2 treatment vs. treatment with AAV5.CYP4V2op, scAAV1.CYP4V2op, or scAAV5.CYP4V2op at an MOI of 1×10e4 GC / cell). P2 (BCD patient 2). Red (dead / diseased cells); Green (live / healthy cells). Figure 10(a): Red only. Figure 10(b): Red and green.

[0231] Figure 11: Cell viability images of BCD patient-derived iPS-RPE samples after 1 hour of exposure to blue light (no AAV.CYP4V2 treatment vs. treatment with scAAV9.CYP4V2op at an MOI of 1×10e5 GC / cell). P1 (BCD patient 1). Red (dead / diseased cells); Green (live / healthy cells). Figure 11(a): Red only. Figure 11(b): Red and green.

[0232] Cell therapy:

[0233] Figure 12 The region showing the CYP4V2 sequence and the position of the guide RNA (gRNA) designed relative to the c.802-8_810del17insGC mutation, as well as the primers used for gRNA activity analysis (orange arrows).

[0234] Figure 13 In vitro surveyor assay shown. Lane 1: amplicon + Cas9; Lane 2: amplicon + g1 + Cas9; Lane 3: amplicon + g2 + Cas9; Lane 4: amplicon + g3 + Cas9; Lane 5: amplicon + g4 + Cas9; Lane 6: amplicon + g5 + Cas9; Lane 7: amplicon alone; Lane M: 1 kb DNA marker.

[0235] Figure 14To confirm the origin of DNA used in the Surveyor analysis for sequence comparison, top: untreated amplicon; middle: fragment from the G2-treated amplicon; bottom: CYP4V2 locus, with mutation sites indicated.

[0236] Figure 15 Schematic illustration of gRNA vector construction.

[0237] Figure 16 This is the vector map of the gRNA (taking g1 as an example), Cas9 and PuroR co-expression plasmid pX459-hSpCas9-2A-Puro.

[0238] Figure 17 The position of gRNA (g1 as an example) relative to the U6 promoter is shown in the pX459-hSpCas9-2A-Puro plasmid. The "G" nucleotide between the U6 promoter and the gRNA is used to enhance the transcription efficiency driven by the U6 promoter. When a different promoter is used or when the gRNA begins with a "G" nucleotide, it is optional and not required.

[0239] definition

[0240] It should be understood that "a" or "an" as used in this specification and claims can mean one or more depending on the context in which they are used. Thus, for example, reference to "a cell" means "at least one cell" or "more than one cell."

[0241] The term "about" or "approximately" or the symbol "~" means within plus or minus 10% (inclusive) of a given value or condition. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term about.

[0242] The term "AAV.CYP4V2" refers to a recombinant adeno-associated virus (AAV) vector comprising a polynucleotide encoding a functional CYP4V2 protein.

[0243] The term "CYP4V2 gene therapy" refers to the introduction of a functional CYP4V2 protein or a polynucleotide encoding a functional CYP4V2 protein into cells and / or subjects. See detailed discussion in this disclosure.

[0244] The term "effective amount" or "effective dose" or "therapeutically effective dose" refers to an amount of a compound (e.g., vector) and / or cell that, when administered to a subject in need of treatment, is sufficient and / or suitable for achieving such treatment. The effective amount will vary depending on the specific activity of the therapeutic agent used, the severity of the patient's disease condition, and the subject's age, physical condition, presence of other disease conditions, and nutritional status. In addition, other medications and / or treatments currently being taken by the patient will also affect the determination of the effective amount of the therapeutic agent to be administered. For a more detailed discussion, see the description herein.

[0245] The terms "treatment" or "treating" refer to administering a composition as disclosed herein (e.g., an AAV and / or cell comprising a transgene) to a subject for purposes including: 1) preventing a disease or condition or providing protection from developing the disease or condition, i.e., so that clinical symptoms do not develop; 2) inhibiting the disease or condition, i.e., arresting, slowing, ameliorating, or arresting the development of clinical symptoms; 3) alleviating the disease or condition, i.e., causing regression of clinical symptoms; and / or 4) replacing cells, tissues, and / or organs affected by the disease and / or restoring their lost function. In some embodiments, the terms "treatment" or "treating" refer to alleviating a disease or condition; i.e., causing regression of clinical symptoms. In some embodiments, the terms "treatment" or "treating" alternately or additionally refer to prophylactic treatment of a subject in need thereof. Prophylactic treatment can be achieved by providing an appropriate dose of a therapeutic agent to a subject at risk of being affected by a disorder, thereby substantially preventing the onset of the disorder. Those skilled in the art will understand that it is not always possible to distinguish between "prevention" and "suppression" because the most important inducing event may be unknown or latent, or the patient may not be diagnosed until long after the event occurs. Therefore, the term "prophylaxis" as used herein is intended as an element of "treatment" to encompass both "prevention" and "suppression" as defined herein.

[0246] The term "subject" refers to an animal, such as a mammal, e.g., a human. The methods described herein can be applicable to human therapeutic, preclinical, and veterinary applications. In some embodiments, the subject is a mammal, and in some embodiments, the subject is a human.

[0247] A "variant" is a protein having sequence homology to a reference biologically active protein that retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity compared to a reference biologically active protein. The term "biologically active protein" includes proteins that have been intentionally modified, such as by site-directed mutagenesis, insertion, or accidental mutation. "Variants" include "fragments," which are truncated forms of native or non-native biologically active proteins that retain at least a portion of their therapeutic and / or biological activity.

[0248] The term "nucleic acid" is used herein to refer to all forms of nucleic acids, polynucleotides and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and RNA. Polynucleotides include naturally occurring, synthetic and intentionally modified or altered polynucleotides. Polynucleotides can be single-stranded, duplexed or triplexed, linear or circular, and can be of any length. The sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in a 5' to 3' direction.

[0249] The term "sequence variant" means a gene or polypeptide that has been modified by insertion, deletion and / or substitution of one or more nucleotides or amino acids compared to its native or original sequence. The insertion can be located at either or both ends of the gene or protein and / or can be located within an internal region of the nucleotide sequence or amino acid sequence. In a deletion variant, one or more nucleotides or amino acid residues in a gene or polypeptide as described herein are removed. In a substitution variant, one or more nucleotides or amino acid residues of a gene or polypeptide are removed and replaced with an alternative residue. In one aspect, substitutions are conservative in nature, and this type of conservative substitution is well known in the art.

[0250] As used herein, the term "therapy" or "treatment" can be administered in vivo to a subject or in vitro to cells.

[0251] As used herein, a plasmid is a type of vector.

[0252] As used herein, the term "genetically repaired" or "gene repair" refers to a cell that originally carried a genetic defect (e.g., a mutation or pathological change) in a gene, but whose genetic defect has been repaired through gene correction or destruction of the cell's genomic DNA or mRNA (defined herein as "gene editing," "gene editing therapy," or "gene correction"), or through gene transfer of an exogenous nucleic acid molecule that expresses a functional protein corresponding to the defective gene into the cell or supplementation of the exogenous nucleic acid molecule to the cell (defined herein as "gene transfer therapy" or "gene therapy").

[0253] As used herein, the term "percent sequence identity" or "sequence identity" should be determined and calculated as follows. When calculating sequence identity (percentage), two sequences are aligned and the number of identically matched nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the alignment region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to obtain the sequence identity percentage value (and rounded up to the next higher integer (e.g., 65.01% will be rounded up to 66% and will be considered 66% for the purposes of this article)). It should be understood that the length of the alignment region can be a portion of one or both sequences to the entire net length of the shorter sequence (no gaps are applied). In order to determine identical matches and calculate sequence identity between two protein-coding nucleotide sequences, any non-coding nucleotide sequences (such as, but not limited to, introns, UTRs, Kozak sequences, promoters, enhancers, or other regulatory sequences) should be removed before submitting the two sequences for alignment and calculating sequence identity. Alignment of two sequences to determine the number of identical matches of nucleotides or amino acid residues between the two sequences can be performed by using Pairwise Sequence Alignment EMBOSS Needle, which uses the Needleman-Wunsch algorithm (available at the European Bioinformatics Institute (EMBL-EBI) and on the World Wide Web: ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html for nucleotide alignment and ebi.ac.uk / Tools / psa / emboss_needle / for protein alignment) and using default parameters (for nucleotide sequences, use: Matrix: EDNAFULL. Gap Open Penalty: 10. Gap Extend Penalty: 0.5. Output Format: Pair. End Gap Penalty: False. End Gap Open Penalty: False. Gap Open Penalty: 10. End Gap Extend Penalty: 0.5. For protein sequences, use: Matrix: EBLOSUM62. Gap Open Penalty: 10. Gap Extend Penalty: 0.5. Output Format: Pair. End Gap Penalty: False. End Gap Open Penalty: 10. End Gap Extend Penalty: 0.5) to create an optimal global alignment of two sequences.

[0254] The term "adeno-associated virus vector" refers to a nucleic acid derived from any AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any other virus or serotype that shares homology with the capsid protein of an AAV serotype in terms of its capsid protein sequence. The term "recombinant adeno-associated virus" or "rAAV" refers to an infectious, replication-defective virus composed of an AAV protein coat that encapsulates a nucleic acid molecule of interest, flanked on one or both sides by AAV ITRs. As used herein, reference to a particular AAV serotype means an AAV having at least one capsid protein of that AAV serotype. For example, the term "AAV2" refers to an AAV having at least one AAV serotype 2 capsid protein.

[0255] The term "CYP4V2" refers to cytochrome P450 4V2 or cytochrome P450 family 4 subfamily V polypeptide 2 (sometimes referred to as CYP4AH1), and its orthologues in other species. Mutations in CYP4V2 are associated with BCD (see, e.g., Li et al., Am J Hum Genet. 74:817-826, 2004) and retinitis pigmentosa (see, e.g., Wang et al., PLOS ONE 7:e33673, 2012). The full-length genomic human CYP4V2 gene is approximately 22,053 bp in length and can be found, for example, on the World Wide Web at genecards.org / cgi-bin / carddisp.pl?gene=CYP4V2&keywords=CYP4V2. As used herein, the term "hCYP4V2" refers to the human CYP4V2 gene or protein. It is understood that hCYP4V2 and CYP4V2 may refer to a gene or protein containing a genetic or epigenetic alteration or a gene or protein without a genetic or epigenetic alteration.

[0256] As used herein, the term "functional CYP4V2" refers to a protein or a nucleotide molecule that produces the protein when expressed that is effective in providing a therapeutic benefit (e.g., improving or rescuing abnormal fatty acid levels (e.g., DHA levels) in a target cell) to an individual (e.g., an individual with a genetic or epigenetic change in the CYP4V2 molecule). A functional CYP4V2 molecule can correspond to a wild-type hCYP4V2 sequence or a naturally occurring variant thereof (e.g., a polymorphic variant; e.g., a variant that does not contain pathological changes) or an optimized sequence. In some embodiments, a functional CYP4V2 molecule is a CYP4V2 molecule from another species (e.g., another mammal, such as a rodent, rabbit, dog, pig, or non-human primate) that shares similar orthology with human CYP4V2. For example, an ortholog of the human CYP4V2 sequence is the murine mCyp4v3 sequence. In some embodiments, the functional CYP4V2 molecule is another P450 molecule (eg, a CYP4 protein).

[0257] The term "ocular cell" refers to any cell in the eye or associated with ocular function, including but not limited to retinal cells, retinal bipolar cells, photoreceptor cells or photoreceptor progenitor cells (including rods and / or cones, collectively referred to as "PRCs"), ganglion cells, retinal pigment epithelial (RPE) cells, choroidal epithelial (CE) cells, corneal epithelial cells (CEC), choroidal cells or corneal cells or optic nerve cells.

[0258] The term "loss of function" or "dysfunctionality" refers to a decrease or loss of cell function (e.g., photoreceptor function, photoreceptor cell function, retinal pigment epithelial cell function, lens function, choroidal function, or corneal function) compared to normal, non-disease-affected cells or compared to the other eye or the same eye at an earlier time point. As used herein, "degeneration," "atrophy," "disorder," "disease," and / or "dystrophy" are used synonymously with respect to loss of function. The term "increased function" means improving function (e.g., function of photoreceptors, photoreceptor cells, retinal pigment epithelial cells, choroidal cells, or keratocytes) or increasing the number or percentage of functional photoreceptors or cells (e.g., photoreceptor cells, retinal pigment epithelial cells, choroidal cells, or keratocytes) compared to a disease-affected eye (having the same ocular disease), the same eye at an earlier time point, the untreated portion of the same eye, or the fellow eye of the same patient.

[0259] The term "transgene" refers to a donor nucleic acid that is intended to be introduced or has been introduced into a cell or organism. Transgenes include any gene, such as the genes or cDNAs set forth in Table 4.

[0260] The terms "pharmaceutically acceptable formulation" and "physiologically acceptable formulation" and "pharmaceutically acceptable carrier" mean a biologically acceptable formulation, gas, liquid or solid or mixtures thereof that is suitable for one or more routes of administration, in vivo delivery, in vitro delivery or contact, and may include formulations or carriers used in therapies for other diseases (e.g., gene therapy or cell therapy for other eye diseases). A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, to administer proteins, polynucleotides, plasmids, viral vectors or nanoparticles to cells or subjects. These compositions include, but are not limited to, solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic agents, and absorption enhancers or delay agents that are compatible with drug administration or in vivo or in vitro contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents, lubricants and thickeners. These pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterials, antivirals and antifungals and immunosuppressants) may also be incorporated into the composition. As described herein or known to those skilled in the art, pharmaceutical compositions can be formulated to be compatible with specific administration or delivery routes. Therefore, pharmaceutical compositions include carriers, diluents or excipients suitable for administration by various routes.

[0261] The term "crRNA" refers to the CRISPR RNA that contains both the protospacer element and additional nucleotides that are complementary to the tracrRNA.

[0262] The term "tracrRNA" refers to transactivating crRNA, which hybridizes with crRNA and binds to the Cas9 protein, activating the complex to create double-strand breaks at specific sites within the genomic sequence.

[0263] The term "sgRNA" refers to a single-guide RNA that combines crRNA and tracrRNA, which are separate molecules in the native CRISPR / Cas9 system in Streptococcus pyogenes, into a single RNA construct.

[0264] The term "PAM" refers to the "protospacer adjacent motif," which is a short sequence in either strand of the genome that is recognized by the CRISPR nuclease as a cleavage site. The PAM varies with the nuclease (e.g., Cas9, Cpf1, etc.). The protospacer element sequence is typically located directly upstream of the PAM site.

[0265] The term "protospacer element" (also called "guide RNA" or "CRISPR gRNA" or "gRNA" or g1, g2, g3, g4, g5, etc.) refers to the portion of the crRNA (or sgRNA) that is complementary to the genomic DNA target sequence.

[0266] DHA: Docosahexaenoic Acid, a polyunsaturated ω-3 fatty acid, also known as 22:6(ω-3) or C22:6n3.

[0267] AA: Arachidonic Acid, a polyunsaturated ω-6 fatty acid, also known as 20:4(ω-6) or C20:4n6 or ARA.

[0268] PBS(+): phosphate buffered saline (PBS) containing calcium and magnesium.

[0269] PBS(-): phosphate-buffered saline (PBS) without calcium or magnesium. [Specific implementation method]

[0270] Detailed description of preferred embodiments

[0271] Methods and compositions for BCD cell disease models

[0272] Developing appropriate BCD disease models and defining the molecular phenotype of BCD are crucial for BCD research, as well as for the development and testing of targeted drugs and treatment options. This is also crucial for understanding CYP4V2 function. As outlined in the Background section of this article, the clinical phenotype of BCD has been characterized, established, and studied for over 80 years, and the genetic mutations that cause BCD have been identified for over a decade. However, a gap remains between the clinical phenotype (e.g., crystalline deposits in the retina of BCD patients) and the underlying CYP4V2 mutations.

[0273] Previous studies on BCD have found abnormal fatty acid levels in BCD patients, including in fibroblasts, lymphocytes, and serum. For example, in Lee et al., The Metabolism of Fatty Acids in Human Bietti Crystalline Dystrophy, Invest Ophthalmol Vis Sci. 2001 Jul;42(8):1707-14, researchers used a pulse-chase method to study abnormalities in fibroblasts and lymphocytes from BCD patients. Fibroblasts and lymphocytes from BCD patients and normal controls were incubated with [(14)C]18:3n-3 or [(14)C]18:2n-6. Fibroblasts from patients with BCD showed that the conversion of 18:3n-3 to polyunsaturated fatty acids (PUFAs) was reduced compared to normal subjects, but the conversion of 18:2n-6 was not reduced. In another study (Lai et al., Alterations in Serum Fatty Acid Concentrations and Desaturase Activities in Bietti Crystalline Dystrophy Unaffected by CYP4V2 Genotypes, Invest Ophthalmol Vis Sci 2010; 51: 1092-7), researchers used GC-MS to analyze serum fatty acid concentrations in serum samples from BCD patients and controls. The study found that the concentration of octadecanoic acid (18:0) in the serum of BCD patients was higher than that in the control subjects, and the concentration of octadecadienoic acid (18:1n-9) was lower than that in the control subjects. In addition, the concentration of total monounsaturated fatty acids in BCD was significantly lower than that in the control subjects. However, in another study that did not use BCD patient samples as study subjects (Nakano et al., CYP4V2 in Bietti's Crystalline Dystrophy: Ocular Localization, Metabolism of omega-3-Polyunsaturated Fatty Acids, and Functional Deficit of the p.H331 P Variety, Mol Pharmacol 82:679-686, 2012), the results showed that the CYP4V2 enzyme has ω-hydroxylase activity towards ω-3-PUFAs.

[0274] It is important to confirm whether the abnormal fatty acid level found in the fibroblast and serum of BCD patient is actually present in the RPE cell of BCD patient, and RPE cell is the pathogenic cell of BCD.Therefore, need a kind of BCD disease model that can allow directly studying the RPE cell of BCD patient to come to more understanding to BCD disease pathology and CYP4V2 function, and assess the effect of potential treatment option.Yet, in view of the position of RPE and the rarity of BCD, it is unrealistic to obtain natural RPE cells from BCD patient.

[0275] The present disclosure provides BCD cell models and methods for generating BCD cell models. BCD cell models are composed of BCD patient-specific stem cells (including but not limited to induced pluripotent stem cells (iPSCs), embryonic stem (ES) cells, somatic (or adult) stem cells, mesenchymal stem cells (MSCs)) and ocular cells derived from any stem cells of BCD patients (including but not limited to RPE cells, photoreceptor (rod or cone) cells, photoreceptor progenitor cells, corneal epithelial cells, lens cells and / or choroidal cells). In addition to patient-specific stem cells, BCD cell models can also be generated by creating artificial CYP4V2 mutations in cells from subjects without BCD, and these cells can be ES cells, iPS cells, or other stem cells, or any cells that can be reprogrammed into stem cells, or any ocular cells (whether or not derived from stem cells).

[0276] Induced pluripotent stem cell technology provides an alternative for disease modeling in animal models. However, not all diseases can be successfully modeled using iPSCs. (Urbach, A., Bar-Nur, O., Daley, GQ & Benvenisty, N. Differential Modeling of Fragile X Syndrome by Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. Cell Stem Cell 6, 407-411 (2010)). In addition, given the reported fatty acid anabolism associated with BCD, it is unclear whether BCD patient-specific iPS or patient-specific iPS-RPE cells can be produced by iPS technology.

[0277] A. Induced Pluripotency

[0278] The method for producing induced pluripotent stem cells (iPSCs) is known in the art to which the present invention belongs. Almost all types of somatic cells can be used as source cells for iPSC reprogramming. In short, iPSCs can be made by introducing a specific set of proteins (e.g., nucleic acids encoding a specific set of proteins or by direct delivery of proteins) into cells. Those skilled in the art will understand that an exemplary non-limiting method is by introducing one or more transgenes encoding one or more of OCT4, SOX2, KLF4 and / or c-MYC (e.g., "Yamanaka factors"). In some embodiments, reprogramming uses all four transcription factors. In some embodiments, one, two, or three transcription factors can be used. Li et al., Stem Cells, 2009; 27: 2992-3000. Zhu et al., Cell Stem Cell 2010; 7: 651-655. In some embodiments, iPSCs can be produced by directly delivering reprogramming proteins. Kim et al., Cell Stem Cell. 2009; 4(6): 472-6. The Examples section provides methods for generating iPSCs using non-integrating methods, such as by Sendai virus (Example 1) or by episomal methods (Example 2). However, any method for generating iPSCs is contemplated within the scope of this disclosure.

[0279] iPSCs can be generated using various methods (e.g., Sendai virus, episomal methods, with or without small molecules), see the Examples section, and see, for example, Hubbard et al., J. Vis. Exp., 2014, 92:52009. In addition, methods for making iPSCs from a variety of different cell types are known in the art. See, for example, Hayashi et al., 2012, PLoS One, 7(9):e45435; Poon et al., 2015, PLoS One, 10(7):e0131288; Lamba et al., 2010, PLoS One, 5(1):e8763. iPSCs typically express detectable levels of at least one marker, including but not limited to Oct-4, Sox-2, SSEA4, TRA-1-60, TRA-1-81, AP, and / or NANOG.

[0280] Any type of stem cell can be used to produce the BCD cell model described herein, including but not limited to induced pluripotent stem cells (iPSC), hematopoietic stem cells (HSC), embryonic stem (ES) cells, mesenchymal stem cells, adult stem cells or tissue-specific stem cells. The stem cells used in the methods described herein can be pluripotent, multipotent or totipotent stem cells.

[0281] As used herein, the term "pluripotent" refers to a cell that is at least capable of developing into one of the ectoderm, endoderm, and mesoderm cells. In one embodiment, the term "pluripotent" refers to a cell that is omnipotent and multipotent. As used herein, the term "totipotent" cell refers to a cell that is capable of developing into cells of all lineages. As used herein, the term "multipotent" refers to a cell that has not undergone terminal differentiation. The multipotent cells disclosed herein can be any stem cell, or produced by non-pluripotent cells (such as fibroblasts) using induction, dedifferentiation, and nuclear transfer methods known in the art to which the present invention belongs. The multipotent cells described herein, whether stem cells or produced by non-pluripotent cells, can be from subjects with BCD or CYP4V2 mutations, or from healthy subjects without BCD to serve as a control group or for creating artificial CYP4V2 mutations.

