Non-coding DNA sequence variants cause retinitis pigmentosa

Abstract

Applicant provides a method for treating or preventing a TMEM216-related disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a gene that supports or restores functional TMEM216 in the patient, such as for example retinitis pigmentosa (RP) or Joubert syndrome 2. The subject to be treated can be identified as harboring a mutation in the 5'UTR of GRCh38: chrll:g.61392563G. Also provided are vectors comprising the gene that supports or restores functional TMEM 216 in the subject. Diagnostic tests to identify these subjects also are provided herein.

Description

[0001] Atty. Dkt. No.: 114198-2110 NON-CODING DNA SEQUENCE VARIANTS CAUSE RETINITIS PIGMENTOSA CROSS-REFERENCE TO RELATED PATENT APPLICATION This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 666,637, filed on July 1, 2024 and 63 / 722,487, filed November 19, 2024, the contents of which are incorporated herein by reference in their entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant Nos. RO1EY21237 and RO1EY031663, awarded by the National Institutes of Health (“NIH”). The government has certain rights in the invention. BACKGROUND TMEM216 is a small gene on chromosome 11 involved in primary ciliogenesis. It has been identified as causative of Joubert and Meckel’s syndrome, which are severe ciliopathies that often involve retinal degeneration. It is a relatively small protein with 4 hydrophobic putative transmembrane domains predicted. It is broadly but relatively lowly expressed, showing expression in the central nervous system, limb bud, kidney, and cartilage. TMEM216 forms a tectonic complex that localizes to the transition zone that regulates ciliogenesis / ciliary membrane composition. Due to its role in ciliary maintenance, mutations in TMEM216 affect ciliogenesis. Fibroblasts derived from patients with TMEM216 mutations had impaired ciliogenesis and centrosomal docking, resulting in shortened cilia. The disruption of ciliogenesis and ciliary maintenance is especially important in the formation and maintenance of photoreceptor sensory cilia. Joubert syndrome 2, retinitis pigmentosa (RP98: OMIM #620996) and other TMEM216-related disorders are inherited, multisystem disorders caused by a defect in the cilia. The diseases are characterized by abnormal development of regions near the back of the brain (molar tooth sign), hypotonia, and developmental delays. There are no current Atty. Dkt. No.: 114198-2110 treatments for these diseases. Thus, a need in the art exists to identify and treat these patients. This disclosure satisfies this need and provides related advantages as well. SUMMARY OF THE DISCLOSURE This disclosure identifies and characterizes the noncoding 5’UTR region of TMEM216 that is linked to TMEM216-related disorders, e.g., inherited retinal degeneration (IRD). In one aspect, the 5’UTR region spans from about -1000 to about -1 of the noncoding region of the TMEM216 gene. Within this region, Applicant has identified 4 nucleotide substitutions (e.g., TMEM216 c.-69G>A) associated with inherited retinal degeneration (IRD) among individuals with various ethnic backgrounds, e.g. in one aspect, of South Asian ancestry. Applicant also observed another variant at the same position in the genome TMEM216 c.-69G>T in patients of African American, African or mixed ancestry. Applicant further identified c.-95G>C in patients of South Asian and European ancestry as well as c.- 41C>T in patients with South Asian, European and mixed ancestry. These variants, located upstream of the TMEM216 gene, are shown to lead to reduced expression of TMEM216, contributing to photoreceptor degeneration. Functional validation of the impact of these variants was established by elucidating the molecular mechanisms underlying IRD pathogenesis. This disclosure provides crucial insights for the development of precision medicine approaches and gene-directed therapies targeting TMEM216 expression regulation to prevent blindness. In one aspect, Applicant provides a method for treating or preventing or delaying progression of a TMEM216-related disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a gene that supports or restores functional TMEM216 in the patient, such as for example retinitis pigmentosa (RP98) or Joubert syndrome 2. The subject to be treated can be identified for treatment as harboring a mutation at the genomic location GRCh38: chr11:g.61392563G, in the 5’UTR region, or alternatively from about 1000 base pairs to 1 base pair from the start codon. In one aspect, the mutation is located 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 (NM_001173991.3). Alternatively, the mutation is at a genomic location upstream of the start codon of the ciliopathy gene TMEM216 (NM_001173991.3: c.-69G>A, c.- Atty. Dkt. No.: 114198-2110 69G>T, c.69G>C., c.-95G>C, and / or c.-41 C>T. Any population or individual subject with these mutations can be treated with the methods described herein. The subject may be of South Asian, African, South Asian, European or a mixed ancestry, or of a different ancestry. Diagnostic tests to identify these patients also are provided herein. In one aspect the gene to be administered to the patient for therapy is TMEM216. The gene may be a long isoform or a short isoform of the TMEM216 gene. In one aspect, the delivery of TMEM216 or its isoforms driven by the native promoter or a modified version thereof, can restore cilia formation / function and / or improve vision. In one aspect, the delivery of TMEM216 or its isoforms is driven by a different promoter, e.g., CAG, a cell specific promoter like BEST1 to deliver TMEM216 to the retina, RPE, or other selected retinal cells. In one aspect, the gene is administered in construct TMEM216_deltaP2 or an equivalent thereof. In one aspect, the gene is administered in construct TMEM216_WT or an equivalent thereof. In another aspect the gene is administered in a different construct. Sequences for the constructs are shown in Table 1 and the partial sequence listing. In another aspect, the gene is administered in the constructs in identified below and FIG. 7 that