[0282] Almost any cell type can be reprogrammed into iPS cells. See the section entitled “Cell Origin” in this article for a discussion.

[0283] B. iPSC Differentiation

[0284] Differentiate BCD patient iPS cells into iPS-RPE cells (or another type of ocular cell (e.g., iPS-CEC, iPS-CE cells, or iPS-PRC). Methods for differentiating iPSCs into RPE cells or another type of ocular cell (e.g., CEC and PRC) are known. See, e.g., the Examples section; Hayashi et al., 2012, PLoS One, 7(9):e45435; Songstad et al., Investigative Ophthalmology & Visual Science, December 2015, Vol. 56, 8258-8267; and Lamba et al., PLoS One. 2010 Jan 20;5(1):e8763. For example, induced pluripotent stem cells (iPSCs) reprogrammed from cells can be generated and these cells can be further differentiated into, for example, RPE cells (referred to herein as "iPS-RPE"), corneal epithelial cells (referred to herein as "iPS-CEC"), photoreceptor cells (or photoreceptor progenitor cells; referred to herein as "iPS-PRC"), or iPS-choroidal endothelial (CE) cells (referred to as "iPS-CE").

[0285] The differentiated cells (eg, iPS-RPE cells) are tested for biochemical function (as described herein and in the Examples section) to assess their biochemical defects / abnormalities compared to healthy control iPS-RPE cells.

[0286] The iPS-RPE cell line produced as described herein shows the form (for example, pigmentation and hexagonal) that can indicate RPE cell and / or expresses one or more biomarkers that can indicate RPE cell.The biomarker of RPE cell (and iPS-RPE cell) is known and includes but not limited to one or more in RLBP1 (also known as CRALBP), RPE65, bestrophin-1 (BESTROPHIN-1), MITF, focal adhesion protein (VINCULIN), LRAT, RDH5, PAX6, MERTK, TYR and / or ZO-1, and can be used to judge or confirm that RPE differentiation has occurred.Similarly, the biomarker of CEC (and iPS-CEC) and PRC (and iPS-PRC) is known and includes for example cytokeratin 12 (cytokeratin 12) and cytokeratin 3 for corneal epithelial cell;And for Crx of photoreceptor, for the recovery protein (recoverin) of rod and cone and for the Nrl of rod.

[0287] Through the iPS reprogramming and RPE differentiation methods as described in the Examples section, BCD patient-specific iPS and iPS-RPE cells were successfully generated.

[0288] C. Biochemical analysis to identify biochemical defects / abnormalities in iPS-RPE cells from BCD patients and cell viability analysis to assess RPE atrophy

[0289] A panel of biochemical assays was developed and used to assess and characterize the phenotypes in BCD patient-specific iPS-RPE cells.

[0290] First, our biochemical analysis included a more comprehensive list of fatty acids. Previous studies have identified abnormal serum fatty acid levels in BCD patients, testing samples for the following fatty acids: 16:0, 16:1, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:3n-6, 20:4n-6, 22:5n-3, 22:6n-3, 24:0, and 24:1. This study found that octadecanoic acid (18:0) concentrations were higher in the serum of BCD patients compared to controls, while octadecadienoic acid (18:1n-9) concentrations were lower. To determine whether the same fatty acid abnormalities were present in BCD patient-specific iPS-RPE cells, as well as whether additional fatty acid abnormalities were present, a biochemical analysis encompassing a larger range of fatty acids was developed using LC-MS (see Table 2).

[0291] In addition, in order to judge whether BCD patient-specific iPS-RPE cells carry other abnormalities except fatty acid, in this analysis, comprise other lipid species, comprise ceramide (Cer), sphingomyelin (SM) and sphingosine and dihydrosphingosine (SOSA), so that the phenotype in the BCD disease model is analyzed and the biochemical function of measuring CYP4V2 protein.Referring to the list of different classes and compound included in the biochemical analysis in order to test BCD patient-specific iPS-RPE cells of Table 2.

[0292] Surprisingly, test results (see Examples section) showed that BCD patient-specific iPS-RPE cells had a fatty acids abnormality profile that was different from that found in the serum of BCD patients.

[0293] The eye is the human body's photoreceptor. BCD begins with RPE atrophy, which in turn causes photoreceptor death and vision loss. The key function of the RPE is light absorption (Strauss, 2005, The retinal pigment epitheliuminvisual function. Physiol Rev 85: 845-81). Exposure to ambient light may affect the development and progression of human retinal degeneration, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Exposure to light in eye disease models is a model system suitable for studying retinal degeneration. Exposure to light, including exposure to blue light, has been widely used in retinal research (Dual roles of polyunsaturated fatty acids in retinal physiology and pathophysiology associated with retinal degeneration, Masaki Tanito & Robert Anderson (2009) Clinical Lipidology, 4:6, 821-827. Seko et al., Graefes Arch Clin Exp Ophthalmol. 2001 Jan;239(1):47-52. Blue light-induced apoptosis in cultured retinal pigment epithelium cells of the rat. Narimatsu et al., Exp Eye Res. 2015 Mar;132:48-51. Blue light-induced inflammatory marker expression in the retinal pigment epithelium-choroid of mice and the protective effect of a yellow intraocular lens material in vivo).Blue light is present in ambient light, such as sunlight and artificial lighting (e.g., office lighting), as well as from electronic display devices such as TVs, monitors, smartphones, laptops, and tablets (Moon et al., Blue light effect on retinal pigment epithelial cells by display devices, Integr Biol (Camb). 2017, 22; 9(5): 436-443. doi: 10.1039 / c7ib00032d).

[0294] In this study, cell viability analysis found RPE atrophy in the BCD cell model. Cell death caused by exposure to (blue) light in iPS-RPE samples of BCD patients was significantly higher than that caused in control group samples. The clinical phenotype of BCD (i.e., RPE atrophy) is obvious in the BCD cell model. AAV.CYP4V2 showed the effect of rescuing RPE atrophy in the BCD cell model.

[0295] Application of D.BCD cell model

[0296] In addition to assessing the cellular level phenotype associated with BCD, the BCD cell model can also be used for other applications of disease models, including but not limited to drug screening, developing therapeutic agents or devices, determining dose ranges, safety and toxicity testing, testing different formulations for BCD or other conditions associated with CYP4V2, or studying CYP4V2 functions and uses, including but not limited to developing and screening drugs comprising or expressing CYP4V2 proteins, such as CYP4V2 gene therapy. In addition, BCD patient-specific iPS-RPE (and other eye cells derived from BCD patient-specific stem cells, including but not limited to iPS-photoreceptor cells, iPS-keratocytes) can be used as cell therapy, used in an unmodified form or after gene repair (e.g., by gene transfer or gene editing as described herein). The Examples section provides examples of non-limiting examples of BCD cell model applications.

[0297] E. Methods for Screening Compounds

[0298] It should be noted that the iPSC-RPE cell lines described herein can provide human cell disease models (for example, BCD, retinitis pigmentosa, IRD). These iPSC-RPE cells, iPSC-CEC cells or iPSC-PRC cells that can be collectively referred to as "iPSC-eye cells" can be used for diagnosis, prognosis, prediction of disease onset, severity and progression rate of BCD patients or retinitis pigmentosa patients or patients with the hereditary retinal disease of another type. For example, these iPSC-eye cell lines can also be used for screening out compounds that may have the therapeutic efficacy of treating or preventing the disease (for example, BCD) related to the heredity or epigenetic changes in CYP4V2 nucleic acid.

[0299] The pluripotent cells described herein, particularly pluripotent cells generated from subjects with genetic or epigenetic alterations in CYP4V2 or subjects with eye diseases (e.g., BCD), can be used as research tools in methods for identifying compounds that can be therapeutic candidates for the treatment, diagnosis, prognosis, or prevention of eye diseases (e.g., BCD). It should be understood that the test compound can be any type of compound. It can be of natural origin or can be produced by chemical synthesis. It can be a library of structurally defined compounds, uncharacterized compounds or substances, or mixtures of compounds. Those skilled in the art will appreciate that the test compound can be, but is not limited to, nucleic acids or analogs thereof, polypeptides or analogs thereof, antibodies, chemicals, and small molecules.

[0300] The cells described herein can be evaluated for their ability to grow and function in an animal model (e.g., in the eye of an animal model) in the presence or absence of a test compound and for their tendency to form tumors or lack thereof. A variety of methods can be used to evaluate the cells, including but not limited to PCR techniques, immunoassays, and / or lipid / fatty acid metabolism analysis.

[0301] Methods and compositions for cell therapy

[0302] As discussed herein, CYP4V2 gene therapy shows the effect of correcting the biochemical abnormalities in BCD patient-specific iPS-RPE cells. However, the prerequisite for gene therapy to work in vivo is that the experimenter still has some RPE and photoreceptor cells to remain in the eye being treated. About late BCD patients with only a small amount or no RPE cells or photoreceptor cells left in the eyes, cell therapy can be used as an alternative or in combination with gene therapy, as a treatment option.

[0303] Cell therapy relates to transplanting new cells to replace dead cells or degenerate cells. About BCD, new cells can be RPE cells, photoreceptor cells (cones and / or rods), photoreceptor progenitor cells, choroid cells, corneal epithelial cells, lens cells or other types of eye cells, which depends on which type of cell in the experimenter shows degeneration and needs to be replaced. The following description and the embodiments herein use iPS-RPE cells to illustrate these methods and processes. It can be applied to other types of eye cells.

[0304] Cell therapies for BCD and other types of eye diseases can be categorized as follows. Other types of eye diseases include, but are not limited to, inherited retinal diseases (IRDs), retinitis pigmentosa (RP), and macular degeneration (including age-related macular degeneration (AMD)).

[0305] (1) Allogeneic transplantation:

[0306] In one embodiment, RPE cells, PRC, CEC, CE cells and other eye cells derived from embryonic stem cells (ESC) or iPSC from healthy donors can be used in allografts as the cell therapy for BCD. It relates to the healthy ESC or iPSC from healthy individuals (i.e., without CYP4V2 mutants) being differentiated into RPE cells and the ESC-RPE cells being transplanted to the eyes of BCD patients. This paper provides a method for reprogramming iPSC and differentiating ESC or iPSC into RPE in the examples section. In previous studies, RPE cells derived from embryonic stem cells (ESC) have been used to treat age-related macular degeneration (AMD), referring to Schwartz et al., Investigative Ophthalmology & Visual Science April 2016, Vol. 57, ORSFc1-ORSFc9. The advantage of allografts (allo-graft) or allografts is that they are cheaper than autologous transplants because a common source can be used to treat multiple patients. However, it also has significant disadvantages, such as immune rejection by the host subject, which may significantly affect its efficacy and duration. In addition, it requires long-term immunosuppressive therapy, which may cause serious systemic side effects. Finally, the use of ESCs may raise ethical issues.

[0307] (2) Autologous transplantation without gene repair:

[0308] In one embodiment, autologous cells can be used in the cell therapy for BCD. One such autologous source is the iPS cell and iPS-RPE cell derived from a BCD patient, which can be transplanted to the eyes of this BCD patient. BCD is a relatively late-onset disease. The symptoms of a BCD patient typically occur between the 2nd, 3rd or even 4th decade of life. In addition, the iPS reprogramming method has a certain degree of "reset clock (reset the clock)" effect on iPS cells and the cell derived from an iPS cell. Therefore, the iPS-RPE cell and other iPS-eye cells derived from a BCD patient can be used as cell therapy and transplanted to a BCD patient, and even do not need to carry out any gene repair to the CYP4V2 mutation in the iPS-RPE cell. iPS reprogramming and RPE differentiation methods are provided in the Examples section herein. As a precautionary measure, whether any pathogenic mutation is produced during iPS reprogramming and RPE differentiation process, whole genome sequencing can be carried out to compare the genomic DNA in iPS or iPS-RPE cells and the genomic DNA in source cells (e.g., fibroblasts or hemocytes).

[0309] (3) Gene-corrected patient-derived autologous cells for cell therapy targeting BCD and other types of IRD and RP

[0310] The present disclosure provides methods and compositions for generating gene-repaired autologous cells for cell therapy. As used herein, "gene-repaired" or "gene repair" refers to correcting CYP4V2 mutations by gene editing the patient's genome (e.g., directly on a chromosome using, for example, CRISPR / Cas9, CRISPR / Cpf1, zinc fingers, TALENs) or by gene transfer of a healthy copy (cDNA, RNA or other form) of the CYP4V2 gene that is not normally integrated into the genome into the patient's cells (e.g., CYP4V2 gene therapy as described herein) or by correcting or compensating for defective mRNA in the patient's cells.

[0311] As for the disease caused by genetic mutation, the autologous cells used in the cell therapy for BCD or another IRD or RP should ideally have repaired their genetic defects (i.e. CYP4V2 mutations) and / or their dysfunctional CYP4V2 proteins before transplantation. In one embodiment, the gene repair can be achieved by gene transfer therapy as discussed herein, including but not limited to AAV-mediated gene therapy transfer of nucleic acid sequences encoding and expressing functional CYP4V2 proteins. The compositions and methods of CYP4V2 gene transfer therapy are provided herein, with reference to the detailed description and examples section herein. BCD patient-specific source cells iPS or iPS-RPE cells can be treated with AAV.CYP4V2 (as provided herein), followed by iPS reprogramming and / or RPE differentiation (if applicable) and verification of improved biochemical function (as provided herein), and then transplanted to the original patient's eyes. In another embodiment, the gene repair can be achieved by gene editing, such as correcting the CYP4V2 mutation in the genome or RNA in BCD patient cells. In addition to being administered in vitro as a part of cell therapy, the gene editing can also be directly administered in vivo as gene therapy. The gene editing can be performed on the patient's source cells (e.g., fibroblasts or blood cells), iPS, iPS-RPE or other types of iPS- eye cells. iPS reprogramming and RPE differentiation for producing patient-specific iPS and iPS-RPE can be performed before or after gene repair (e.g., gene transfer therapy or gene editing).

[0312] The present disclosure provides compositions and methods for correcting CYP4V2 mutations via gene editing. The description in the Examples section herein illustrates compositions and methods for correcting the most common mutation, c.802-8_810del17insGC, in BCD patients using CRISPR / Cas9 constructs. The invention can also be used for other gene editing methods (e.g., CRISPR / Crp1, TALEN, zinc finger) and other IRD mutations (e.g., other CYP4V2 mutations in Table 1), in combination with methods known in the art to which the invention belongs.

[0313] The most common CYP4V2 mutation in BCD patients is c.802-8_810del17insGC (referring to a 17-base deletion and a two-base (GC) insertion eight bases from the end of intron 6 of the CYP4V2 gene, also known as IVS6-8del / insGC; see SEQ ID NO: 46, which shows the sequence of the human CYP4V2 genomic DNA region containing the c.802-8_810del17insGC mutation, and SEQ ID NO: 47, which shows the corresponding wild-type sequence). The c.802-8_810del17insGC mutation is shown in the sequence below showing the human CYP4V2 intron 6-exon 7 junction. The intron 6 sequence is shown in lowercase letters, and the exon 7 sequence is shown in uppercase letters. 17 bp deletion and GC insertion are in brackets): caa aca gaa gca tgt gat tat cat tca aa(tca tac agG TCATCG CT)(GC)GAACGG GCC AAT GAA ATG AAC GCC AAT GA (SEQ ID NO: 46), which results in the prediction of skipping exon 7. (Xiao et al., Biochem Biophys Res Commun. 409: 181-6, 2011; Meng et al., 2014, Mol. Vis., 20: 1806-14; Wada et al., Am J Ophthalmol. 139: 894-9, 2005; Jiao et al., European Journal of Human Genetics (2017) 25, 461-471). Recent studies estimate that the c.802-8_810del17insGC mutation exists between 1,040 and 8,200 generations in Chinese populations and between 300 and 1100 generations in Japanese populations. Human Genetics(2017)25,461-471.

[0314] Cell therapy (also known as cellular therapy or cytotherapy) can be used as described herein to treat or prevent an eye disease in a subject. As described herein, BCD, certain RP, IRD, and other eye diseases mentioned herein are associated with genetic or epigenetic changes in the CYP4V2 nucleic acid sequence.

[0315] Cell therapy generally involves injecting, implanting, transplanting, or otherwise delivering a composition comprising cells into a subject, e.g., into a patient's tissue or organ (e.g., the eye). The methods described herein are unique in that they allow for autologous cell therapy with gene repair in subjects with ocular diseases.

[0316] Methods described herein include: obtaining cells from subjects with eye diseases (e.g., associated with genetic or epigenetic changes in CYP4V2 nucleic acid sequences) and using, for example, gene editing to repair mutations in CYP4V2 nucleic acids (e.g., DNA or RNA), or repairing (e.g., gene transfer) via delivery of nucleic acid sequences encoding functional CYP4V2 proteins. Before cells are administered back into the subject (e.g., into the subject's eyes), cells can be made into pluripotent cells (e.g., by inducing pluripotency, such as to make iPSC) and can be differentiated into one or more eye cells (e.g., iPS-RPE, iPS-CEC, iPS-PRC). It should be understood that cells can be genetically repaired before or after they become pluripotent cells or after they are differentiated into eye cells.

[0317] A. Cellular Origin

[0318] In some cases, autologous cells (e.g., subject (e.g., patient)-specific cells) can be used in the cell therapy methods described herein. For example, cells such as fibroblasts or peripheral blood mononuclear cells (PBMCs) can be obtained from a subject and these cells can be used to produce iPSCs, as described in the Examples section. Almost all types of cells can be used to produce iPSCs and therefore can be used as source cells. In some cases, cells obtained from urine (see, for example, Zhou et al., 2012, Nat. Protoc., 7: 2080-9) or hair follicles or dermal papilla cells (dermal papilla cells) (see, for example, Muchkaeva et al., 2014, Acta Naturae, 6: 45-53) can be used to produce iPSCs.

[0319] B. Induced Pluripotency

[0320] Methods for producing induced pluripotent stem cells (iPSCs) are known in the art to which the present invention pertains. In short, iPSCs can be produced by introducing a specific set of proteins (e.g., nucleic acids encoding a specific set of proteins) into cells. Those skilled in the art will appreciate that an exemplary, non-limiting method is by introducing one or more transgenes encoding OCT4, SOX2, KLF4, c-MYC (e.g., "Yamada factors"). In some embodiments, reprogramming uses all four transcription factors. In some embodiments, one, two, or three transcription factors can be used. Li et al., Stem Cells, 2009; 27: 2992-3000. Zhu et al., Cell Stem Cell 2010; 7: 651-655. In some embodiments, iPSCs can be generated by directly delivering reprogramming proteins. Kim et al., Cell Stem Cell. 2009; 4(6): 472-6. The Examples section provides methods for generating iPSCs using non-integrating methods, such as by Sendai virus (Example 1) or by episomal methods (Example 2). However, any method for generating iPSCs is contemplated to be within the scope of the present disclosure.

[0321] iPSCs can be generated using various methods (e.g., Sendai virus, episomal methods, with or without small molecules), see the Examples section, and see, for example, Hubbard et al., J. Vis. Exp., 2014, 92:52009. In addition, methods for making iPSCs from a variety of different cell types are known in the art. See, for example, Hayashi et al., 2012, PLoS One, 7(9):e45435; Poon et al., 2015, PLoS One, 10(7):e0131288; Lamba et al., 2010, PLoS One, 5(1):e8763. iPSCs typically express detectable levels of at least one marker, including but not limited to Oct-4, Sox-2, SSEA4, TRA-1-60, TRA-1-81, AP, and / or NANOG.

[0322] Any type of stem cell can be used in the cell therapy methods described herein, including but not limited to induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), embryonic stem (ES) cells, mesenchymal stem cells, adult stem cells, or tissue-specific stem cells. The stem cells used in the methods described herein can be multipotent, pluripotent, or totipotent stem cells.

[0323] As used herein, the term "pluripotent" refers to a cell that is at least capable of developing into one of ectoderm, endoderm, and mesoderm cells. In one embodiment, the term "pluripotent" refers to a cell that is omnipotent and multipotent. As used herein, the term "totipotent" cell refers to a cell that is capable of developing into cells of all lineages. As used herein, the term "multipotent" refers to a cell that has not undergone terminal differentiation. The pluripotent cells of the present invention may be any stem cell, or may be produced by non-pluripotent cells (such as fibroblasts) using induction, dedifferentiation, and nuclear transfer methods known in the art to which the present invention pertains. The pluripotent cells described herein, whether stem cells or those produced by non-pluripotent cells, may be derived from subjects or healthy individuals with BCD or CYP4V2 mutations.

[0324] iPSCs can be characterized by one or more of the following: a. unique morphology of iPSCs; b. one or more pluripotency markers, such as Oct-4, Sox-2, SSEA-4, TRA-1-60, TRA-1-81, Nanog, and AP; c. the ability to differentiate into a desired cell type (e.g., RPE cells); and / or d. teratoma analysis. Not all of the above are necessary to characterize iPSCs and verify pluripotency (e.g., teratoma; see, e.g., Buta et al., 2013, Stem Cell Res., 11(1):552-562).

[0325] C.Gene editing

[0326] In the methods described herein, a variety of gene editing techniques can be used to repair genetic or epigenetic changes present in a subject's CYP4V2 nucleic acid. Gene editing can be performed using any number of techniques, including clustered regularly interspaced short palindromic repeats (CRISPR) technology (see, e.g., U.S. Patent Nos. 8,697,359; 8,889,418; 8,999,641; and US2014 / 0068797), transcription activator-like effector nucleases (TALEN) technology (see, e.g., Li et al., 2011, Nucleic Acids Res., 39(14): 6315-25), or zinc finger nuclease technology (see, e.g., Wright et al., 2005, The Plant J., 44: 693-705).

[0327] In order to complete gene editing using CRISPR technology, it can be by encoding nuclease (for example, often Cas9 nuclease, but other nucleases (for example, other Cas nucleases, such as Cpf1, or non-Cas nucleases)) nucleic acid is incorporated into one or more vectors, and applied to subject as described herein.For example only, cell described herein (for example, subject cell before being reprogrammed into iPSC, before being differentiated into RPE, corneal epithelial cell or photoreceptor cell or subject iPSC (referred to herein as " iPSCs-RPE ", " iPSC-CEC " or " iPSC-PRC ") after being differentiated into RPE, corneal epithelial cell or photoreceptor cell)) can be with one or more containing and / or encoding at least one guide RNA (gRNA), at least one CRISPR associated protein (for example, Cas9 or Cpf1) and at least one donor template nucleic acid construct (for example, carrier, RNP, mRNA) transduction or transfection.In some embodiments, donor template nucleic acid is not needed, for example, when reaching gene repair via knocking out (knock out).

[0328] Similarly, in order to complete gene editing using TALEN technology, nucleic acids encoding TALEN (e.g., dimeric transcription factor / nuclease) can be incorporated into a vector and applied to a subject as described herein. Similarly, in order to complete gene editing using zinc finger nuclease technology, nucleic acids encoding custom DNA endonucleases (custom DNA endonucleases) (e.g., heterodimers, wherein each subunit contains a zinc finger domain (zinc finger domain) and a FokI endonuclease domain (FokI endonuclease domain)) can be incorporated into one or more vectors and applied to a subject as described herein.

[0329] The components required to perform each of these techniques are commercially available and can be customized for a specific target sequence. See, for example, Caribou Biosciences; GenScript, CRISPR Therapeutics; Editas Medicine; Cellectis Bioresearch; Life Technologies; Sangamo BioSciences; or Sigma Aldrich Chemical Co.

[0330] Where appropriate, gene editing can occur such that the genetic or epigenetic changes in the subject's CYP4V2 nucleic acid are repaired, thereby allowing functional CYP4V2 protein to be expressed. The CYP4V2 nucleic acid sequence is repaired when the presence of the CYP4V2 nucleic acid (e.g., CYP4V2 mRNA) is restored, the presence of the CYP4V2 protein is restored, or the function of the CYP4V2 protein is restored. Similarly, "repair" or "correction" can refer to restoring the affected sequence (e.g., genetic or epigenetic changes) to a wild-type sequence or another non-mutated sequence as described herein.

[0331] There may be some situations where one or more mutations are introduced into cells (e.g., in CYP4V2 nucleic acids) using gene editing. This is a way of creating a cell model of a disease (e.g., BCD). For example, embryonic stem cells (ES cells) can be gene edited to produce a cell line with an artificial CYP4V2 mutation, which is then differentiated into RPE cells. Alternatively, iPS cell lines from healthy subjects (e.g., non-BCD subjects) or RPE cell lines (e.g., ARPE-19 cell lines) can be gene edited to produce CYP4V2 mutant iPS or RPE cell lines.

[0332] In some cases, it is desirable to screen cells (e.g., using whole genome sequencing) after the gene editing step is complete to confirm that the targeted mutation has been repaired and that no significant off-target editing has occurred.

[0333] CRISPR and CRISPR-associated protein 9 (Cas9), known as CRISPR-Cas9, consist of an RNA-guided nuclease (Cas9) and a guide RNA (guide RNA), generating site-specific DNA breaks that are repaired by endogenous cellular mechanisms. Possible outcomes of this approach include mutation of specific sites via mutagenic non-homologous end-joining (NHEJ), insertions or deletions (indels) at the break site, and precise alteration of the genomic sequence via homologous recombination (HR) using a donor template introduced from an exogenous source. CRISPR guide RNA consists of two RNAs, referred to as CRISPR targeting RNA (crRNA, also referred to herein as CRISPR RNA) and trans-activating crRNA (tracrRNA). crRNA is typically about 20 nucleotides (nt) long. It hybridizes to the target DNA sequence via Watson-Crick base pairing and guides the Cas endonuclease to cleave the target genomic DNA.

[0334] In order to repair the most common CYP4V2 mutations via gene editing, various CYP4V2 mutation CRISPR correction constructs (see Examples) have been developed. CRISPR is used because it is relatively simple to implement and its editing efficiency is higher than other forms of gene editing, such as TALEN and zinc finger nucleases. The CRISPR construct contains optimized and in vitro validated gRNA sequences and different construct options, which can be easily used to correct the c.802-8_810del17insGC mutation in BCD patient cell lines, generating gene-repaired cells that can be used in cell therapies for BCD, including but not limited to autologous cell therapy.

[0335] CRISPR gene editing therapy involves the use of CRISPR-associated proteins (Cas), which are nucleases and CRISPR guide RNAs. The role of the CRISPR guide RNA is to guide Cas to the sequence targeted by the CRISPR guide RNA, which is a protospacer element contained in the CRISPR guide RNA that is complementary to the target sequence (or specific to the target sequence). In order for Cas (e.g., Cas9 or Crf1) to bind to and cut the target sequence or near the target sequence, a protospacer adjacent motif (PAM) sequence is also required. The PAM sequence is a short stretch of DNA (usually 2-6 nucleotides) that serves as a binding signal for Cas. Different Cas can have different PAMs and cutting patterns. For example, for Streptococcus pyogenes Cas9 (SpCas9), the typical PAM sequence is NGG. For Staphylococcus aureus (SaCas9), the PAM sequence is NGRRT or NGRRN. For Neisseria meningitidis (NM) and Treponema denticola (Td), the PAM sequences are NNNNGATT and NAAAC, respectively. Engineered or mutated Cas sequences can also cause changes in the PAM sequence. For example, the SpCas9 VQR variants (D1135V, R1335Q, and T1337R) have PAM sequences of NGAN or NGNG. The SpCas9 EQR variants (D1135E, R1335Q, and T1337R) have PAM sequences of NGAG. The SpCas9 VRER variants (D1135V, G1218R, R1335E, and T1337R) have PAM sequences of NGCG. For Cpf1, the PAM sequence is TTTN. Cas normally generates double-stranded breaks (DSBs), but modified Cas enzymes can either generate single-strand breaks (e.g., SpCas9 Nickase (Cas9n D10A)) or no breaks (dCas9). Cas9 generates blunt ends 3 nt upstream of the PAM site, while Cpf1 cleaves in a staggered manner, generating a 5-nucleotide 5' overhang 18-23 bases away from the PAM.

[0336] The CRISPR guide RNA of Cas9 generally comprises CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). The crRNA comprises a protospacer element sequence designed to be complementary to (or specific for) the targeted sequence within or near the gene targeted for correction, destruction, or replacement, and a sequence corresponding to the complementary region of tracrRNA. The tracrRNA comprises a region complementary to the corresponding region of the crRNA and a sequence that interacts with CRISPR-associated protein 9 (Cas9). Cpf1 does not require tracrRNA.

[0337] The length of the protospacer element is typically about 20 nucleotides. Longer or shorter protospacer element sequences (about 16-24 nt) can also be used. The protospacer element can be 100% complementary to the target sequence or can contain mismatches with the target sequence. In some embodiments, a "G" nucleotide can be optionally added at the beginning of the protospacer element sequence.

[0338] After the DNA molecule is cut by Cas, it can be repaired by one of two ways. Error-prone non-homologous end joining (NHEJ) repairs can cause insertion-deletion mutations, which can destroy the function of the protein encoded by the gene. NHEJ can be used to produce artificial mutations in cell lines. In some embodiments, it can be used to produce mutations (for example, insertion-deletions in exons or splicing acceptor regions) in the CYP4V2 gene of a cell line (for example, ES cells, iPS cells or ARPE-19 cell line) without endogenous CYP4V2 mutations, thereby producing a disease cell model (for example, BCD cell model). In addition, two other CRISPR guide RNAs can be used together to knock out the targeted region or the entire target gene of the target gene, thereby producing a knockout model. In some embodiments, CRISPR-based gene silencing is used to destroy (or silence) or defective genes, for example, to treat dominant genetic diseases. During gene silencing, cells attempt to repair broken DNA, but NHEJ often confers errors in destroying genes when doing so, thereby effectively silencing the gene. In some embodiments, NHEJ may also result in correction of mutations, for example, particularly when the mutation is a single nucleotide variation or does not exceed about 10 nucleotides. Alternatively, if a donor nucleic acid sequence is available, the DNA break can be repaired by homology-directed repair (HDR) for correction or replacement of the target gene. The donor nucleic acid sequence can be provided in the form of single-stranded DNA (ssDNA), or single-stranded oligoDNA nucleotides (ssODN) or a vector. In some embodiments, for a donor nucleic acid sequence provided in the form of ssODN, the donor nucleic acid sequence is no more than about 1 kb, 800 bp, 600 bp, 500 bp, 400 bp, 300 bp, 280 bp, 260 bp, 240 bp, 220 bp, or 200 bp. In some embodiments, for a donor nucleic acid sequence provided in the form of a vector, the donor nucleic acid sequence is no more than about 25 kb, 20 kb, 15 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, 4.5 kb, 4 kb, 3.5 kb or 3 kb. In some embodiments, the donor nucleic acid sequence is symmetrical. In some embodiments, the donor nucleic acid sequence is asymmetric. In some embodiments, the length of the donor nucleic acid sequence can be adjusted for a higher HRD rate. In some embodiments, if the PAM targeted by the Cas used in CRISPR gene editing is also present in the donor nucleic acid sequence, it can be mutated (changed to a different nucleotide) so that the PAM is no longer present in the donor nucleic acid sequence, thereby avoiding the donor template or the DNA sequence repaired by the donor template from being cut and destroyed by Cas.In addition to correcting or replacing a mutated or defective gene or a portion thereof, HDR can also be used to generate artificial mutations (e.g., insertion of mutations in exons or splice acceptor regions) in the CYP4V2 gene of a cell line without endogenous CYP4V2 mutations (e.g., ES cells, iPS cells, or ARPE-19 cell line), thereby generating a disease cell model (e.g., a BCD cell model).

[0339] The CRISPR guide RNA and Cas used in CRISPR gene editing therapy can be provided in the form of a vector (e.g., a plasmid (e.g., pX330, pX458, pX459), a recombinant AAV vector, or a recombinant lentivirus vector) or mRNA and / or RNA and protein encoding such components.

[0340] The donor template can be provided in the form of ssDNA (eg, ssODN) or cloned in a plasmid or other type of vector (eg, an AAV vector (eg, AAV2 or AAV6)) for use in HDR.

[0341] A variety of compositions and methods can be used to improve on-target editing or repair efficiency and / or reduce potential off-target effects. For example, different Cas (e.g., Cas9 or Cpf1) or Cas from different species (e.g., SpCas9, SaCas9, NMCas9) or variants (SpCas9, SpCas9VQR) can be used to broaden the PAM options available for the target sequence, thereby enhancing specificity. If the target sequence region lacks an NGG PAM site for SpCas9 but is rich in AT, Cpf1 can be considered instead. Cas9 nickase (e.g., Cas9 D10A) only produces single-strand breaks in the target DNA, so two paired CRISPR guide RNAs are required to produce double-strand breaks. This requirement significantly increases target specificity because it is unlikely that two off-target nicks will be produced within a sufficiently close range to cause DSBs. In addition, asymmetric donor templates can enhance HDR rates. Catalytically inactive dCas9 will not cut the target DNA, but sequence replacement can still be achieved without any error-prone repairs that are typically associated with Cas9 cutting. See Richardson et al., Nature Biotechnology 34, 339-344 (2016).

[0342] Achieving targeted gene correction and simultaneously avoiding or minimizing off-target editing are two purposes of gene editing. Previous studies have revealed off-target mutations caused by gene editing technologies, including but not limited to CRISPR and TALEN, see, for example, Tsai et al., Nature Biotechnology 33, 187-197 (2015); Wang et al., Nature Biotechnology 33, 175-178 (2015); Wu, WH et al. CRISPR repair reveals causative mutation in a preclinical model of retinitis pigmentosa. Mol. Ther. 24, 1388-1394 (2016). For gene editing used in vivo or in cell therapy (for example, first in vitro in cells and then transplanted into the body), the second purpose, avoiding or minimizing off-target editing, is as important as achieving targeted gene correction, because off-target editing may cause disease or induce tumor formation. It should be noted that not all off-target editing can be predicted using computer software or algorithms.

[0343] Therefore, meticulous design, validation, and refinement were used to develop and validate the CYP4V2 mutation CRISPR gene correction construct:

[0344] (1) Based on the mutant CYP4V2 nucleic acid sequence containing the c.802-8_810del17insGC mutation, multiple candidate gRNAs were generated;

[0345] (2) The top 5 gRNAs were selected using the following criteria (see SEQ ID NO: 48-52, Table 5 and Figure 12 ):

[0346] a. The proximity of the gRNA cleavage site to the modification site, and

[0347] b. Off-target profile of gRNA;

[0348] (3) Validation of the activity of the first five gRNAs in genomic DNA from a BCD patient with the homozygous c.802-8_810del17insGC mutation (see Figure 13 );

[0349] (4) Three gRNAs were selected according to (2) and (3). Each of the three gRNAs was cloned into the pX459 plasmid together with the nucleic acid sequence encoding Cas9 and the puromycin resistance gene (Puro) (see Figure 15 ) to allow selection of transfected cells using puromycin.

[0350] (5) Generate two donor templates (both positive and negative complementary) that provide HDR donor nucleic acid sequences. Synthesize ssODN containing the donor template sequences by IDT (see SEQ ID NOs: 56 and 57).

[0351] (6) In addition to plasmid constructs, CRISPR RNP constructs have also been developed. RNP constructs have certain advantages over other constructs. Detailed discussion is provided below and in the Examples section.

[0352] (7) Validation of the CYP4V2 CRISPR correction construct in iPS cells derived from a BCD patient with the homozygous c.802-8_810del17insGC mutation.

[0353] (8) Whole-genome sequencing was performed in unmodified cells and iPS cells genetically repaired with the CYP4V2 mutation CRISPR correction construct to confirm correction of the c.802-8_810del17insGC mutation and assess off-target editing.

[0354] Provide for determining the optimal conditions for transfection in iPSC and for selecting the method for transfected cells. Detailed description is given in the Examples section. It is expected that these constructs can be used not only for in vitro treatment of BCD patient-specific iPS cells, but also for in vitro treatment of source cells (e.g., fibroblasts or PBMC) or iPS-RPE, iPS-PRC, iPS-CE cells or iPS-CEC cells or other eye cells derived from BCD patient-specific iPS cells, and can be used in the body of the patient with c.802-8_810del17insGC mutation. In one embodiment, the components of the construct can be used directly. In some embodiments, the components in the construct can be modified, or cloned into different vectors to achieve higher in vivo transduction efficiency or higher specificity to target cell types or to achieve other purposes. For example, Cas9 can be modified to Cas9 nickase (Cas9n D10A), which contains a mutation that allows endonuclease to produce single-stranded nicks rather than double-stranded breaks. Pairing two opposite-facing gRNA sequences with the SpCas9 nickase is an efficient gene editing method that prevents the formation of undesirable indels. In addition to plasmids, other common vectors used to package CRISPR components include lentiviral vectors and adeno-associated virus (AAV) vectors. When using AAV vectors, the Staphylococcus aureus Cas9 ortholog (SaCas9) can be used as the nuclease because SaCas9 is approximately 1 kb shorter than SpCas9 and provides additional flexibility to AAV packaging constraints.

[0355] Various improvements have been made to the CRISPR RNP construct. Synthetic sgRNAs are used instead of IVT sgRNAs or crRNA:tracrRNA duplexes. Synthetic gRNAs have higher purity than IVT sgRNAs, thus reducing the risk of off-target editing caused by impurities in the sgRNA. Furthermore, chemical modifications to the sgRNAs prevent intracellular degradation, which can improve editing efficiency. Further details can be found in the Examples section.

[0356] It is contemplated that in addition to the plasmid constructs and CRISPR RNP constructs described herein, mRNA constructs comprising Cas9-encoding mRNA and guide RNA oligonucleotides may also be used.

[0357] After BCD patient-specific iPS cells were transfected with CYP4V2 mutation CRISPR correction constructs, puromycin was used to select transfected cells. It should be understood that other markers such as GFP can be incorporated into the construct and used as markers instead of puromycin or as markers in addition to puromycin. Single cell cloning was performed after selection, and some cells were collected from the single cell clones for sequencing. After sequencing results confirmed that the target gene editing was successful and no pathogenic gene editing was found, the remaining cells of the same clone were used to differentiate into desired eye cell types, such as iPS-RPE cells.

[0358] D. Differentiation of iPSCs

[0359] Differentiate gene-corrected BCD patient iPS cells into iPS-RPE cells (or another type of ocular cell (e.g., iPS-CEC, iPS-CE cells, or iPS-PRC). Methods for differentiating iPSCs into RPE cells or another type of ocular cell (e.g., CEC and PRC) are known. See, e.g., Hayashi et al., 2012, PLoS One, 7(9):e45435; Songstad et al., Investigative Ophthalmology & Visual Science, December 2015, Vol. 56, 8258-8267; and Lamba et al., PLoS One. 2010 Jan 20;5(1):e8763. For example, induced pluripotent stem cells (iPSCs) reprogrammed from cells can be generated and these cells can be further differentiated into, for example, RPE cells (referred to herein as "iPS-RPE"), corneal epithelial cells (referred to herein as "iPS-CEC"), photoreceptor cells (or photoreceptor progenitor cells; referred to herein as "iPS-PRC"), or iPS-choroidal endothelial (CE) cells (referred to as "iPS-CE").

[0360] The differentiated cells (eg, iPS-RPE cells) are tested for biochemical function (as described in the Examples section) to confirm that their biochemical function is improved compared to iPS-RPE cells from patients without gene repair.

[0361] The iPSC-RPE cell line produced as described herein shows the form (for example, pigmentation and hexagonal) that can indicate RPE cell and / or expresses one or more biomarkers that can indicate RPE cell.The biomarker of RPE cell (and iPS-RPE cell) is known and includes but not limited to one or more in RLBP1 (also known as CRALBP), RPE65, spot withering protein-1, MITF, focal adhesion protein, LRAT, RDH5, PAX6, MERTK, TYR and / or ZO-1, and can be used to judge or confirm that RPE differentiation has occurred.Similarly, the biomarker of CEC (and iPS-CEC) and PRC (and iPS-PRC) is known and includes for example cytokeratin 12 and cytokeratin 3 for corneal epithelial cell; And for the Crx of photoreceptor, for the recovery protein of rod and cone and for the Nrl of rod.

[0362] E. Administration / Delivery

[0363] The iPS-RPE cells of gene repair can be used for autologous transplantation to the patient who derives this iPS-RPE cell.The patient with BCD or another kind of ophthalmological condition caused by CYP4V2 sudden change can be treated with the cell therapy method provided herein.Similarly, the method can be used for providing an autologous cell therapy through gene repair for the eye diseases caused by one or more genetic mutations.

[0364] The method for using or delivering cells is known, and the method for using or delivering cells to eyes is known, referring to, for example, Wert et al., J Vis Exp.2012; (69): 4286; WO 2016 / 179496; Schwartz et al., Investigative Ophthalmology&Visual Science April 2016, Vol. 57, ORSFc1-ORSFc9. In one embodiment, eye cells can be transplanted via an injection cell suspension (such as RPE cell suspension). In another embodiment, cells can be transplanted as a part for lamina or scaffold, such as using natural and / or synthetic scaffolds to produce the in vitro tissue of polarized functional RPE monolayer.

[0365] The therapeutically effective amount of cells administered to the eye is known to those skilled in the art and will vary depending on the type of cells to be transplanted, the maturity of the cells to be transplanted and whether they are expected to divide after transplantation, the size of the area targeted for replacement or the number of cells, and the subject to be treated (e.g., the age, sex, weight, stage of disease and condition of the subject to be treated); the route of administration; and the desired regimen. In a single administration, the therapeutically effective amount of cells used in ocular cell therapy can be about 1*10 3 to about 1*10 8 within the range of cells.

[0366] Although iPSC cell lines can be generated for individual subjects, iPSC cell banks with common HLA haplotypes (or in which HLA haplotypes have been genetically manipulated) can also be generated that will be designed to be immune-matched with a large proportion of the patient population. See, for example, Turner et al., Cell Stem Cell, 13:382-384, 2013. In addition, iPSC cell lines can be generated to be immune-silent, regardless of the subject's genotype (see, for example, Riolobos et al., Mol. Ther., 21:1232-41, 2013). When combined with these methods, patient-specific iPS cells and iPS-ocular cells can be used not only for narrow autologous use, but also for transplantation into other patients.

[0367] The cell therapy administration step typically occurs after the onset of disease symptoms or after the subject has shown signs of retinal degeneration or corneal degeneration (if applicable). In one embodiment, the ocular cell therapy provided herein can be used independently to treat ocular diseases (e.g., BCD). In another embodiment, the ocular cell therapy provided herein can be used in combination with one or more other treatment options, including but not limited to the CYP4V2 gene transfer therapy and / or CYP4V2 CRISPR gene editing therapy provided herein.

[0368] Likewise, administration can occur once or multiple times (e.g., over several weeks, months, or years) and can be administered to the same eye or to the fellow eye. Additionally, one or more types of cells can be administered in a single dose or in separate doses.

[0369] Post-treatment assessment can use the methods described in the CYP4V2 gene therapy section of this article, which include but are not limited to eye examinations, such as visual function, for example, as measured by visual acuity, visual field, dark adaptation, visual function and / or optical coherence tomography (OCT, such as spectral domain-OCT (SD-OCT)) and ERG.

[0370] Methods for using CRISPR RNA in ocular cell therapy and gene therapy

[0371] CRISPR RNP is a gene editing ribonucleoprotein (RNP) complex that includes a guide RNA complexed with a Cas protein (e.g., Cas9 protein). Guide RNA is composed of two RNAs referred to as CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). In one embodiment, crRNA and tracrRNA are provided as two separate nucleic acid molecules. In another embodiment, crRNA and tracrRNA can be combined in a chimeric single guide RNA (sgRNA). The length of sgRNA can be about 100 nucleotides (nt), or shorter or longer as needed or necessary. Twenty nt (crRNA) at the 5' end hybridizes with the target DNA sequence by Watson-Crick base pairing and guides the Cas endonuclease to cut the target genomic DNA, and the double-stranded structure remaining on the 3' side is used for Cas9 recognition.

[0372] Compared with traditional Cas9 / gRNA constructs (e.g., plasmid constructs incorporating nucleic acid sequences of CRISPR guide RNA and Cas9 protein), CRISPR RNP has advantages and disadvantages. For example, guide RNA (crRNA and tracrRNA) and Cas9 protein can be delivered to target cells as a complete complex, thereby not requiring the cell's own transcriptional machinery to express the CRISPR components. Therefore, CRISPR RNP can be edited quickly after transfection. In addition, CRISPR components are consumed faster from cells, which can reduce the possibility of off-target editing. In addition, it can reduce the possibility of integration mutagenesis caused by plasmids. In view of these advantages, RNP is also advantageous in in vivo gene editing. However, on the other hand, since RNP is quickly cleared from cells via protein degradation, the on-target editing efficiency of RNP may be lower than that of plasmid constructs with a longer expression duration in cells.

[0373] To evaluate this hypothesis and demonstrate whether CRISPR RNP constructs can achieve the dual goals of gene editing and gene therapy in ocular cell therapy, two sets of constructs were designed. One construct was a plasmid construct, and the other was an RNP construct. Both constructs were transfected into iPS cells from the same BCD patient, which were then sequenced to analyze on-target gene repair and off-target editing in each construct. Off-target editing was confirmed by comparison with genomic DNA from unmodified fibroblasts from the same patient. Results from the plasmid construct and the RNP construct were comparable.

[0374] Detailed descriptions of RNPs, methods of forming RNPs, and using RNP constructs to generate genetically repaired cells (iPS and iPS-RPE cells) for BCD patients are provided in the Examples section.

[0375] It should be noted that similar CRISPR RNP constructs can be used to correct or inactivate other mutations of BCD and other RPs and IRDs. In one aspect, the crRNA sequence used herein is changed to another crRNA sequence that specifically targets different target mutation sequences. On the other hand, the guide RNA or sgRNA in the RNP construct can be modified to enhance gene editing efficiency. See Hendel et al., Nat Biotechnol. 2015 September; 33 (9): 985-989. In some embodiments, the CRISPR RNP construct can be transfected using electroporation. In some embodiments, the CRISPR RNP construct can be transfected using lipofection or nucleofection. In some embodiments, the CRISPR RNP construct can be delivered via microinjection.

[0376] In addition to in vitro gene repair and treatment of patient cells, CRISPR RNP constructs can also be used in vivo to treat eye diseases caused by genetic mutations and have advantages over other types of CRISPR constructs (e.g., plasmids and / or mRNA encoding CRISPR components) in in vivo applications. For example, compared with in vitro transcribed Cas9 mRNA and sgRNA, CRISPR RNP constructs have higher efficacy, lower off-target risk and / or lower toxicity or innate immune response activation. In one embodiment, a CRISPR RNP construct comprising a Cas9 protein complexed with a guide RNA targeting a mutant DNA sequence region can be directly injected into the subject's eye (e.g., subretinal injection, intravitreal injection, or injection into the cornea). In another embodiment, a Cas9 variant engineered to have multiple SV40 nuclear localization sequences (NLS) can be used, which has been shown to improve editing efficiency in brain cells in vivo (Staahl et al., Nat Biotechnol. May 2017; 35(5): 431-434) to achieve higher editing efficiency in ocular cells. Cas9 proteins with one or more NLS (at the N-terminus and / or C-terminus) are commercially available from various CROs such as IDT and Feldan. In some embodiments, the CRISPR RNP construct is delivered "as is". In some embodiments, the CRISPR RNP construct is formulated with a pharmaceutically acceptable carrier at the time of delivery. In some embodiments, the CRISPR RNP construct is delivered in a packaged form, for example, in nanoparticles.

[0377] It should be understood that the ratio between CRISPR RNP components (e.g., guide RNA and Cas9 protein) can be adjusted and optimized by testing different ratios in patient cell lines in vitro before in vitro or in vivo treatment (e.g., BCD patient-specific iPS cells or iPS-RPE cells). The CRISPR RNP construct can be used independently or in combination with another CRISPR construct, and another CRISPR construct includes but is not limited to a plasmid or vector encoding CRISPR guide RNA or crRNA, or Cas protein or a combination thereof; Cas9 encoding mRNA; guide RNA oligonucleotides; another CRISPR RNP construct; or a combination thereof or hybrid. In addition, the CRISPR RNP construct can be used to correct or inactivate one or more than one mutation associated with one or more than one eye disease.

[0378] Combination of gene therapy and cell therapy

[0379] The disclosure herein provides a variety of treatment options for BCD and other eye diseases caused by CYP4V2 mutations, including but not limited to CYP4V2 gene transfer therapy and CYP4V2 CRISPR gene editing therapy. CYP4V2 gene transfer therapy and CYP4V2 gene editing therapy can be used in vivo or in vitro or both in vivo and in vitro. When implemented in vivo, CYP4V2 gene transfer therapy and / or CYP4V2 CRISPR gene editing therapy can be used as gene therapy to treat the remaining ocular cells affected by BCD. When implemented in vitro in patient cells or patient-derived cells, as cell therapy, cells treated with CYP4V2 gene transfer therapy and / or CYP4V2 CRISPR gene editing therapy can be transplanted into the patient to replace dead or degenerated ocular cells. It is worth noting that the gene therapy and cell therapy compositions and methods provided herein can be combined to provide patients with additional benefits that cannot be achieved by using gene therapy or cell therapy alone. "Combination therapy" can also expand the eligible patient base. For example, for late-stage patients who do not leave or hardly leave photoreceptors or RPE cells, gene therapy is not as effective as in early-stage patients. In this case, cell therapy can benefit by providing new cells (for example, RPE or photoreceptor cells), and gene therapy can improve the effect of cell therapy by rescuing remaining RPE or photoreceptor cells and / or by improving the condition of choroidal cells (its health affects the situation of eye cells). Combining gene therapy and cell therapy's respective "rescue" and "replacement" effects makes combined therapy become the improvement of gene therapy or cell therapy. This combined treatment method can be applied to other eye diseases caused by one or more genetic mutations.

[0380] Methods and compositions for CYP4V2 gene therapy

[0381] The present disclosure relates to various compositions comprising nucleic acid molecules encoding functional CYP4V2 proteins and various methods of using these compositions to treat ocular cells and / or ocular diseases. In one embodiment, the functional CYP4V2 protein can be used directly for therapeutic purposes. In some embodiments, a nucleic acid molecule encoding a functional CYP4V2 protein is used. In some embodiments, an expression cassette is used to direct and control the expression of the product of the nucleic acid molecule, which comprises such a nucleic acid molecule encoding a functional CYP4V2 protein operably linked to one or more regulatory sequences. In some embodiments, a vector is used to package the CYP4V2 expression cassette comprising a nucleic acid molecule encoding a functional CYP4V2 protein and one or more regulatory sequences to enhance delivery to target cells and achieve desired expression of the product of the CYP4V2-encoding nucleic acid molecule and the expression cassette.

[0382] In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is another type of viral vector or a non-viral vector. The treatment method includes administering or delivering an effective amount (or effective concentration) of these vectors to the subject's eyes and / or target cells. In one embodiment, the treatment is directly administered in vivo. In another embodiment, the treatment includes ex vivo treatment in target cells (e.g., ocular cells) and transplanting the treated target cells into the subject (e.g., transplanted into the subject's eyes). These treatment methods are directed to eye diseases and other conditions associated with CYP4V2 mutations. In one embodiment, the eye disease is crystalline retinitis pigmentosa (BCD).

[0383] A. Functional CYP4V2 Proteins and Nucleic Acids Encoding Functional CYP4V2 Proteins

[0384] CYP4V2 (cytochrome P450, family 4, subfamily V, polypeptide 2, (MIM 608614), synonym: CYP4AH1) is one of the proteins in the cytochrome P450 superfamily (P450) and a member of cytochrome P450 subfamily 4 (CYP4). Cytochrome P450 (CYP) is an important heme-containing protein known for its role as an oxidase. The term P450 is derived from the spectrophotometric peak at the wavelength of maximum absorption (450 nm) of the enzyme when in the reduced state and complexed with carbon monoxide. It is involved in the metabolism of xenobiotics and endogenous compounds, such as steroids and fatty acids. CYP enzymes have been identified in all kingdoms of life: animals, plants, fungi, protists, bacteria, archaea, and even viruses. However, they are not ubiquitous; for example, CYP enzymes have not yet been found in Escherichia coli.

[0385] P450 proteins serve as key structural elements. For example, P450 proteins can be identified by their characteristic sequence element FXXGXXXCXG (SEQ ID NO:30), in which a cysteine ​​serves as the axial ligand for the heme iron. Sequence identity among P450 proteins is relatively low, but their general topography and structural fold are highly conserved. The conserved core consists of a coil called the 'meander,' a four-helix bundle, helices J and K, and two sets of β-sheets. These comprise the heme-binding loop (with an absolutely conserved cysteine ​​serving as the fifth ligand for the heme iron), the proton-transfer groove, and the conserved EXXR motif in helix K (SEQ ID NO:31). P450 proteins are primarily membrane-associated proteins located in the inner mitochondrial membrane or the endoplasmic reticulum of cells.

[0386] In addition to structural similarities, P450 proteins also share functional similarities. The most common reactions catalyzed by P450 enzymes are monooxygenase reactions, i.e., the insertion of one oxygen atom into an aliphatic position of an organic substrate (RH) while the other oxygen atom is reduced to water:

[0387] RH+O2+NADPH+H + →ROH+H2O+NADP +

[0388] Many hydroxylation reactions (insertion of a hydroxyl group) use P450 enzymes. Many P450 enzymes have steroids and / or fatty acids as substrates.

[0389] The human CYP4V2 protein (NCBI RefSeq: NP_997235.3) has 525 amino acids (the amino acid sequence is shown in SEQ ID NO: 4). Variants of the human CYP4V2 protein exist, including pathological variants (i.e., mutations) (see Table 1 herein for a selected list of CYP4V2 mutations in BCD patients) and non-pathological (i.e., functional) variants.

[0390] In one aspect, the functional CYP4V2 protein is a human CYP4V2 protein (SEQ ID NO: 4). In other aspects, the functional CYP4V2 protein is a functional variant or fragment of a human CYP4V2 protein, including but not limited to a protein having the amino acid sequence shown in SEQ ID NO: 5.

[0391] A functional CYP4V2 protein can also be a variant of another functional CYP4V2 protein. The following is a discussion based on altering the amino acids of the polypeptides described herein to produce equivalent or even improved second-generation molecules. For example, within a protein structure, certain amino acids can be substituted for other amino acids without significantly losing the ability to interact with structures such as, for example, binding sites on substrate molecules (e.g., fatty acid binding sites). Because it is the interaction ability and properties of a protein that determine its biological functional activity, certain amino acid substitutions can be made in the protein sequence and its underlying DNA or RNA coding sequence while still producing a protein with similar properties. Therefore, it is contemplated that various changes can be made in the amino acid sequence of a functional CYP4V2 protein or the DNA or RNA sequence of the gene or its coding region without significantly losing its biological utility or activity, as discussed herein. For example, SEQ ID NO:5 is the amino acid sequence of a CYP4V2 protein variant having one amino acid change compared to the human CYP4V2 protein sequence shown in SEQ ID NO:4.

[0392] Various techniques, algorithms, software, and tools can be used to design or engineer functional derivatives, variants, and / or fragments of functional CYP4V2 proteins (e.g., human CYP4V2 proteins). For example, the structure and function or alteration of various polypeptides can be modeled, solved, or predicted by NMR, x-ray crystallography, or computer modeling (e.g., ClustalW, SWISS-MODEL server, Swiss-Pdb Viewer, Polyphen-2, PROVEAN, SIFT, Condel, MutationAssessor, and FatHMM).

[0393] A functional CYP4V2 protein can also be a fragment of a functional CYP4V2 protein or be derived from a fragment thereof. For example, the human CYP4V2 protein (SEQ ID NO:4) and its variant (SEQ ID NO:5) both have a transmembrane domain between approximately the 13th and 35th amino acid residues from the N-terminus. The backbone of the human CYP4V2 protein (SEQ ID NO:4) is located between approximately 36 and 525 amino acids. Therefore, a functional CYP4V2 can be derived from a human CYP4V2 protein (SEQ ID NO:6) by deleting the first approximately 35 amino acids and replacing this deletion with an alternative transmembrane domain sequence. Another source of a functional CYP4V2 protein is a splice variant of a functional CYP4V2 protein.

[0394] The predicted transmembrane segment of CYP4V2 is located near the N-terminus, followed by a globular domain typical of the CYP450 family. The globular domain of CYP4V2 consists of 18 helices and a β-structural segment. The heme group is located near the surface of the protein, coordinating with the I helix facing the interior of the protein and the L helix on the surface. Li et al., Am J Hum Genet. 74:817-826, 2004. The CYP4V2 protein is primarily active in fatty acid metabolism. Many other P450 enzymes are also involved in fatty acid metabolism. CYP4V2 is ubiquitously expressed in almost all tissues and organs. CYP4V2 expression has been found in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, retina, retinal pigment epithelium, cornea, and lymphocytes (Li et al., Am J Hum Genet. 74:817-826, 2004). However, most other P450 enzymes are not present in ocular cells. For example, CYP4V2 and CYP1B1 are the only P450 enzymes expressed at high levels in the ARPE-19 cell line; CYP2E1, CYP2J2, and CYP3A4 are transcribed only at low levels (5% of CYP4V2 mRNA expression), and transcripts for CYP4A11, CYP4B1, CYP4F2, CYP4F3, and CYP4F12 are undetectable (Nakano et al., Mol Pharmacol 2012;82:679-686). In fact, the symptoms of CYP4V2 mutations are limited to the eye, where CYP4V2 is the only major P450 enzyme expressed besides CYP1B1 and the only P450 subfamily 4 (CYP4) enzyme expressed, while these symptoms are not manifested in organs where CYP4V2 is present alongside other P450 enzymes, suggesting that other P450 enzymes, particularly CYP4 enzymes, may be used to replace all or part of the function of CYP4V2. Indeed, the CYP4 subfamily has been found to have a common role in fatty acid metabolism, including but not limited to being a hydroxylase of PUFAs. See Hardwick, Biochem. Pharmacol., 75(12):2263-75; Fer et al., J. Lipid Res., 49(11):2379-89; Nakano et al., Mol. Pharmacol., 2012, 82:679-686. The protein sequences of human CYP4 proteins are shown in SEQ ID NOs: 8-18.

[0395] In addition to sharing substrates and functions with other proteins of the CYP4 subfamily, computational analysis showed that CYP4V2 was formed by duplication of an ancestor of CYP46A (SEQ ID NO: 7), which then duplicated to give rise to the entire CYP4 family. Pan et al., Int. J. Mol. Sci., 2016, 17(7) pii:E1020. doi:10.3390 / ijms17071020.

[0396] In addition, the CYP4V2 gene (or its ortholog, such as Cyp4v3 in mice) is conserved in many species, including but not limited to humans, chimpanzees, rhesus monkeys, dogs, cows, mice, rats, chickens, frogs, horses, rabbits, and fruit flies (SEQ ID NOs: 19-29). Orthologs of the human gene CYP4V2 have been found in 196 organisms.

[0397] Functional CYP4V2 proteins may comprise, be designed, engineered, or derived from, the following, including but not limited to:

[0398] (i) human CYP4V2 protein (SEQ ID NO: 4);

[0399] (ii) a variant (e.g., altered amino acid and / or splice variant) of a human CYP4V2 protein or a functional CYP4V2 protein (e.g., SEQ ID NO: 5);

[0400] (iii) one or more fragments of a functional CYP4V2 protein (e.g., SEQ ID NO: 6);

[0401] (iv) CYP4V2 (or orthologs) from other species;

[0402] (v) another CYP4 protein or CYP46A1;

[0403] (vi) a polypeptide capable of ameliorating, treating, or suppressing one or more biochemical abnormalities of one or more compounds listed in Table 2 in patient cells (e.g., iPS-RPE cells from a BCD patient); and / or

[0404] (vii) derivatives, hybrids or variants of any one or more of (i) to (vi) above.

[0405] It is contemplated that the compositions and methods disclosed herein can be used to express any functional CYP4V2 protein as described above. In one embodiment, the functional CYP4V2 protein is a polypeptide comprising all or a portion of the amino acid sequence set forth in SEQ ID NO: 4, 5, or 6. In some embodiments, the functional CYP4V2 protein is a polypeptide comprising all or a portion of the amino acid sequence selected from the group consisting of CYP4V2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1, and CYP46A (SEQ ID NO: 4-18), and CYP4V2 of chimpanzee, rhesus monkey, dog, cow, mouse, rat, chicken, frog, horse, rabbit, and fruit fly (SEQ ID NO: 19-29), and derivatives, hybrids, variants, and / or fragments thereof. In some embodiments, the functional CYP4V2 protein can have at least 80% amino acid sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any of the sequences selected from the group consisting of SEQ ID NOs: 4-29. In one embodiment, the functional CYP4V2 protein is a polypeptide comprising sequence elements of FxxGxxxCxG and ExxR (SEQ ID NOs: 30 and 31).

[0406] In some embodiments, the functional CYP4V2 protein is a compound or agent that is capable of ameliorating, treating, or suppressing one or more biochemical abnormalities in a patient's cells (eg, iPS-RPE cells from a BCD patient).

[0407] In one embodiment, a functional CYP4V2 protein can be used directly to treat BCD, similar to protein-based drugs for other diseases. In another embodiment, a nucleic acid molecule encoding a functional CYP4V2 protein is used to express a functional CYP4V2 protein in a targeted cell. In one embodiment, the nucleic acid molecule is RNA. In another embodiment, the nucleic acid molecule is DNA, including but not limited to complementary DNA (cDNA), for long-term expression. The cDNA can be positive-sense or negative-sense, single-stranded or double-stranded. In some embodiments, the nucleic acid encoding the functional CYP4V2 protein is operably linked to one or more regulatory sequences to form a CYP4V2 expression cassette. In some embodiments, this expression cassette is packaged in a vector to enhance delivery and / or expression efficiency.

[0408] A codon consists of a group of three nucleotides and encodes a specific amino acid or causes translation to terminate (i.e., a stop codon). Most amino acids (typically all except methionine) are encoded by multiple codons. Therefore, the same protein can be expressed using different nucleic acid sequences. The sequence identity between two nucleic acid molecules encoding the same protein sequence can range from 0% to over 99%. For example, a nucleic acid sequence (SEQ ID NO:1) and another nucleic acid sequence (SEQ ID NO:2), both encoding the human CYP4V2 protein (SEQ ID NO:4), share only 77% sequence identity.

[0409] Codon optimization of nucleic acid sequences can improve and / or stabilize protein expression without changing the encoded amino acid sequence. Codon optimization is the replacement of codons present in a nucleic acid sequence with preferred codons encoding the same amino acids, such as preferred codons for mammalian expression. Various strategies and parameters can be used in codon optimization, including but not limited to codon usage preference, GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splicing sites, premature PolyA sites, internal chi sites, and ribosome binding sites, negative CpG islands, RNA instability motifs (AREs), repeat sequences (direct repeats, reverse repeats, and dyad repeats), and restriction sites that may interfere with cloning. Codon optimization methods are known in the art, such as U.S. Patent No. 6,114,148 and US 20110081708. Codon-optimized nucleic acid sequences of a given amino acid sequence or polypeptide-encoding nucleic acid sequence can be generated by the methods described herein and / or by using various codon-optimization software, including generation via online software.

[0410] Should be understood that, depending on the codon optimization method, configuration, algorithm or software used, different codon optimized nucleic acid sequences encoding the same protein can be produced. However, codon optimization does not always result in improved expression, compared to the wild-type unmodified nucleic acid sequence. See Alexeyev MF, Winkler HH: Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP / ADP translocase. Biochim Biophys Acta.1999,1419:299-306.10.1016 / S0005-2736(99)00078-4; Curran KA, Leavitt JM, Karim AS,Alper HS:Metabolic engineering of muconic acid production in Saccharomyces cerevisiae.Metab Eng.2013,15:55-66;Agashe D,Martinez-Gomez NC,Drummond DA,Marx CJ:Good codons,Bad transcript:large reductions in gene expression and fitness arising from synonymousmutations in a Key enzyme.Mol Biol Evol.2013,30(3):549-560.10.1093 / molbev / mss273.doi:10.1093 / molbev / mss273.

[0411] Provided herein is a codon-optimized nucleic acid sequence (SEQ ID NO:2) encoding a human CYP4V2 protein (SEQ ID NO:4). SEQ ID NO:1 and SEQ ID NO:2 both encode the same human CYP4V2 protein (SEQ ID NO:4). The codon-optimized nucleic acid sequence (SEQ ID NO:2) has a codon adaptation index (CAI) of 0.95, an improvement over the CAI of 0.94 for the nucleic acid sequence shown in SEQ ID NO:1. A CAI of 1.0 is considered perfect in the desired expression organism. It should be understood that the present disclosure encompasses all forms and types of codon-optimized nucleic acid sequences, such as those represented by the cDNA sequence shown in SEQ ID NO:2, including any RNA, DNA, or other nucleic acid sequence corresponding to or derived from such a cDNA sequence, and which may be single-stranded or double-stranded, and / or sense, negative-sense, antisense, or complementary-sense sequences provided herein.

[0412] In addition to codon optimization, other methods can also be used to improve translation performance. For example, a Kozak sequence or a Shine-Dalgarno sequence can be used to improve translation initiation efficiency. Different stop codons (e.g., TGA) can be used to improve translation termination efficiency. In addition to the ORF sequence, the nucleic acid sequence encoding a functional CYP4V2 protein can also include one or more non-coding sequences (such as UTRs) and / or one or more introns to improve protein expression. A Kozak sequence (an exemplary sequence is shown in SEQ ID NO: 36) can be inserted immediately before the CYP4V2-encoding cDNA to enhance expression.

[0413] As discussed herein, it is contemplated that functional variants and / or fragments of the human CYP4V2 protein may be employed.The nucleic acid sequence encoding a functional variant (SEQ ID NO:5) of the human CYP4V2 protein (SEQ ID NO:4) is provided in SEQ ID NO:3.

[0414] In some embodiments, the CYP4V2 nucleic acid molecule is a polynucleotide molecule encoding any functional CYP4V2 protein, including but not limited to SEQ ID NOs: 4-30, or encoding a polypeptide having at least 80% amino acid sequence identity with any of the sequences shown in SEQ ID NOs: 4-30. In some embodiments, the CYP4V2 nucleic acid molecule is a polynucleotide that shares at least 60% sequence identity with any of SEQ ID NOs: 1, 2, or 3.

[0415] As described herein, vectors (e.g., viral vectors or non-viral vectors) and CYP4V2 expression cassettes typically contain one or more CYP4V2 nucleic acid molecules or fragments thereof. It should be understood that nucleic acid molecules can take a variety of forms, including but not limited to DNA or RNA, single-stranded nucleic acids (e.g., ssDNA, ssRNA), double-stranded nucleic acids (e.g., dsDNA, dsRNA), plus-strand or minus-strand nucleic acids, complementary DNA (cDNA), genomic DNA, messenger RNA (mRNA), small interfering RNA (siRNA), and / or DNA-guided RNA interference (ddRNAi). Nucleic acid molecules can also include one or more nucleotide analogs or backbone modifications. In addition, it should be understood that cDNA can be synthesized from an mRNA template in a reaction catalyzed by reverse transcriptase, or can be designed and synthesized based on the protein it is to encode, including but not limited to codon-optimized cDNA, or can be synthesized from another nucleic acid molecule via mutagenesis. It should also be understood that the cDNA may contain only exons, or may contain exons plus other sequences, such as untranslated regions (UTRs) and / or introns. In some cases, the vectors and CYP4V2 expression cassettes described herein may include a nucleic acid molecule having a sequence encoding a human CYP4V2 protein or a functional variant or fragment thereof.

[0416] Suitable nucleic acid sequences can be any nucleic acid sequence encoding a functional CYP4V2 protein. This nucleic acid sequence may or may not contain non-coding elements, such as UTRs, introns, or Kozak sequences. It may include a wild-type sequence, a synthetic sequence, or a modified sequence (e.g., a codon-optimized sequence). Nucleic acid sequences encoding functional CYP4V2 proteins can be generated as described herein or by other methods known in the art to which the present invention pertains.

[0417] A nucleic acid molecule having a sequence encoding a human CYP4V2 protein as shown in SEQ ID NO: 1 is referred to herein as "CYP4V2st." A nucleic acid molecule having a codon-optimized sequence encoding a human CYP4V2 protein as shown in SEQ ID NO: 2 is referred to herein as "CYP4V2op." A nucleic acid molecule having a sequence encoding a functional variant of a human CYP4V2 protein as shown in SEQ ID NO: 3 is referred to herein as "CYP4V2fv." In some embodiments, the nucleic acid sequence encoding a functional CYP4V2 protein has at least 60% sequence identity to one of SEQ ID NOs 1, 2, or 3.

[0418] Functional CYP4V2 proteins and nucleic acid molecules encoding these proteins can be synthesized or isolated, purified, and detected using methods known in the art. Protein synthesis, isolation, purification, and detection are also commercially available through contract research organizations (CROs) including Wuxi Apptec (Shanghai, China) and GenScript (Piscataway, New Jersey). Nucleic acid molecule synthesis, isolation, purification, cloning, and detection are also commercially available through contract research organizations (CROs) including GenScript (Piscataway, New Jersey) and Integrated DNA Technologies (Coralville, Iowa).

[0419] Polypeptides can be synthesized (e.g., via recombinant protein expression or chemical synthesis) or isolated. As used herein, a "purified" polypeptide is one that has been separated or purified from cellular components with which it naturally accompanies it. Generally, a polypeptide is considered "purified" when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) free, by dry weight, from polypeptides with which it is naturally associated and naturally occurring molecules. Because chemically synthesized polypeptides are essentially separated from components with which they naturally accompany them, synthetic polypeptides are "purified."

[0420] Polypeptides can be purified from natural sources (e.g., biological samples) by known methods, such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified, for example, from nucleic acids expressed in expression vectors. In addition, purified polypeptides can be obtained by chemical synthesis. The purity of a polypeptide can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0421] Polypeptides are typically detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. Antibodies may be polyclonal or monoclonal. Antibodies having specific binding affinity for a polypeptide or a portion of a polypeptide may be produced using methods well known in the art to which the present invention belongs. Antibodies may be attached to a solid support, such as a microtiter plate, using methods known in the art to which the present invention belongs. In the presence of a polypeptide, an antibody-polypeptide complex is formed.

[0422] An "isolated" nucleic acid molecule generally refers to a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid molecule is derived (e.g., cDNA or genomic DNA fragments generated by PCR or restriction endonuclease digestion). Such isolated nucleic acid molecules are generally introduced into constructs (e.g., cloning constructs or expression constructs for use in gene therapy), usually for ease of manipulation, for expression of proteins, for production of fusion proteins, or for other purposes, including but not limited to, for packaging into vectors (e.g., viral or non-viral vectors).

[0423] Nucleic acid can be separated using conventional techniques in the art to which the present invention pertains. For example, nucleic acid can be separated using any method, including but not limited to recombinant nucleic acid technology, site-specific mutagenesis, polymerase chain reaction (PCR) and / or other genetic engineering methods. General PCR techniques are described in, for example, PCR Primer: A Laboratory Manual, compiled by Dieffenbach & Dveksler, Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid technology includes, for example, restriction enzyme digestion and ligation, which can be used to separate nucleic acids. Mutagenesis protocols are described in, for example, In Vitro Mutagenesis Protocols, compiled by Braman, Humana Press, 2002.

[0424] Isolated nucleic acids can also be chemically synthesized as a single nucleic acid molecule or as a series of oligonucleotides.

[0425] Constructs containing nucleic acids are known in the art to which the present invention belongs. Constructs, including cloning constructs and expression constructs, can be commercially customized or can be produced by conventional recombinant DNA techniques in the art to which the present invention belongs. Constructs can have regulatory sequences operably linked to the nucleic acid to be expressed and can further include sequences such as sequences encoding selectable markers (e.g., antibiotic resistance genes). Regulatory sequences are discussed herein. Constructs containing nucleic acids can encode chimeric or fusion polypeptides (i.e., polypeptides operably linked to a heterologous polypeptide, which can be located at the N-terminus or C-terminus of the polypeptide). Representative heterologous polypeptides are polypeptides that can be used in the purification or detection of the encoded polypeptide (e.g., 6×His tag, glutathione S-transferase (GST), CFP, Fc, FLAG, HA, Myc, RFP, Strep, VSV, GFP, and YFP).

[0426] The construct carrying nucleic acid sequence can be introduced into host cells.As used herein, " host cell " refers to the specific cells to be introduced into nucleic acid and also includes the progeny of the carrier of this construct of such cells.Host cell can be any prokaryotic cell or eukaryotic cell.For example, host cell can be bacterial cell, such as Escherichia coli cell, or insect cell, yeast or mammalian cell (such as Chinese hamster ovary cell (Chinese hamster ovary cell, CHO), COS cell, HEK293 cell, HeLa, Vero, V27, A549, K562, B50, WI38 and BHK cell).Other host cells include but are not limited to iPS cell, ES cell, RPE cell, iPS-RPE cell, iPS-photoreceptor cell, ES-RPE cell, ARPE-19 cell, keratocyte, photoreceptor cell, choroid cell, optic nerve cell, any other type of eye cell discussed herein, neuronal cell, epithelial cell, hemocyte, fibroblast, lymphocyte and the cell derived from stem cell. Many methods for introducing nucleic acids, vectors, or expression cassettes carrying nucleic acid transgenes into host cells in vivo and in vitro are well known to those skilled in the art, and include, but are not limited to, electroporation, sonoporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer.

[0427] Nucleic acids can be detected using any number of amplification techniques (see, for example, PCR Primer: A Laboratory Manual, 1995, Dieffenbach & Dveksler, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188) with a pair of appropriate oligonucleotides (e.g., primers). Many modifications of the original PCR have been developed, and these modifications can be used to detect nucleic acids. Nucleic acids can also be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. disclose suitable Southern blot conditions for oligonucleotide probes less than about 100 nucleotides (Sections 11.45-11.46) and suitable Southern blot conditions for oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.54).

[0428] B. Carrier

[0429] In some embodiments, a nucleic acid molecule encoding a functional CYP4V2 protein or fragment thereof is delivered to ocular cells in need of treatment with the aid of a vector. Regarding delivery to ocular cells, the desired therapeutic vector is non-toxic and can effectively deliver nucleic acid molecules (e.g., DNA, RNA) to target cells. Gene therapy vectors are known in the art and can be viral or non-viral.

[0430] One method for introducing nucleic acids into cells in vivo is through the use of viral vectors containing nucleic acid molecules (e.g., cDNA). The advantage of using viral vectors to infect cells is that a large proportion of target cells will receive the nucleic acid molecule. Furthermore, molecules encoded within the viral vector (e.g., cDNA contained within the viral vector) can be efficiently expressed in cells that have received the viral vector containing the nucleic acid molecule.

[0431] Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, herpes virus (HV) vectors, such as herpes simplex virus (HSV) vectors, papillomavirus vectors, poxvirus vectors, human foamy virus (HFV) vectors, Epstein-Barr virus (EBV) vectors, poxvirus vectors, Sendai virus vectors, and retroviral vectors. Plasmids can also be used to deliver nucleic acid molecules to target cells. In some cases, the viral vector is a recombinant viral vector, such as a recombinant AAV (rAAV) vector. Those skilled in the art will appreciate that certain vectors will integrate or be more inclined to integrate into the genome of the host cell (e.g., a subject's cell), while other vectors will not integrate or be less inclined to integrate into the genome of the host cell (e.g., extrachromosomal expression).

[0432] Recombinant AAV (rAAV) vectors are commonly used in gene therapy methods. AAV belongs to the Parvoviridae family and each contains single-stranded DNA. rAAV vectors are currently considered to be the safest and most effective gene transfer platform in mammalian cells (Salganik et al., 2015, Microbiol. Spectr., 3(4): doi: 10.1128 / microbiolspec.MDNA3-0052-2014). To date, 12 AAV serotypes (AAV1 to AAV12) and more than 100 variants have been isolated from human and non-human primate tissue samples (see, for example, Gao et al., 2005, Curr. Gene Ther., 5: 285-97) and other species. Both naturally occurring and modified AAV types can be used in the methods described herein.

[0433] Wild-type AAV contains a linear single-stranded DNA genome enclosed in a capsid composed of three proteins, VP1, VP2, and VP3. In recombinant AAV (rAAV), the rep and cap genes from the wild-type AAV genome are typically replaced with a transgene expression cassette flanked by AAV inverted terminal repeats (ITRs) required for packaging. As used herein, "rAAV vector" refers to a recombinant AAV vector containing one or more capsid elements of or derived from one or more AAV viruses.

[0434] Although AAV and other viral vector-mediated gene therapies have these advantages, not all viral vectors and not all AAV types are suitable for treating specific diseases. Gene therapies using viral vectors (e.g., AAV vectors) face two major challenges. First, it is expected that AAV vectors have sufficient transduction efficiency in the cell types targeted for treatment. Second, the potential immune response triggered by the viral vector needs to be considered. See Madsen et al., Adeno-associated virus serotype 2 induces cell-mediated immune responses directed against multiple epitopes of the capsid protein VP1. J Gen Virol 90, 2622-2633 (2009); Mingozzi et al., CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med 13, 419-422 (2007). Although the eye is considered an immune-privileged organ relative to most other organs and tissues, and the immune response in AAV-mediated gene therapy in the eye can be controlled by the use of immunosuppressants, the role of immune responses such as neutralizing antibodies (NAB) in AAV transduction of the eye in large animals is unclear. In addition, intravitreal AAV administration is more likely to interact with the immune system than subretinal administration. Therefore, the viral vectors used in ocular gene therapy will trigger minimal or no immune response, thereby avoiding potential side effects and ensuring that the transduction / expression efficiency of the viral vector is not substantially reduced due to immune responses (e.g., pre-existing NAB in the subject), and / or thereby reducing the dose of rAAV vector.

[0435] Various compositions and methods related to AAV vector design and selection can be used to address these challenges. About using CYP4V2 gene therapy to treat BCD, when the cells of targeted therapy are mainly RPE cells, a vector with sufficient transduction efficiency in RPE cells is desired. When treating the corneal cells of BCD patients, a vector with sufficient transduction efficiency in corneal cells is desired. In some embodiments, a vector with sufficient transduction efficiency in RPE cells is used. In some embodiments, a vector with sufficient transduction efficiency in corneal cells is used. In some embodiments, a vector with sufficient transduction efficiency in RPE and photoreceptor cells is used. In some embodiments, a vector with sufficient transduction efficiency in RPE, photoreceptors and choroidal cells is used. In some embodiments, a vector with sufficient transduction efficiency in retinal cells is used. In some embodiments, a vector with sufficient transduction efficiency in ocular cells is used. In some embodiments, a vector with sufficient transduction efficiency in ocular cells and / or blood cells is used. To address potential immune responses (e.g., NAB and cell-based immune responses to gene therapy vectors), different AAV serotypes and variants, modified AAV vectors, and / or immunosuppressive regimens can be used.

[0436] The rAAV vector used herein can be based on or derived from wild-type AAV (e.g., one of AAV1 to AAV12 or other wild-type AAV variants isolated from humans or other species, including but not limited to AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11 and AAV12) or a modified AAV. Modified AAV can be generated in many different ways, including but not limited to pseudotyped AAV (e.g., AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 4, AAV2 / 6, AAV2 / 7, AAV2 / 9, AAV2 / 12, AAV8 / 2), chimeric AAV (e.g., AAV-DJ), AAV with modified capsids (e.g., capsid mutant AAV (e.g., AAV with YF, KR, TA, SA and / or TV mutations, and AAV-DJ / 8 or AAV-DJ / 9, which are capsid mutant AAVs derived from AAV-DJ), capsid variant AAV (e.g., AAV 7m8 and derivatives), ancestral AAV (e.g., AAV-DJ), and AAV-DJ / 9. AAV, e.g., Anc80), recombinant AAV including any alteration of the genome and / or capsid of naturally occurring AAV or variants, and any combination thereof. It should be understood that modified AAV can be referred to in different ways, including but not limited to artificial, modified, synthetic, reconstructed, engineered, evolved, designed, derived or enhanced AAV, or AAV generated through rational design and / or directed evolution and / or DNA shuffling, or AAV variants. The use of modified AAV may have certain advantages over unmodified AAV, including but not limited to higher transduction efficiency, higher tissue or cell specificity, less immune response and / or better suitability for certain types of administration (e.g., intravitreal injection or delivery via the bloodstream).

[0437] In some embodiments, the modified AAV vector used herein is a pseudotyped AAV. AAV pseudotyping refers to mixing capsids and genomes from different viral serotypes. These serotypes are indicated by slashes, so AAV2 / 5 represents a virus containing the genome (e.g., ITRs) of serotype 2 packaged in a capsid of serotype 5. In some embodiments, the AAV vector is an AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 8, AAV2 / 6, AAV2 / 9, AAV2 / 4, AAV2 / 7, AAV2 / 10, or AAV2 / 12 vector.

[0438] In some embodiments, the modified AAV vector used herein is a chimeric (sometimes also referred to as hybrid or rearranged) AAV derived from different AAV serotypes, including different AAV serotypes isolated from different species. In some embodiments, the AAV vector is AAV-DJ, AAV-DJ / 8, or AAV-DJ / 9. AAV-DJ is an AAV variant generated by a DNA rearrangement method from a hybrid library of eight AAV serotypes. Grimm, D. et al. (2008). J. Virol. 82:5887-5911. It is capable of effectively transducing a wide range of cell types, including ocular cells. In addition, chimeric AAV has a stronger ability to evade immune neutralization than naturally occurring AAV, and therefore can effectively deliver higher amounts of therapeutic transgenes. Hybrid AAV can be further modified. For example, AAV-DJ / 8 and AAV-DJ / 9 were generated by creating point mutations in the heparin binding domain (HBD) of AAV-DJ. Grimm, D. et al. (2008). J. Virol. 82:5887-5911.

[0439] In some embodiments, the modified AAV used herein is a capsid mutant AAV. It involves generating one or more mutations (e.g., point mutations) in the AAV capsid protein. Capsid mutant AAV has advantages over unmodified AAV. For example, point mutations of surface-exposed tyrosine (Y) residues of the AAV capsid protein have been reported as a simple and effective method for avoiding phosphorylation and subsequent ubiquitination, resulting in higher transduction efficiency both in vitro and in vivo (Zhong et al., Proc Natl Acad Sci US A. 2008; 105(22): 7827-32; Markusic et al., Mol Ther. 2010; 18(12): 2048-56; Li et al., Hum Gene Ther. 2010 November; 21(11): 1527-1543). For example, site-directed mutagenesis of each of the seven AAV2 capsid tyrosine residues (Y252, Y272, Y444, Y500, Y700, Y704, and Y730) by substitution with phenylalanine residues resulted in increased vector transduction and transgene expression by circumventing EGFR-PTK phosphorylation and the ubiquitin-proteasome pathway in human cells in vitro and in murine hepatocytes in vivo (Zhong et al., Virology. 2008 Nov 25;381(2):194-202). It has also been reported that point mutations at specific tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on the AAV capsid can significantly improve transduction both in vitro and in vivo (Gabriel et al., Hum Gene Ther Methods. 2013; 24(2): 80-93; Sen et al., Hum Gene Ther Methods. 2013; 24(2): 104-16; Sen et al., Sci Rep. 2013; 3: 1832; Wu et al., J Virol. 2006; 80(22): 11393-7). Modified AAVs can also be subjected to capsid mutations to generate another modified AAV. For example, AAV-DJ / 8 and AAV-DJ / 9 were generated by creating point mutations in the heparin binding domain (HBD) of AAV-DJ, a hybrid AAV. Grimm, D. et al. (2008). J. Virol. 82:5887-5911. Capsid mutations can also enable AAV to evade NAB and produce less immune response. In addition, certain capsid mutations can make AAV more suitable for intravitreal delivery. Kay et al., PLoS One, 8:e62097, 2013.In some embodiments, the AAV vector used herein is a modified AAV having one or more capsid mutations, including but not limited to tyrosine to phenylalanine (YF), threonine to valine (TV), lysine to arginine (KR), threonine to alanine (TA), serine to alanine (SA), and / or mutations affecting the heparin binding domain (HBD) of AAV, and / or mutations in its antigenic region, including but not limited to mutations at positions 459, 493, and 551. In some embodiments, the AAV vector is an AAV2 having one or more capsid mutations of Y444F, Y500F, Y730F, Y252F, Y272F, Y700F, Y704F, and T491V, where the number (e.g., 444) indicates the position of the point mutation in the AAV capsid. In some embodiments, the AAV vector is an AAV5 having one or more capsid mutations of Y263F and Y719F. In some embodiments, the AAV vector is AAV8 having one or more capsid mutations of Y447F, Y733F, and T494V. In some embodiments, the AAV vector is AAV1 having a capsid mutant of Y731F. In some embodiments, the AAV vector is AAV6 having one or more capsid mutations of Y445F and Y731F. In some embodiments, the AAV vector is AAV9 having a capsid mutation of Y731F. In some embodiments, the AAV vector is AAV-DJ, AAV-DJ / 8, or AAV-DJ / 9 having one or more capsid mutations of K137R, T251A, and S503A.

[0440] In some embodiments, the modified AAV vector is an AAV with a variant AAV capsid protein. Variant AAV capsid proteins are known in the art to which the present invention belongs. In some embodiments, the non-naturally produced capsid protein may include a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) combined with a heterologous sequence (e.g., from different selected AAV serotypes, non-continuous parts of the same AAV serotype, sequences obtained from non-AAV viral sources, or from non-viral sources). In some embodiments, the modified AAV vector includes one or more amino acid insertions (e.g., about 5 amino acids to about 11 amino acids) in the capsid protein GH loop. Compared to the infectivity of non-variant AAV (e.g., wild-type AAV) to retinal cells, variant AAV capsid proteins can confer increased retinal cell infectivity. In some embodiments, the modified AAV is one that can deliver transgenes across the blood-ocular barrier (BOB), a property that makes it suitable for delivery via the bloodstream, thereby providing an alternative administration / delivery route to conventional administration methods used in ocular gene therapy (e.g., subretinal injection or intravitreal injection). In some embodiments, the AAV with a variant AAV capsid protein is AAV 7m8 or a derivative or variant thereof (Dalkara et al., Science Translation Medicine, 5:189ra76, 2013; PCT Application No. PCT / US2012 / 034413, PCT Application No. PCT / US2014 / 039015, U.S. Application No. 14 / 214,011, and U.S. Application No. 13 / 899,481). In some embodiments, the AAV with a variant AAV capsid protein is AAV-PHP.B.

[0441] In some embodiments, an AAV vector can be reconstructed or synthesized by reconstructing a viral revolutionary lineage. The reconstruction can produce an ancestral, ancient, or parental AAV. In one embodiment, the AAV vector is Anc80 (ancestor of AAV1, 2, 8, and 9) or a derivative thereof. Zinn et al., Cell Rep. 2015 Aug 11;12(6):1056-68.

[0442] In some embodiments, one or more AAV and / or other viral vectors can be modified (for example, for intravitreal delivery, enhancing transduction in target cell types (for example, RPE cells) or for delivery via bloodstream and optimized) by means of techniques known in the art to which the present invention pertains (including, for example, " directed evolution " and / or " rational design "). See, for example, Asuri et al., Mol Ther. 20: 329-338, 2012 and Yang et al., Methods Mol Biol. 709: 127-139, 2011. Modified AAV or other viral vectors can be described as, for example, "engineered," "hybrid," "evolved," "enhanced," or "designed" vectors. These modifications can, for example, improve vector targeting (for example, improving intravitreal delivery or suitability for delivery via bloodstream), transduction efficiency, and / or reduce immune response, resulting in, for example, a lower required dose. In some embodiments, the rAAV vector is AAV serotype rh10 (EP 20100178940) or ShH10. In some embodiments, the rAAV vector is AAV-PHP.B (US 20150079038).

[0443] In some embodiments, AAV vectors can be generated and / or selected from a combination of more than one of the strategies described herein. For example, AAV-DJ / 8 and AAV-DJ / 9 were generated by making point mutations in the heparin-binding domain (HBD) of AAV-DJ, a hybrid AAV.

[0444] It is known in the art that some AAVs may be more suitable for intravitreal delivery than some other AAVs. Many of these AAVs for intravitreal delivery involve modifications of the AAV capsid protein (e.g., AAV2 (quad Y-F + TV) (Kay et al., PLoS One. 2013 Apr 26; 8(4)) or variant AAV capsid proteins (e.g., AAV 7m8) via mutations. In addition, there are AAVs suitable for delivery via the bloodstream, such as AAV-PHP.B. However, the use of these AAVs is not limited to intravitreal delivery or delivery via the bloodstream, for example, they can also be used as AAV vectors for subretinal and other routes of administration.

[0445] In some embodiments, a self-complementary AAV vector (scAAV) is used. Wild-type AAV has a single-stranded DNA genome. One disadvantage of AAV is its single-stranded DNA genome. Because the single-stranded AAV genome relies on the cell's DNA replication mechanism to synthesize the complementary chain, transgene expression is delayed and not as robust as double-stranded DNA. Regarding CYP4V2 gene therapy, we have developed a scAAV design (see Figure 7) to avoid the rate-limiting second-strand synthesis in conventional single-stranded AAV vectors and promote robust transgene expression. scAAV.CYP4V2 includes an intramolecular self-complementary CYP4V2 DNA structure, which eliminates the need for host cell DNA synthesis and results in faster and more robust expression during transduction. However, the self-complementary structure of scAAV reduces the packaging limit of the scAAV vector from about 4.7-5.0 kb of ssAAV to about 2.4-2.5 kb of scAAV. Therefore, a shorter length of regulatory sequence (e.g., promoter, enhancer and / or polyA signal) is required in the scAAV design. To ensure that the expression cassette does not exceed the vector packaging limitations and depending on the length of the cDNA and other regulatory sequences used, it may be necessary to exclude certain optional regulatory sequences, such as enhancers, from the scAAV construct. One of the two ITRs in the scAAV design is a truncated ITR and has a mutation in the terminal resolution site (TRS). For a detailed discussion of scAAV structure, purification, and production, see McCarthy, Molecular Therapy, Vol. 16, No. 10, pp. 1648-1656, October 2008.

[0446] Many other vector designs can be used. For example, a dual vector system (e.g., a dual vector system based on AAV, such as trans-splicing or hybrid dual AAV vectors) can be used to express nucleic acid sequences (e.g., CYP4V2 nucleic acid sequences). See, for example, Colella et al., Gene Ther. 21, 450-456, 2014. For example, a dual vector system can include (i) a first AAV vector polynucleotide, having an inverted terminal repeat at each end (5' end and 3' end) of the polynucleotide, and having a suitable promoter between the inverted terminal repeats, which is operably linked to a partial coding sequence of the N-terminal portion of a protein encoded by the nucleic acid sequence of interest; and ii) a second AAV vector polynucleotide, having an inverted terminal repeat at each end (5' end and 3' end) of the polynucleotide, and having a partial coding sequence of the C-terminal portion of a protein encoded by the nucleic acid sequence of interest between the inverted terminal repeats, followed by a polyadenylation (pA) signal sequence.

[0447] For our study, various rAAV vectors were designed and produced, including scAAV2 / 1, AAV2 / 2, AAV2 / 5, scAAV2 / 5, AAV2 / 8, scAAV2 / 9, and AAV2 / 2 (Y444F+Y500F+Y730F) (see the schematic diagram and annotations in Figure 7 of this article). This demonstrates that rAAV vectors of various vector designs can be used in CYP4V2 gene therapy. In addition, given the pre-existing neutralizing antibodies and other individual immune responses against certain AAV types in the patient population, the inclusion of multiple rAAV vectors as options can help reduce potential immune responses in CYP4V2 gene therapy. It will also provide more options if subsequent administration to the same eye or administration to the contralateral eye of the same subject is required.

[0448] Methods for producing viral delivery vectors, including using helper-free systems, are known in the art. See, for example, PCT / US2007 / 010055; US Patent No. 6,458,587; and US Patent No. 6,428,988 B1. The manufacture of various vectors used in gene therapy, including but not limited to AAV vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors, is also commercially available through contract research organizations (CROs) and contract manufacturing organizations (CMOs), such as Vector Biolabs (Malvern, PA) and Cell Biolabs, Inc. (San Diego, CA).

[0449] In some embodiments, recombinant AAV vectors suitable for use in the methods described herein can be produced by culturing host cells (e.g., HEK293 cells) containing: nucleic acid molecules encoding AAV serotype capsid proteins or fragments thereof; rep genes; minigenes comprising at least AAV inverted terminal repeats (ITRs) and nucleic acid molecules of interest (e.g., having CYP4V2 nucleic acid sequences); and auxiliary functions sufficient to allow the nucleic acid of interest to be packaged into AAV capsid proteins. Components that need to be cultured in host cells to package nucleic acids into AAV capsids can be provided to the host cells in cis or trans. Alternatively, any one or more of the required components (e.g., nucleic acid molecules of interest, rep sequences, cap sequences, and / or auxiliary functions) can be provided by stable host cells that have been engineered to contain one or more of the required components. Any of these components can be selected from any suitable serotype. For example, rAAV vectors are produced by co-transfecting producer cells (e.g., HEK 293 cells) with: (a) a plasmid (AAV cis plasmid) containing a cloned recombinant AAV genome consisting of a gene of interest (e.g., a cDNA encoding CYP4V2) and other desired regulatory sequences flanked by two AAV ITRs; (b) another construct expressed in trans-acting AAV viral Rep and Cap genes; and (c) adenoviral helper factors, which are provided by a third plasmid providing these adenoviral helper factors into the producer cells via adenoviral infection or transfection. In addition to HEK293 cells, other cell lines can also be used to produce rAAV vectors, including but not limited to HeLa, Vero, A549, B50, WI38, and BHK cells.

[0450] In some embodiments, the viral delivery vector is rAAV2 virus, rAAV2 / 5 virus, rAAV2 / 8 virus, rAAV2 / 1 virus, rAAV2 / 4 virus, rAAV2 / 6 virus, rAAV2 / 9 virus, rAAV2 / 12 virus, or rAAV virus having capsid elements from one or more of AAV1, AAV2, AAV5, AAV8, AAV9, and / or AAV12 viruses. In one embodiment, the viral delivery vector is a rAAV virus having one or more YF mutations, including but not limited to AAV2 (Y444F+Y500F+Y730F) or AAV8 (Y733F).

[0451] In some embodiments, the viral delivery vector is a single-stranded rAAV (ssAAV) virus. In some embodiments, the viral delivery vector is a self-complementary rAAV (scAAV) virus.

[0452] In addition to AAV vectors, other viral vectors can also be used in CYP4V2 gene therapy. For example, adenovirus vectors have also been shown to be suitable for gene delivery. For example, Mori et al., 2002. IOVS, 43: 1610-1615 disclosed the use of an adenovirus vector, which is an E-1-deleted and partially E-3-deleted type 5 Ad, in which the transgene (green fluorescent protein) is driven by a CMV promoter. 7 to 10 8 Peak expression levels were observed after 10 viral particles were injected, and subretinal injection provided higher levels of expression than intravitreal injection.

[0453] In some embodiments, the delivery vector is a plasmid containing a nucleic acid molecule encoding a human CYP4V2 protein or a functional variant or fragment thereof.

[0454] Non-viral vectors can also be used in CYP4V2 gene therapy. Examples of non-viral vectors include, but are not limited to, naked nucleic acids, dendrimers, liposomes (e.g., cationic or anionic liposomes), polymers (e.g., polyplexes), lipid-polymer systems, and nanoparticles (e.g., inorganic or synthetic nanoparticles). For example, Farjo et al., 2006, PLoS 1: e38, demonstrated efficient non-viral ocular gene transfer using compacted DNA nanoparticles as a system for non-viral gene transfer to ocular tissue. As proof of concept, the pZEEGFP5.1 (5,147 bp) expression construct, encoding an enhanced green fluorescent protein (GFP) cDNA transcriptionally controlled by the CMV immediate-early promoter and enhancer, was used. DNA nanoparticles were formulated by mixing plasmid DNA with CK3OPEG10K, a 30-mer lysine peptide with an N-terminal cysteine, conjugated to 10 kDa polyethylene glycol via a maleimide bond using known methods. The nanoparticles were concentrated in saline to 4 mg / ml of DNA. The compacted DNA was delivered to the vitreous cavity at a dose of 0.6 μg. GFP expression was observed in the lens, retina, and pigment epithelium / choroid / sclera by PCR and microscopy.

[0455] In addition, a number of patents have been issued regarding ocular gene transfer methods, including but not limited to U.S. Patent No. 7,144,870, which provides a hyaluronic acid-mediated adenoviral transduction method; U.S. Patent Nos. 7,122,181 and 6,555,107, which provide lentiviral vectors and their uses for ocular gene delivery; U.S. Patent No. 6,106,826, which provides herpes simplex virus vectors and their uses for ocular gene delivery; and U.S. Patent No. 5,770,580, which provides DNA expression vectors and their uses for ocular gene delivery.

[0456] The Examples section herein provides a method for screening and selecting suitable vectors for use in CYP4V2 gene therapy from among different vectors. The examples illustrate this method using different AAV vectors. It should be understood that one skilled in the art can also use this method to compare and select among different types of vectors, such as viral vectors versus non-viral vectors, adenovirus versus AAV, lentivirus versus AAV, HSV versus AAV, and the like.

[0457] C. CYP4V2 expression cassette and regulatory sequences

[0458] The present invention also provides an expression cassette comprising a nucleic acid sequence encoding a functional CYP4V2 protein (e.g., a nucleic acid sequence of SEQ ID NO: 1, 2, or 3) and an expression control sequence operably linked to the CYP4V2 encoding nucleic acid sequence. In addition to the nucleic acid molecule encoding the functional CYP4V2 protein, other key elements of the expression cassette used in CYP4V2 gene therapy include one or more regulatory sequences for controlling the expression of the nucleic acid molecule. In some embodiments, the expression cassette is packaged in a delivery vector (e.g., an rAAV vector flanked by AAV ITRs) to enhance delivery, transduction, and / or expression efficiency. Any AAV ITR can be used in the methods described herein. The ssAAV vector described in the examples herein contains two AAV2 ITRs, each approximately 141 bp (exemplary sequences are shown in SEQ ID NOs 42 and 43). The scAAV vector described in the examples contains two AAV2 ITRs, one of which is a truncated ITR (exemplary sequences are shown in SEQ ID NOs 44 and 45). AAV2 ITRs typically have a length of about 132 to about 167 bp, depending on the parental vector used.

[0459] As used herein, the term "regulatory sequence" refers to any genetic element (e.g., a polynucleotide sequence) that is capable of regulating the replication or expression (transcription or translation) of a nucleic acid sequence or otherwise directing, influencing, and / or regulating the expression of a nucleic acid sequence. Common expression control sequences include promoters, polyadenylation (polyA) signals, enhancers, upstream regulatory domains, introns, UTRs, response elements or inducible elements, origins of replication, internal ribosome entry sites (IRES), transcription initiation sequences, termination sequences, RNA processing sequences such as splicing and polyadenylation (polyA) sequences, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, or sequences that enhance secretion of the encoded protein. Regulatory sequences may be of bacterial, yeast, insect, mammalian, or viral origin, or may be derivatives, hybrids, or variants thereof, or may be synthetic, and vectors may contain combinations of regulatory sequences from different sources. For example, regulatory sequences can be heterologous (e.g., of a different origin or from a different gene; e.g., from a non-CYP4V2 gene) or homologous (e.g., from the same gene; e.g., from a CYP4V2 gene) to the coding sequence whose expression is regulated by these regulatory sequences. As used herein, "operably linked" means that the promoter and / or other regulatory sequences are positioned relative to the nucleic acid coding sequence in such a manner that they are able to direct, influence, or regulate the expression of the nucleic acid coding sequence. Regulatory sequences can be "operably linked" to nucleic acid coding sequences in the same vector or in different vectors. One or more regulatory sequences operably linked to a nucleic acid coding sequence can be continuous and / or can act in trans or at a distance to direct, influence, or regulate the expression of the nucleic acid coding sequence. Among regulatory sequences, promoters are essential, while other regulatory sequences, such as enhancers, introns, and terminators, can be beneficial but optional.

[0460] Various promoter sequences can be used to drive expression of nucleic acid coding sequences. Some promoters are constitutive promoters, which direct expression in almost all tissues and most cell types, while other promoters are more controlled. Regulated promoters may only work in certain tissues or cells (i.e., tissue- or cell-specific promoters) or at certain times during development (i.e., developmental stage-specific promoters) and / or may depend on environmental conditions or external stimuli, such as chemicals, oxygen levels, heat, or light (i.e., inducible promoters).

[0461] In some cases, it may be desirable to use a constitutive promoter (or ubiquitous promoter).Exemplary constitutive promoters include, but are not limited to, the cytomegalovirus (CMV) promoter (Gray et al., Hum Gene Ther. 2011 Sep;22(9):1143-1153; Norman et al., PLoS ONE 5(8):e12413, August 2010), the chicken β-actin promoter, the hybrid CAG (also known as CAGGS, CBA, or CB) promoter derived from CMV / chicken β-actin / rabbit β-globulin (Miyazaki J, Takaki S, Araki K, Tashiro F, Tominaga A, Takatsu K, Yamamura K. 1989. Expression vector system based on the chicken β-actin promoter directs efficient production of interleukin-5. Gene 79:269-277; Acland, GM et al. Mol. Genet. 2010;14 ... Then, 2005, 12: 1072-1082), small CBA (smCBA) promoter (~953 bp, see Mah et al. 2003, Hum. Gene Ther. 14: 143-152; Haire et al. 2006 IOVS, 2006, 47: 3745-3753), CBh promoter (~800 bp, see Gray et al., Hum Gene Ther. 2011 Sep; 22(9): 1143-1153), human β-actin promoter (ACTB) (Norman et al., PLoS ONE 5(8): e12413, August 2010), elongation factor 1 alpha (EF-1alpha) promoter (see Gill et al., Gene Ther. 2001; 8(20): 1539-1546; Norman et al., PLoS ONE 5(8):e12413, August 2010), phosphoglycerate kinase (PGK, human or mouse) promoter (Norman et al., PLoS ONE 5(8):e12413, August 2010), ubiquitin C (UBC) promoter (Norman et al., PLoS ONE 5(8):e12413, August 2010), GUSB (glucuronidase β) promoter, GUSB minimal promoter (hGBp) (Husain, Gene Therapy (2009) 16, 927-932), UCOE promoter, elongation factor 1 alpha short form (EFS) promoter, simian virus 40 (SV40) promoter, Rous sarcoma virus (RSV) promoter.For a general comparison and discussion of various promoters, see, for example, Powell, Discov Med. 2015 Jan; 19(102): 49-57. It is understood that in some cases, "constitutive" or "ubiquitous" promoters may tend to silence or promote differential expression intensities in selected cell types, see, for example, McCown et al., Brain Res. 1996; 713(1-2): 99-107; Gray et al., Hum Gene Ther. 2011; 22: 1143-1153.

[0462] In some cases, it is desired to use cell-specific or tissue-specific promoters, which instruct the expression of nucleic acid coding sequences in specific types of cells or tissues. Based on the disclosure herein, it should be understood that cell-specific or tissue-specific promoters can be specific to eye cells or tissues or to lymphocytes. Eye cell types include but are not limited to retinal cells, retinal bipolar cells, photoreceptor cells, rod cells and cone cells, ganglion cells, retinal pigment epithelium (RPE) cells, choroid cells or corneal epithelial cells. Therefore, as described herein, cell-specific promoters can be retinal-specific promoters (for example, RPE specificity, photoreceptor specificity (for example, cone-specificity and / or rod-specificity) and / or choroid-specific) or corneal-specific promoters.Exemplary ocular cell-specific promoters include, but are not limited to, the human G protein-coupled receptor protein kinase 1, also known as rhodopsin kinase 1 (GRK1) promoter (Genbank accession number AY327580), a 292 nt fragment of the GRK1 promoter (positions 1793-2087) (see Beltran et al., Gene Therapy 17:1162-74, 2010), the human interphotoreceptor retinoid-binding protein (IRBP) proximal promoter, a 235 nt fragment of the hIRBP promoter, the RPGR proximal promoter, the red opsin promoter, the red-green opsin promoter, the blue opsin promoter, the mouse opsin promoter (both long and short forms, Le et al., Molecular Vision 2006;12:389-398; Beltran et al., Gene Therapy 17:1162-74, 2010), the rhodopsin (Rho) promoter (Mussolino et al., Gene Therapy, 18:637-45, 2011), the α-subunit of cone transducin (Morrissey et al., BMC Dev, Biol, 11:3, 2011), the β phosphodiesterase (PDE) promoter, the retinitis pigmentosa (RP1) promoter (Nicord et al., J. Gene Vied. 9:1015-23, 2007), the NXNL2 / NXNL1 promoter (Lambard et al., PLoS One, 5:e13025, 2010), the RPE65 promoter (Li et al., Investigative Ophthalmology & Visual Science, December 2002, Vol. 43, 3640), the retinal degeneration slow / peripherin 2 (Rds / perphZ) promoter (Cai et al., Exp Eye Res, 91:186-94, 2010), VMD2 promoter (vitelliform macular dystrophy 2, also known as BEST1, Kachi et al., Human Gene Therapy, 20:31-9, 2009), IRBP / GNAT2 promoter (hIRBP enhancer fused to cone transducin alpha promoter), Rds (retinal degeneration slow) promoter, hPDE6b promoter, or VEcad promoter (VE-cadherin / cadherin 5 (CDH5) / CD144 promoter).It should be understood that based on the basic principles and discussions provided herein, other promoters known in the art to which the present invention pertains can be used in place of or in addition to any exemplary promoter provided herein.

[0463] Exemplary inducible promoters include, but are not limited to, calcium-sensitive promoters (e.g., NFAT promoter, see GeneTher. 2013 Mar; 20(3): 248-54), zinc-inducible sheep metalloihionine (MX) promoter, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system, ecdysone insect promoter, tetracycline-repressible system, tetracycline inducible system, RU486 inducible system, rapamycin inducible system, various commercially available inducible promoters, and inducible promoters that are regulated by specific physiological states (e.g., temperature, acute phase, specific differentiation state of cells, or only in replicating cells). In some embodiments, the inducible promoter is a promoter that is tightly regulated and specific for a specific ocular cell type.

[0464] A promoter can be a hybrid of another promoter and / or another regulatory sequence, or a truncated / shortened or modified version thereof, or otherwise derived from another promoter and / or another regulatory sequence, for example, the CAG promoter is a hybrid of the CMV immediate early enhancer, the chicken β-actin promoter, and the rabbit β-globin gene, and the smCBA promoter is a truncated version of the CBA promoter. A promoter can contain other elements, such as introns, exons, and / or enhancers, such as the CAG promoter. More than one promoter can be used together in an expression cassette.

[0465] In some cases, to increase expression and / or stabilize expression above that occurring due to the promoter, it is desirable to use enhancer sequences. Representative enhancer sequences include, but are not limited to, post-transcriptional regulatory elements (e.g., woodchuck hepatitis virus post-transcriptional regulatory element (also known as WPRE) or hepatitis B virus post-transcriptional regulatory element (also known as HPRE or HBVPRE, Donello et al., J Virol. 1998 Jun;72(6):5085-92; Sun et al., DNA Cell Biol. 2009 May;28(5):233-240), or various shortened, mutated, or modified WPREs, such as a shortened WPRE of ~247 bp containing minimal gamma and alpha elements of WPRE (Choi et al., Mol Brain. 2014;7:17; Donello et al., J Virol. 1998 Jun;72(6):5085-5092; Zanta-Boussif et al., Gene Therapy (2009) 16, 605-619), or an IRBP enhancer (Nicord et al., J. Gene Biol. 2009 May;28(5):233-240). Vied.9:1015-23,2007), a constitutive transport element (CTE) enhancer (e.g., Mason-Pfizer Monkey Virus CTE or avian leukosis virus CTE), a cytomegalovirus (CMV) immediate early enhancer, an enhancer derived from an immunoglobulin gene or an SV40 enhancer, or a cis-acting element identified in the mouse proximal promoter, an intronic regulatory sequence, such as a mini-intron derived from SV-40 called SD-SA, a splice donor / splice acceptor, an internal ribosome entry site (IRES), which can be used to produce more than one polypeptide from a single gene transcript, such as a protein containing more than one polypeptide chain or two different proteins, can be a poliovirus internal ribosome entry sequence, which supports transgene expression in RPE, photoreceptors, and ganglion cells.

[0466] Polyadenylation of transcripts is important for nuclear export, translation, and mRNA stability. Therefore, the efficiency of transcript polyadenylation is very important for transgenic expression. Representative PolyA signal sequences include, but are not limited to, SV40 polyA signal, SV40 late polyA signal, SV40 early polyA signal, bovine growth hormone polyadenylation (bGH polyA) signal, small polyA or human growth hormone polyadenylation signal (hGH polyA). In some cases, an upstream enhancer (USE) sequence can be used to increase the efficiency of the polyA signal, such as SV40 late 2xUSE, HIV-1 USE (human immunodeficiency virus 1), GHV USE (Ground squirrel hepatitis virus), adenovirus (L3) USE (adenovirus), hTHGB USE (human prothrombin), or hC2 USE (human C2 complement gene) (Schambach A, Galla M, Maetzig T, Loew R, Baum C. Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors. Mol Ther. 2007; 15(6): 1167-1173).

[0467] As with promoter sequences, other regulatory sequences used in expression cassettes can be hybrids, shortened / truncated, modified, or otherwise derived versions of regulatory sequences. For example, shortened WPRE, SV40 late 2×USE, SV40 late polyA. In addition to the elements described herein, expression cassettes may also contain other regulatory sequences, such as introns, UTRs, and linker sequences. The inclusion of splice sites (i.e., an exon flanked by two introns) has been shown to be useful for increasing gene expression of proteins from the expression cassette.

[0468] It is known in the art to which the present invention belongs that regulatory sequences or hybrid regulatory sequences often have multiple versions and have more than one name. For example, various promoters, enhancers and polyA signals have multiple versions, including but not limited to CMV promoter, EF1α promoter, WPRE enhancer and SV40 polyA signal. The CAG promoter has multiple alternative names, including but not limited to CBA promoter, CB promoter or CAGGS promoter. In addition, it is also known in the art to which the present invention belongs that regulatory sequences can be shortened, modified or combined with other sequences to produce derivatives or variants, for example, the CAG (also known as CBA, CB or CAGGS) promoter is a hybrid of the CMV immediate early enhancer, the chicken β-actin promoter and the rabbit β-globin gene, the smCBA promoter is a truncated CAG promoter, and the CB promoter is a truncated CAG promoter. SB The promoter is a shortened CAG promoter that differs by approximately 152 bp from the 5' end of the CMV immediate early enhancer. In addition, regulatory sequences may be referred to by different names, for example, post-transcriptional regulatory elements such as HPRE or WPRE may also be referred to as enhancers. Any regulatory sequence described herein encompasses all variants, derivatives, and / or hybrids of such regulatory sequences. Any exemplary sequences provided herein relating to regulatory sequences are exemplary in nature and do not limit the definition or scope of such regulatory sequences to those shown in the exemplary sequences.

[0469] In some embodiments, microRNA (miRNA) technology can be used in expression cassette design to achieve targeted expression specificity, for example, by suppressing off-target transgene expression. By way of example only and not limitation, a target sequence for miR181 (a miRNA that has been shown to be expressed only in ganglion cells and inner retina) can be added immediately downstream of the CYP4V2 cDNA to inhibit the expression cassette-mediated synthesis of CYP4V2 protein in ganglion cells and inner retinal cells. Similarly, target sequences for miRNAs that are expressed only in certain cell types can be used to suppress expression cassette-mediated CYP4V2 protein expression in these cell types to achieve targeted tissue- or cell-specific expression.

[0470] D. Design of effective expression cassettes and delivery vectors for CYP4V2 gene therapy

[0471] A detailed discussion of the CYP4V2 expression cassette and delivery vector design methods and various designs for these studies is provided in the Examples section herein.

[0472] Treatment of ocular diseases using the EFS promoter and / or small PolyA signal (SPA)

[0473] As discussed herein, gene delivery vectors have packaging size limitations. For example, the packaging limit of single-stranded AAV vectors is approximately 4.7-5.0 kb. Beyond this size, transduction and expression efficiency will drop significantly. For self-complementary AAV (scAAV), the packaging limit is halved to approximately 2.4-2.5 kb. Therefore, for vector-mediated gene delivery and gene therapy, size is very important. For double-stranded self-complementary vectors, it is desirable and sometimes critical to use small-sized regulatory sequences to leave enough space for transgenes (e.g., cDNA). Because of this size limitation, large promoters are also not suitable for use with scAAV. Regarding CYP4V2 gene therapy, given that the size of cDNA is approximately 1578 bp and AAV ITR (containing mutations) is approximately 258 bp, only about 500-600 bp is left for regulatory sequences. Because CYP4V2 is expressed almost ubiquitously, in some embodiments, it is desirable to use a constitutive promoter to drive CYP4V2 transgenic expression. However, there is no room for the constitutive CAG promoter of about 1.7 kb or the shortened CBA promoter (smCBA) of about 953 bp or the CBh promoter of about 800 bp that we used in the single-stranded AAV design. The same is true for many other constitutive promoters, such as the CMV promoter of about 600 bp. Instead, the shorter EFS promoter (exemplary sequence is shown in SEQ ID NO: 34) can be used for scAAV design. The same size restrictions apply to other regulatory sequences, such as the polyA signal. bGH poly A is about 225 bp, SV40 poly A is about 240 bp and SV40 late poly A is about 120 bp. Any of these will take up most of the ~500 bp length left for regulatory sequences including the promoter. Therefore, the scAAV design uses a small polyA signal (SPA), which is only about 54 bp (exemplary sequence is shown in SEQ ID NO: 35).

[0474] The design using the EFS promoter and SPA occupies only about 300 bp and, together with the AAV ITR, occupies a total of about 600 bp, leaving about 1.8-1.9 kb of remaining packaging space for the nucleic acid sequence encoding the desired protein and any other sequences in the expression cassette designed for scAAV, and about 4.1-4.4 kb of remaining packaging space for the nucleic acid sequence encoding the desired protein and any other sequences in the expression cassette designed for ssAAV. Thus, compared to using larger promoters and polyA signal sequences, including but not limited to CMV promoter, CAG promoter, smCBA promoter, CBh promoter, EF1α promoter, bGH polyA, SV40 polyA, and SV40 late polyA, larger cDNA and / or other sequences can be packaged in rAAV vectors containing the EFS promoter and SPA.

[0475] A schematic diagram of an expression cassette comprising the EFS promoter and SPA is provided in Figure 7c. The construct shown in Figure 7b includes the CYP4V2 cDNA. For expression cassettes used for other transgene expression, the CYP4V2 cDNA can be replaced with another gene of interest.

[0476] In this study, the use of EFS promoter and SPA in expression cassettes and delivery vectors was tested to drive nucleic acid coding sequences for the treatment of eye diseases. ScAAV2 / 1 vectors containing EFS promoter, CYP4V2 cDNA and SPA were generated, referred to as scAAV1.EFS.CYP4V2op.SPA. ScAAV1-EFS-CYP4V2op-SPA was administered in iPS-RPE cells of BCD patients. Although the length of EFS promoter and SPA was very short, scAAV1-EFS-CYP4V2op-SPA showed rapid and robust effects (see Table 3) in iPS-RPE cells of BCD patients within just 4 days. This shows that EFS promoter and / or SPA are small-sized regulatory sequences that are very suitable for scAAV systems of eye gene therapy. In addition, the robust expression of scAAV vectors makes scAAV design also suitable for other routes of administration (e.g., intravitreal delivery) in addition to subretinal delivery.

[0477] The use of the EFS promoter and / or SPA is not limited to CYP4V2 gene therapy or scAAV constructs. They can be used in gene therapies involving other genes where transgene size and / or scAAV design require the use of a shorter promoter and polyA signal to drive rapid and sufficient protein expression.

[0478] E. Treatment Options, Subject Selection, and Administration

[0479] CYP4V2 gene therapy can be used in a variety of ways. In some cases, the cell, tissue or organ of the targeted therapy of the experimenter can be effectively delivered to the delivery vector containing CYP4V2 expression cassette, such as RPE, photoreceptor, choroid, cornea, lymphocyte, retina or eyes, so as to give treatment in experimenter (for example, BCD patient). In some cases, treatment can be given in vitro in institute's targeted cells (for example, patient's iPS-RPE cells, patient's iPS-photoreceptor cells, iPS-photoreceptor progenitor cells, iPS-CEC, lymphocyte).Then the treated cell can be transplanted to the experimenter (for example, BCD patient) in need. In some cases, treatment can be given via combination in vivo method and in vitro method. In some cases, CYP4V2 gene therapy can be used independently. In some cases, CYP4V2 gene therapy can be used together with another treatment option.

[0480] Subjects that are candidates for the treatment methods of the present invention include subjects diagnosed with BCD. Subjects with other clinically defined ophthalmic conditions caused by mutations in the CYP4V2 gene (e.g., inherited retinal degeneration (IRD), retinitis pigmentosa (RP), or corneal degeneration) can also be treated using the methods described herein. Diagnosis of BCD, IRD, RP, corneal degeneration, or another ophthalmic condition caused by a mutation in the CYP4V2 gene can be performed using methods known in the art to which the present invention pertains. The methods described herein may include: identifying a subject, such as a child, adolescent, or adult subject, who has BCD or another ophthalmic condition caused by a mutation in the CYP4V2 gene or who is suspected of having BCD or another ophthalmic condition caused by a mutation in the CYP4V2 gene (e.g., based on the presence of symptoms of the condition and no other obvious cause); and obtaining a sample comprising genomic DNA from the subject and detecting the presence of a mutation in the CYP4V2 gene using known molecular biology methods.

[0481] Numerous mutations have been identified in the CYP4V2 gene that cause BCD, with at least one mutation in each of the gene's 11 exons. Genotyping analysis shows that the most common CYP4V2 mutation in BCD patients is c.802-8_810del17insGC (a 17-base deletion with a two-base (GC) insertion eight bases from the end of intron 6 of the CYP4V2 gene, also known as IVS6-8del / insGC; this insertion-deletion mutation is located at the intron 6-exon 7 junction, and the 17-base deletion encompasses the exon 7 splice acceptor site, resulting in an in-frame deletion of exon 7, encoding 62 amino acids), which results in the skipping of exon 7. (Xiao et al., Biochem Biophys Res Commun. 409:181-6, 2011; Meng et al., 2014, Mol. Vis., 20:1806-14; Wada et al., Am J Ophthalmol. 139:894-9, 2005; Jiao et al., European Journal of Human Genetics (2017) 25, 461-471). Various types of mutations have been found in CYP4V2 mutations associated with BCD, including but not limited to missense, splice site, frameshift, deletion, insertion, indel, nonsense, polymorphism (e.g., single nucleotide polymorphism), and premature termination. A summary of select CYP4V2 mutations in human BCD patients is provided in Table 1 and can be found in various publications and online databases, such as LOVD (databases.lovd.nl / shared / genes / CYP4V2 on the World Wide Web), OMIM (omim.org / allelicVariant / 608614 on the World Wide Web), and ClinVar (ncbi.nlm.nih.gov / clinvar?term=608614[MIM] on the World Wide Web).

[0482] It should be noted that the human CYP4V2 mutations in Table 1 are not exhaustive. More CYP4V2 mutations may be identified in the future. It should be understood that not all variations relative to the reference sequence are mutations. Some variations are non-pathological. The method for confirming whether a genetic variation is a pathological variation (i.e., whether it is a mutation) is known in the art to which the present invention belongs, including but not limited to comparing the variation with the known mutation previously identified clinically, and / or determining whether there is a corresponding functional change. For example, determining whether a genetic variation is a pathological variation (i.e., a mutation) is to test the biochemical function of the iPS-RPE cell line derived from the experimenter as described herein, and compared with the biochemical function of the iPS-RPE cell line of the healthy control group, assess whether there is any abnormality.

[0483] Patients with BCD or another ophthalmological condition due to a CYP4V2 mutation who can be treated using the methods described herein preferably retain some photoreceptor and visual function, e.g., as measured by visual acuity, visual field, visual function, and / or optical coherence tomography (OCT, e.g., spectral domain-OCT (SD-OCT)).

[0484] Before administration, the final product will undergo a series of steps (e.g., ultrapurification) to meet clinical-grade standards. Clinical-grade production is commercially available through various GMP facilities, including but not limited to facilities in the NIH Gene Therapy Resource Program (GTRP) and contract manufacturing organizations (CMOs).

[0485] Prior to administration, the subject can be tested for pre-existing neutralizing antibodies (NAb) to the type of AAV vector to which the subject is to be administered. In one embodiment, if the subject has pre-existing NAbs for this AAV type, an alternative AAV vector or an AAV vector with a modified capsid structure that has low cross-reactivity to the subject's pre-existing NAbs can be used to administer to the subject, thereby reducing the immune response and retaining sufficient transduction efficiency via the AAV vector. Other methods for minimizing the immune response are known in the art to which the present invention pertains, including but not limited to administering immunosuppressants and regimens before, during, and / or after treatment.

[0486] Viral vectors or non-viral vectors or their combination (e.g., hybrid vectors) can be delivered to the eye cells of the subject using one or more physical means. As used herein, eye cells refer to, but are not limited to, retinal pigment epithelium (RPE) cells, photoreceptor cells, corneal epithelial cells, retinal cells, retinal bipolar cells, rod cells, cone cells, ganglion cells, choroid cells and / or lens cells. In addition or as a further option, the vector can be delivered to nearby or adjacent cells or cells that can be contacted with the targeted cells, including but not limited to cells in the brain or cells or blood cells in the optic nerve.

[0487] In vitro treatment can use any method or multiple methods and / or the combination of medicament that can effectively deliver vector to the cell (for example, the iPS-RPE cell of BCD patient) of targeted therapy.In vitro treatment can be carried out via one or more than one round of infection.In some cases, directly in cultured cells, vector is applied to transfect or transduce these cells.In some cases, other methods of being delivered to cell and / or enhancing the transfection / transduction efficiency in cell can be used, including but not limited to multiple transfection / transduction, electroporation, magnetic transfection (magnetofection) or sonoporation.Used method and medicament when infecting / transfecting cell with vector or expression cassette are known in the art of the present invention, including but not limited to as described in the Examples section herein.

[0488] The cells treated in vitro can then be transplanted into the subject's eye. For example, gene-repaired iPS-RPE cells from a BCD patient can be transplanted into the patient via subretinal injection. Methods, agents, and devices for transplanting cells into the eye are known in the art, see, for example, Wert et al., J Vis Exp. 2012; (69): 4286; WO 2016 / 179496; Schwartz et al., Investigative Ophthalmology & Visual Science April 2016, Vol. 57, ORSFc1-ORSFc9.

[0489] For in vivo treatment, the vector and / or expression cassette can be delivered to the cells targeted for in vivo treatment (e.g., via administration to the eye of a subject in need of treatment, thereby delivering to the targeted cells). Methods for in vivo delivery of nucleic acid molecules, expression cassettes, and vectors to target ocular cells are known in the art. For example, administration to the eye can be performed using any method (or combination of multiple methods and / or agents) that effectively delivers the vector to the retina, subretinal space, choroid, or generally the posterior segment of the eye, cornea, lens, or vitreous, depending on the cells targeted for treatment. Administration can be by any suitable means, including but not limited to injection (e.g., subretinal injection, intravitreal injection, direct retinal injection, direct injection into the suprachoroidal space at the back of the eye), eye drops, and can be administered in combination with other delivery techniques (e.g., electrically assisted delivery to the corneal epithelium). CYP4V2 nucleic acids, expression cassettes, and / or delivery vectors can also be introduced into cells using, for example, DNA particle impaction (e.g., by gene gun), hydrodynamic gene transfer, eye drops, electroporation, magnetic transfection, or sonoporation. Methods and techniques for administration and delivery to the eye are known in the art to which the present invention pertains. See, by way of example only, but not limitation, Wert et al., J Vis Exp. 2012;(69):4286; WO 2016 / 179496; Mohan et al., Prog Retin Eye Res. 2012 Jan;31(1):43-64.

[0490] In addition to using subretinal injection to carry out known delivery to RPE cells, one aspect of the method discussed herein is to deliver nucleic acid molecules (for example, with non-mutated CYP4V2 nucleic acid sequence) in the vitreous to treat or prevent eye diseases. Some vectors (for example, AAV2 (four Y-F+TV) and AAV 7m8) show the special prospect of effective transduction in the retina via intravitreal administration. In addition, AAV or other viral vectors can be modified by known technical means in the art of the present invention, including for example " directed evolution " and " rational design ", so as to improve or optimize its suitability as a vector for gene delivery to one or more types of cells or tissues (for example, intravitreal injection) in a manner different from conventional subretinal injection. In addition to subretinal delivery, scAAV vectors can also be used in intravitreal delivery because of its rapid and robust expression profile (expression profile). Because CYP4V2 is almost ubiquitously distributed and has particularly high expression in the retina, the heredity and epigenetic changes of CYP4V2 are particularly suitable for repairing via intravitreal administration of one or more vectors. Current gene therapy methods generally require subretinal administration of vectors. Therefore, one of the technological advances achieved by the materials and methods disclosed herein is the intravitreal delivery of nucleic acid sequences (e.g., wild-type or non-mutated nucleic acid sequences, or nucleic acid sequences encoding gene-editing polypeptides) and / or polypeptides to treat and prevent eye diseases associated with genetic or epigenetic changes in the nucleic acid sequence of CYP4V2.

[0491] Certain techniques and agents can be used to facilitate the administration or delivery process. Non-limiting examples include the use of lubricants to prevent the carrier from adhering to the delivery vehicle (e.g., needle). In addition, the use of immunosuppressive drugs before, during, and / or after the administration or delivery process can increase infection or transduction efficiency.

[0492] Various pharmaceutically and / or physiologically acceptable vehicle excipients, diluents and / or carriers can be used to prepare the carrier for delivery to the eye cells of the experimenter. Suitable vehicle excipients, diluents and / or carriers for eye administration can be referred to as pharmaceutically acceptable carriers, examples of which include sterile pyrogen-free water and sterile pyrogen-free buffered saline (for example, using phosphate or other buffer buffered saline, such as HEPES, which keeps pH at appropriate physiological levels), isotonic sodium chloride solution, balanced salt solution, emulsion (for example, oil / water emulsion) and various types of wetting agents. In some cases, preparations can include other medicaments (medicinal agent), pharmaceutical agent (pharmaceutical agent), stabilizer, buffer, carrier, adjuvant and diluent. In some cases, preparations can include DBPS, glycerol or Tween20 for long-term storage.

[0493] Methods for determining the most effective means of administration and therapeutically effective doses are known to those skilled in the art and will vary with the following: the vector, its capsid structure, vector design (e.g., ssAAV vs. scAAV), the composition of the expression cassette, the expression level of the vector, the promoter, other regulatory sequences or nucleic acid molecules, the vector titer, the target cell type, the target expression level, the area size or number of targeted cells and the subject to be treated (e.g., the age, sex, weight, stage of development of the disease and condition, and potential immune response of the subject to be treated); the route of administration; the location of the cells targeted for treatment (e.g., retina vs. cornea); the nature and expression level of the relevant gene in wild-type cells and / or tissues; and the desired regimen. The therapeutically effective dose can be determined and evaluated in disease models (e.g., BCD cell models (e.g., iPS-RPE cell lines from BCD patients) or animal models) and confirmed or improved by clinical trials. For in vitro treatment of cells, the dose is usually expressed as an MOI, which is then multiplied by the number of cells to be treated. The MOI is generally about 1×10 3 GC / cell is about 1×10 6 The range of GC / cell or infection MOI is about 100 to about 10,000 GC / cell (GC: genome copies, measuring AAV particles containing the genome (also known as vector genomes (vg) or genome particles (gp)). With respect to in vivo treatment, in addition to the factors described above, the actual administered dose will also be affected by the individual circumstances unique to each patient during administration. For example, in the case of choroideremia described below, the dose of patient 6 was reduced during subretinal administration. Therefore, the therapeutically effective dose for a single in vivo administration may be approximately about 1×10 6 to 2×10 13 GC and including end values ​​(e.g., about 1×10 11 GC to about 1×10 12 The high dose range of GC, about 1×10 10 GC to about 1×10 11 The medium dose range of GC is about 1×10 9 GC to about 1×10 10 The low dose range of GC is about 1×10 6 GC to about 1×10 9 The extremely low dose range of GC and about 1×10 12 GC to about 2×10 13 GC), or any dose within these ranges sufficient to provide the desired effect. In one embodiment, the composition is administered at a dose of about 1×10 6 to 2×10 13In another embodiment, the in vivo dose is determined by multiplying the number of cells targeted for treatment by the target MOI (e.g., 1×10 3 GC / cell is about 1×10 6 The volume of the rAAV vector-containing dose in any single administration to the eye can range from about 1 μL (0.001 mL) to about 1000 μL (1 mL).

[0494] Compositions as described herein can be formulated into single dose or multiple dosage.Similarly, administration can occur once or repeatedly (for example, in a few weeks, months or years) and can be applied to same eye or contralateral eye.In the case of multiple administration, identical or different AAV serotypes and / or administration routes can be considered.Administration also can be used for treating different tissues and cells, for example, one administration targeting RPE and another administration targeting cornea.

[0495] Methods for producing viral vectors for gene therapy (including ocular gene therapy), GMP production methods, purification methods, formulation methods, and dosages are known to those skilled in the art, and methods for preparing viral vectors can be performed by any of a variety of companies and methods, as shown below in the various gene therapy studies for LCA-2. The expression cassettes provided herein can be inserted into any of the exemplary viral vectors listed below. Alternatively, viral vectors can be generated according to the examples provided below. See Bainbridge et al., 2008. N Engl J Med. 358:2231-9; Maguire et al., 2008. N Engl J Med. 358:2240-8; Hauswirth et al., Hum Gene Ther. 2008 Oct; 19(10):979-990.

[0496] For example, in the Bainbridge study, gene delivery was performed using the tgAAG76 vector, a recombinant adeno-associated virus serotype 2 vector. This vector contains the human RPE65 coding sequence, driven by the human RPE65 promoter and terminated by a bovine growth hormone polyadenylation site, as described elsewhere. The vector was produced by Targeted Genetics Corporation according to Good Manufacturing Practice guidelines using the B50 packaging cell line, an adenovirus-adeno-associated virus hybrid shuttle vector containing the tgAAG76 vector genome, and an adenovirus 5 helper virus. The vector was injected into the 1×10 11The titer of 10 vector particles / ml was filled in a buffered saline solution and frozen in 1 ml aliquots at -70°C.

[0497] Maguire used a recombinant AAV2.hRPE65v2 viral vector, a replication-defective AAV vector containing the RPE65 cDNA, to demonstrate long-lasting (>7.5 years, observed) visual function recovery in a canine LCA2 model following a single subretinal injection of AAV2.RPE65. The cis-plasmid used to generate AAV2.RPE65 contains a kanamycin resistance gene. The virus was produced at the Center for Cellular and Molecular Therapeutics after triple transfection of HEK293 cells and isolated and purified by microfluidization, filtration, cation exchange chromatography (POROS 50HS; GE Healthcare, Piscataway, NJ), density gradient ultracentrifugation, and diafiltration in PBS. This combination provides an AAV vector product of optimal purity, including efficient removal of empty capsids and residual cesium chloride. A portion of the product was supplemented with PF68 NF Prill Poloxamer 188 (PF68; BASF, Ludwigshafen, Germany) to prevent subsequent loss of the vector from product-contacting surfaces. The purified virus, with or without PF68, was then passed through a 0.22-μm filter using a 60-ml sterile syringe and syringe filter and stored frozen (-80°C) in sterile tubes until use. 1.5×10 10 Injections of 10 AAV2.hRPE65v2 vector genomes in phosphate-buffered saline supplemented with Pluronic F-68NF Prill poloxamer 188 were administered into the subretinal space in a volume of 150 μl.

[0498] The viral vector used by Hauswirth was a recombinant adeno-associated virus serotype 2 (rAAV2) vector modified to carry the human RPE65 gene (rAAV2-CB SB -hRPE65), which has previously been shown to restore vision in animal models with RPE65 deficiency. RPE65-LCA viral vectors were delivered by subretinal injection (5.96×10 10 vector genomes).

[0499] Methods and protocols for administering therapeutic agents (e.g., proteins, nucleic acid molecules, expression cassettes, gene therapy vectors, cells), including but not limited to administration to the eye, and other procedures and protocols (including but not limited to immunological tests, eye examinations, and immunosuppressants) are known in the art to which the present invention relates. For example, the following is an example of subretinal injection of AAV vectors used by MacLaren in the treatment of choroideremia. The surgery begins by detaching the retina using a balanced salt solution (Alcon Laboratories, Fort Worth, TX, USA) through a 41G Teflon cannula (DORC International BV, Zuidland, Netherlands). Once the target area of ​​the retina is detached from the underlying retinal pigment epithelium, a 1×10 10 A fixed volume (0.1 mL) of AAV2.REP1 genome particles was injected via a new syringe into the subretinal space created in the first five patients. In patient 6, up to 6 × 10 9 The detachment was further extended by slowly injecting the vector through the same retinotomy. In the first five patients, the surgery was not difficult or complicated, but in patient 6, the retina was difficult to detach from the peripheral macula, necessitating the induction of detachment from a point close to the fovea, which resulted in visible stretching of the papillomacular bundle. Because of concerns about stretch-related damage to this important structure in patients with 6 / 7.5 vision, a smaller volume of vector (up to 0.06 mL) was injected in a second step. In all patients, the remaining vector in the syringe was drained via cannula into a polypropylene vial and then frozen. The efficacy of this remaining vector was subsequently tested by Western blotting after transduction of the human HT1080 cell line. Patients were treated with a 10-day course of oral prednisolone, starting 2 days before surgery at 1 mg / kg (70-100 mg) for 7 consecutive days, then reduced to 40 mg for 1 day, 20 mg for 1 day, and 10 mg for 1 day. Blood samples were obtained for immunological testing before surgery and at 1 and 5-6 weeks post-surgery. See MacLaren et al., Lancet. 2014 Mar 29;383(9923):1129-1137.

[0500] In the Hauswirth study, drug administration was performed as follows. After light intravenous sedation, the operated eye received retrobulbar anesthesia and was then prepared and draped in a standard aseptic manner. A standard three-port 23-gauge core and peripheral vitrectomy were performed. The conjunctiva at the right sclerotomy was dissected using Westcott scissors and 0.3 forceps. Hemostasis was maintained by eraser-tipped cautery. The sclerotomy was enlarged with a No. 20 MVR blade to allow easy insertion of the subretinal cannula into the eye. The vector was drawn into a 39-gauge injection cannula (Synergetics, O'Fallon, MO) and introduced into the subretinal space. At the end of the operation, the sclerotomy was secured with 7.0 Vicryl sutures and the conjunctiva was closed with interrupted sutures. Antibiotics and steroids were applied subconjunctivally. Postoperatively, topical antibiotics and steroids were applied for 20 days. See Hauswirth et al., Hum Gene Ther. 2008 Oct;19(10):979-990.

[0501] With respect to in vitro CYP4V2 gene therapy treatment, post-treatment assessment can compare the cell morphology and / or biochemical dysfunction of the patient's cells, for example, comparing the levels of compounds that show abnormalities in iPS-RPE cells (or iPS-PRC or iPS-CEC cells, if applicable) of BCD patients before and after treatment to assess whether the cell morphology and / or biochemical function are improved after treatment.

[0502] For in vivo CYP4V2 gene therapy treatment, post-treatment assessment can use eye and retinal examinations (and corneal tests, if applicable) known in the art for retinal and corneal diseases, including but not limited to dark adaptation, contrast sensitivity, visual field tests, visual acuity tests, color vision tests, ERG, OCT, fundus imaging, corneal examinations, functional tests such as mobility, etc. Efficacy can be verified by one of the following: improvement in vision, cessation of disease progression, or a slower than expected rate of retinal degeneration or vision loss.

[0503] One challenge of viral vector-mediated gene therapy is the immune response of the recipient. In addition to the known risks associated with the recipient, immune responses can significantly reduce the transduction efficiency of viral vectors and / or prevent the establishment of long-term transgene expression. Mingozzi F, Meulenberg JJ, Hui DJ, Basner-Tschakarjan E, Hasbrouck NC, Edmonson SA, Hutnick NA, Betts MR, Kastelein JJ, Stroes ES, High KA. AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells. Blood. 2009 Sep 3;114(10):2077-86.

[0504] Perhaps, in part, because of the unique immune environment of the eye, the immune effects of various recombinant viral vectors (e.g., AAV, lentivirus, adenovirus) in ocular gene therapy appear to be quite benign. Despite this, a significant cell-mediated immune response develops after intraocular administration of adenovirus. However, neither AAV nor lentivirus triggers a cell-mediated response and is therefore a promising vector for the treatment of chronic ocular (retinal) diseases. J Bennett, Immune response following intraocular delivery of recombinant viral vectors, Gene Therapy (2003) 10, 977-982. doi: 10.1038 / sj.gt.3302030. However, on the other hand, previous studies have shown that intravitreal administration of AAV vectors causes increased levels of anti-AAV antibodies in the vitreous humor and serum of non-human primates. In addition, the presence of pre-existing neutralizing antibody titers in monkey serum is strongly correlated with weak transgene expression, diminished transgene expression, or no transgene expression after intravitreal administration of AAV. Kotterman et al., Antibody Neutralization Poses a Barrier to Intravitreal Adeno-Associated Viral Vector Gene Delivery to Non-Human Primates, Gene Ther. 2015 Feb; 22(2): 116-126. Therefore, it is desirable to reduce the immune response in ocular gene therapy, especially the immune response of neutralizing antibodies (NAbs), so as to maintain the desired transduction efficiency and / or long-term transgene expression.

[0505] Historically, it has been common practice for companies in the gene therapy field to use a single serotype of AAV vector. Vector types that have good transduction efficiency and extensive safety data in animal studies and / or clinical trials of other gene therapies are typically used. For example, AAV2 is the AAV serotype most commonly used for ocular gene therapy in clinical trials. However, due to individual differences in the immune system, such as individual differences in pre-existing anti-AAV antibodies, the best serotype for one patient may not always be the best for another. For example, the prevalence of pre-existing anti-AAV neutralizing antibodies to specific AAV serotypes varies across countries and populations. In addition, immune responses can significantly reduce transduction efficiency, which can reduce the efficacy of the administered gene therapy and / or require the administration of higher doses.

[0506] Provided herein is a method for reducing the immune response to viral vectors, maintaining transduction efficiency, reducing the dosage of viral vectors and / or immunosuppressants, and / or maximizing the therapeutic effect on different patients with the same genetic disease in viral vector-mediated gene therapy, comprising:

[0507] (a) establishing a library of more than one recombinant viral vector (e.g., rAAV) with sufficient transduction efficiency in the target cell type for the gene therapy. The library of viral vectors can be expanded by generating variants with mutations in the antigenic region or other mutations or variants on the capsid of these viral vectors, and after confirming that these mutations or variants have sufficient transduction efficiency in target cells related to the disease (e.g., in iPS-RPE or RPE cell lines for CYP4V2 gene therapy for BCD).

[0508] (b) detecting pre-existing neutralizing anti-viral vector antibodies (NAb) against different viral vector serotypes and / or capsid mutations or variants in a subject in need of the gene therapy, and / or testing and comparing different viral vectors in patient-specific disease target cells (e.g., iPS-RPE cells) derived from the subject.

[0509] (c) selecting a viral vector from the library of viral vectors that (i) has sufficient transduction efficiency in the disease target cells and (ii) has low cross-reactivity with the pre-existing Nabs in the subject, and / or (iii) has good phenotypic rescue results in the subject's patient-specific disease target cells (e.g., for CYP4V2 gene therapy for BCD, it is a patient-specific iPS-RPE or RPE cell line), wherein the library of viral vectors comprises viral vectors of different serotypes and / or capsids modified (e.g., including but not limited to capsid mutant AAV and / or capsid protein variant AAV).

[0510] (d) administering to the subject a viral vector selected from (c).

[0511] (e) Repeat (b) to (d) above (repeating only the portion relevant to pre-existing NAbs) each time the subject requires administration of gene therapy, including but not limited to subsequent administration to the same organ (e.g., the eye or the contralateral eye) or to other organs.

[0512] Potential benefits of this approach include reduced use of immunosuppressants, lower doses of rAAV vector, higher transduction efficiency with longer periods of transgene expression, and / or a higher percentage of patients eligible for gene therapy.

[0513] It will be appreciated that this approach can be used in conjunction with other viral vectors. Furthermore, this approach can be used in all types of ocular and non-ocular gene therapies, whether the therapy is related to the CYP4V2 gene or other genes.

[0514] Methods for detecting pre-existing anti-AAV antibodies are known in the art to which the present invention belongs. It is worth noting that anti-AAV antibodies include neutralizing antibodies and non-neutralizing antibodies. Methods for detecting pre-existing anti-AAV antibodies and other immune responses to AAV are known in the art to which the present invention belongs. Melvin Y Rincon et al., JMIR Res Protoc. 2016 Apr-Jun;5(2):e102; Hauswirth et al., Hum Gene Ther. 2008 Oct;19(10):979-990. Although neutralizing antibodies have the most significant effect, even non-neutralizing antibodies can trigger vector clearance by the immune system. Non-neutralizing antibodies can be detected by ELISA. Boutin S,Monteilhet V,Veron P,LeborgneC,Benveniste O,Montus MF,Masurier C,Prevalence of serum IgG and neutralizing factors against adeno-associated virus(AAV)types 1,2,5,6,8,and 9in the healthy population:implications for gene therapy using AAV vectors.Hum GeneTher. 2010 Jun;21(6):704-12.

[0515] Unless further defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject methods and compositions belong. In addition to the definitions of terms provided herein, definitions of common terms in molecular biology can also be found in the following: Glossary of Genetics: Classical and Molecular, Rieger et al., 1991, 5th edition, Springer-Verlag; Current Protocols in Molecular Biology, Ausubel et al., ed., 1998 supplement, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.; Current Protocols in Cell Biology, Bonifacino et al., ed., 1999 supplement, John Wiley & Sons, Inc.; and Current Protocols in Neuroscience, Crawley et al., ed., 1999 supplement, John Wiley & Sons, Inc.

[0516] Representative methods and materials are described herein; other suitable methods and materials known in the art to which the present invention pertains can also be used. These methods and materials are illustrative only and are not intended to be limiting.

[0517] Example

[0518] The present invention is further described in the following examples, which do not limit the scope of the invention and the claims.

[0519] These studies were initiated, designed, organized, and funded by Reflection Biotechnologies Limited ("ReflectionBio"), a biotechnology company founded and driven by patients and families living with rare retinal diseases. Rare disease patients bear the inevitable burden of human genetic mutations, yet are often overlooked by society and receive insufficient public support. As a patient-driven biotechnology company, ReflectionBio employs a "By Patients, For Patients" approach to empower patients and play a more active role in advancing scientific and medical research for rare and other challenging diseases.

[0520] Patients diagnosed with BCD and harboring various biallelic CYP4V2 mutations (including homozygous CYP4V2 mutations or compound heterozygous CYP4V2 mutations) were included in this study. Specifically, one patient (referred to herein as Patient 1, P1, or RB001) harbored the homozygous c.802-8_810del17insGC mutation. The c.802-8_810del17insGC mutation causes an in-frame deletion of exon 7, encoding 62 amino acids. The c.802-8_810del17insGC mutation is the most common mutation in BCD patients. Patient 2 (P2 or RB002) harbored compound heterozygous CYP4V2 mutations, each of which is a single nucleotide change that alters only one amino acid in the 525-amino acid-long CYP4V2 protein.

[0521] Informed consent was obtained. Procedures followed the guidelines of the Declaration of Helsinki and were approved by the Institutional Review Board.

[0522] Example of BCD human cell disease model

[0523] Clinically, BCD is associated with RPE atrophy, which in turn leads to photoreceptor death and visual loss. Therefore, it is crucial to establish and use a human RPE model to study BCD and develop treatments for it.

[0524] Example 1 - Generation and Characterization of Induced Pluripotent Stem Cells (iPSCs) Derived from BCD Patients

[0525] In this study, iPSCs were generated from BCD patients using a non-integration method. Conventional techniques for iPSC reprogramming (e.g., lentivirus, retrovirus) are integrated into the genome of the target cell. The resulting iPSCs and cells differentiated from those iPSCs will contain exogenous DNA and may be unsafe and may be problematic when used in cell therapy and drug discovery applications. In addition, integration may occur in key regions of the genome, causing problems in unrelated developmental processes. Compared to traditional reprogramming methods, non-integrati...

Claims

1. A recombinant adeno-associated virus (rAAV) vector, comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a non-mutant or functional CYP4V2 protein as shown in SEQ ID NO: 4, and the nucleic acid sequence is operably linked to at least one regulatory sequence, wherein the regulatory sequence comprises a promoter, and the promoter is a CAG promoter or an elongation factor 1α short EFS promoter.

2. The rAAV vector of claim 1, wherein the rAAV vector comprises a VP1, VP2 or VP3 capsid protein selected from any serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or an isolate of AAV, or a chimera or mutant thereof.

3. The rAAV vector of claim 1, wherein the rAAV vector comprises a 5′ AAV inverted terminal repeat ITR or a 3′ AAV ITR selected from any one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or an isolate of AAV, or a chimera or mutant thereof.

4. The rAAV vector of claim 1, wherein the rAAV vector is a chimeric AAV, a rearranged AAV, or an AAV with a modified capsid.

5. The rAAV vector of claim 1, wherein the rAAV vector is a pseudotyped AAV.

6. The rAAV vector of claim 1, wherein the rAAV vector comprises one or more modifications to one or more native AAV capsid proteins.

7. The rAAV vector of claim 1, wherein the rAAV vector is selected from the group consisting of AAV2 / 5, AAV2 / 8, AAV2 / 2, AAV2, AAV2 / 1, AAV2 / 9, AAV2 / 6, AAV2 / 4, AAV2 / 7, AAV5, AAV8, AAV1, AAV9, AAV6, AAV10, AAV3, AAV4, AAV7, AAV11, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV-PHP.B, rh10, and chimeras or mutants thereof.

8. The rAAV vector according to claim 7, wherein the modification in the AAV2 is Y444F+Y500F+Y730F, or any one or more modifications of Y444F, Y500F, and Y730F.

9. The rAAV vector of claim 7, wherein the modification in AAV2 / 8 is Y733F.

10. The rAAV vector according to claim 1, wherein the rAAV vector is a single-stranded AAV vector, i.e., an ssAAV vector, or a self-complementary AAV vector, i.e., a scAAV vector.

11. The rAAV vector according to claim 1, wherein the nucleic acid sequence encoding a non-mutant or functional CYP4V2 protein as shown in SEQ ID NO: 4 has at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

12. The rAAV vector according to any one of claims 1 to 11, wherein the nucleic acid sequence of the CAG promoter is as shown in SEQ ID NO: 32, and the nucleic acid sequence of the elongation factor 1α short EFS promoter is as shown in SEQ ID NO:

35.

13. The rAAV vector according to any one of claims 1 to 11, wherein the regulatory sequence comprises a Kozak sequence or a variant thereof.

14. The rAAV vector of any one of claims 1 to 11, wherein the regulatory sequence comprises an enhancer.

15. The rAAV vector of claim 14, wherein the enhancer comprises a post-transcriptional regulatory element PRE, an internal ribosome entry site IRES, an intronic regulatory sequence, a small intronic splice donor / splice acceptor SD-SA, or a constitutive transport element CTE.

16. The rAAV vector of claim 14, wherein the enhancer is selected from the group consisting of: woodchuck hepatitis virus post-transcriptional regulatory element WPRE enhancer, hepatitis B virus post-transcriptional regulatory element HPRE or HBVPRE enhancer, IRBP enhancer, Mason-Fischer monkey virus CTE enhancer, cytomegalovirus CMV immediate early enhancer, SV40 enhancer, avian leukosis virus CTE enhancer, chimeras or mutants thereof.

17. The rAAV vector of any one of claims 1 to 11, wherein the regulatory sequence comprises a polyadenylation polyA signal.

18. The rAAV vector of claim 17, wherein the polyA signal is selected from the group consisting of bovine growth hormone polyadenylation signal bGH polyA, small polyA signal SPA, human growth hormone polyadenylation signal hGHpolyA, SV40 polyA signal, SV40 early polyA signal, SV40 late polyA signal, or mutants thereof.

19. The rAAV vector according to any one of claims 1 to 11, wherein the regulatory sequence comprises an upstream enhancer USE operably linked to a polyA signal.

20. The rAAV vector of claim 19, wherein the USE is selected from the group consisting of: SV40 late 2xUSE, human immunodeficiency virus 1 USE, ground squirrel hepatitis virus USE, adenovirus USE, human prothrombin USE, human C2 complement gene USE, and derivatives, variants and hybrids thereof.

21. The rAAV vector of any one of claims 1 to 11, wherein the regulatory sequence comprises an intron sequence, a UTR sequence, a splice site sequence, an upstream regulatory domain sequence, a response element sequence, an inducible element sequence, a replication origin sequence, an internal ribosome entry site IRES sequence, a transcription start sequence, a termination sequence, a terminator, an RNA processing sequence, a junction sequence, a linker sequence, a derivative, a hybrid or a variant thereof.

22. The rAAV vector of any one of claims 1 to 11, wherein the regulatory sequence is heterologous or homologous to the CYP4V2 coding sequence whose expression is regulated by the regulatory sequence.

23. The rAAV vector of any one of claims 1 to 11, wherein the regulatory sequence comprises a target sequence for a tissue-specific or cell-specific microRNA, i.e., miRNA.

24. The rAAV vector according to any one of claims 1 to 11, wherein the nucleic acid molecule has at least 80% sequence identity with any one of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63 and SEQ ID NO:

64.

25. The rAAV vector of any one of claims 1 to 11, contained in a pharmaceutically acceptable carrier and additional components suitable for a particular route of administration or delivery device.

26. A cell comprising the rAAV vector according to any one of claims 1 to 25, wherein the cell is a non-plant cell and a non-animal fertilized egg, germ cell and embryonic stem cell.

27. The cell of claim 26, wherein the cell is a mammalian cell or an insect cell.

28. The cell according to claim 26, wherein the cell is a HEK293 cell, a 293T cell, an A459 cell or a Sf9 cell.

29. cells according to claim 26, wherein the cell is retinal pigment epithelium RPE cell, photoreceptor cell, photoreceptor progenitor cell, choroid cell, retinal cell, retinal bipolar cell, induced pluripotency dry iPS cell, adult stem cell or mesenchymal stem cell MSC, or is derived from the induced pluripotency dry iPS cell or the iPS-derived retinal pigment epithelium RPE cell of the human subject suffering from crystalline sample retinal degeneration.

30. The cell of claim 29, wherein the human subject carries biallelic CYP4V2 mutations, comprising homozygous mutations or compound heterozygous mutations.

31. Use of a cell according to any one of claims 26 to 30 in the preparation of a medicament for treating, slowing progression, arresting or preventing crystalline retinal degeneration in a human subject by a method comprising delivering a therapeutically effective amount of a cell according to any one of claims 26 to 30 to the retina of the human subject.

32. Use of the rAAV vector according to any one of claims 1 to 25 in the preparation of a medicament for treating, suppressing or preventing crystalline retinal degeneration in a human subject by the following method, the method comprising: delivering a therapeutically effective amount of the rAAV vector according to any one of claims 1 to 25 to the retina of the human subject, wherein one or more retinal pigment epithelial RPE cells, one or more choroidal cells, and / or one or more photoreceptor cells of the human subject are transduced with the rAAV vector, thereby treating, suppressing or preventing crystalline retinal degeneration in the human subject.

33. The use according to claim 32, wherein the human subject carries biallelic CYP4V2 mutations, wherein the biallelic CYP4V2 mutations comprise homozygous mutations or compound heterozygous mutations.

34. The use according to claim 32, wherein the photoreceptor cell is a rod cell or a cone cell.

35. according to the purposes of the rAAV vector described in any one of claims 1 to 25 in the preparation of a medicine for treating, curbing or preventing a disease associated with atrophy, degeneration, dysfunction, loss of function, illness, degeneration or death of retinal pigment epithelial RPE cells of a human subject by the following method, the method comprising: delivering a therapeutically effective amount of the rAAV vector described in any one of claims 1 to 25 to the retina of the human subject, wherein one or more RPE cells of the human subject are transduced with the rAAV vector, thereby treating, curbing or preventing the disease in the human subject.

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