optionally exclude sequences coding for a marker, for clinical use. The subject to be treated is a mammal, optionally a human patient. The gene can be delivered by any appropriate therapy, e.g., the effective amount of the gene is administered in an adeno-associated viral (AAV) particle or a derivative or variant thereof. In one aspect, the AAV is selected from an AAV serotype selected from the group of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, or AAV-11. In one aspect, the AAV serotype is AAV-8 or AAV-9. In another aspect, the AAV serotype is a different serotype. According to one aspect, the AAV has retinal tropism. In one aspect, the gene is delivered or administered through intravitreal injection, sub- retinal injection or sub-RPE injection. This disclosure focuses on non-coding variants in the TMEM216 gene, providing a novel perspective on IRD pathogenesis. Unlike existing reports that primarily target coding mutations in known IRD genes, Applicant’s disclosure uncovers the regulatory mechanisms influencing TMEM216 expression, offering unique insights into the genetic basis of IRD. Atty. Dkt. No.: 114198-2110 While previous studies have mainly focused on coding mutations, Applicant’s disclosure identifies non-coding variants with significant implications for disease pathology and treatment strategies. Applicant analyzed genome data from individuals affected by IRD to identify rare nucleotide substitutions upstream of the TMEM216 gene. Through comprehensive genetic analyses and functional assays, it was demonstrated that these non-coding variants lead to reduced TMEM216 expression, contributing to photoreceptor degeneration. By elucidating the regulatory mechanisms governing TMEM216 expression, Applicant has provided insights into the molecular basis of IRD and therapeutic targets for gene-directed therapies. Applicant provides herein in-depth genome analysis of individuals affected by IRD, identification of rare nucleotide substitutions upstream of the TMEM216 gene, identification of shared haplotype analysis, clinical phenotype analysis, and functional assays demonstrating reduced TMEM216 expression. These data collectively support the role of non-coding variants in TMEM216 as the underlying cause of non-syndromic inherited retinal degeneration in affected individuals of African and South Asian ancestry. Thus, this disclosure provides a method for treating, preventing or delaying the comprising, or consisting essentially of, or consisting of administering to the subject an effective amount of at least one gene that supports or restores functional TMEM216 in the patient. Also provided is a nucleic acid including the TMEM216_Native promoter-Short Isoform-AAV genome as shown in FIG. 7, optionally without the HA tag or a marker sequence. In a further aspect, a nucleic acid comprising the TMEM216_Native promoter-Long Isoform-AAV genome as shown in FIG. 7 is provided, optionally without the HA tag, antibiotic resistance gene, or a marker sequence. In another aspect, the disclosure provides a nucleic acid including the TMEM216_Short Isoform-AAV genome as shown in FIG. 7, optionally without the HA tag, antibiotic resistance gene, or marker sequence. Atty. Dkt. No.: 114198-2110 In one embodiment, the disclosure provides a nucleic acid including the TMEM216_Long Isoform-AAV genome as shown in, optionally without the HA tag or marker sequence. In some aspects, the disclosure provides a method for diagnosing or prognosing a TMEM216-related disorder in a subject, the method comprising, or consisting essentially of, or consisting of detecting a mutation at the genomic location GRCh38: chr11:g.61392563G, 69 in the 5’UTR region, or alternatively, a region comprising, or consisting essentially of, or further consisting of, about -1000 to about -1 base pairs upstream of the start codon of the ciliopathy gene TMEM216, wherein a mutation at the location from about -1000 to about -1 is a positive diagnosis or prognosis for the TMEM216-related disorder in the subject. BRIEF DESCRIPTION OF THE DRAWINGS 1: Segregation of TMEM216 Variants Segregation analysis of TMEM216 upstream sequence variants in representative pedigrees of African (A-Q) and South Asian (R-W) origin. M1 = c.-69G>T. M2 = C.61382891-61393975del. M3 = c.35-2A>G. M4 = c.-69G>A. * , +, and # indicates individuals that underwent whole genome sequencing, whole exome sequencing, or targeted sequencing, respectively. Additional details of pedigrees are provided in Table 2. FIGS. 2A – 2B: Homozygosity mapping of Family A-4 and across families with TMEM216 c.-69G>A. ( 2A) Homozygosity in affected and unaffected members of A-4 across the genome identified an 8.25 Mb (GRCh38: chr11:g.55000000 — 63258298) region on chromosome 11 shared by three affected individuals. Red indicates variants in the region that are homozygous, blue indicates alleles that are heterozygous, and white indicates homozygous and heterozygous alleles in the same frequency. ( 2B) Homozygosity mapping across families with TMEM216 c.-69G>A: Homozygous regions identified on chromosome 11 in pedigrees A-4, A-5, A-7 and A-6 and 11 genes located within the 330 kb shared homozygous interval are shown. FIGS. 3A – 3H: Clinical Findings for Individuals with TMEM216 c.-69G>T and TMEM216c.-69G>A variants of 72 and 24 years of age, respectively. (FIGS. 3A-3D). En face pseudo color images (A / B) and green (532nm) autofluorescence (C / D) from an Optos Atty. Dkt. No.: 114198-2110 wide angle fundus camera. The 72-year-old patient shows a greater amount of pigment and further reduction in autofluorescence (A / C). In the 24-year-old patient, there is typical bone- spicule pigment in the peripheral retina. This patient showed loss of autofluorescence, with retention of autofluorescence within a 10-degree area centered on the fovea (B / D). (FIGS. 3E-3H). En face infrared and OCT images of the right (E / F) and left (G / H) eyes centered on the fovea. The 72-year-old patient showed atrophy of both outer retina and RPE on OCT with some preservation of the foveal layers. In the 24-year-old patient, the region of preserved retinal anatomy on OCT imaging matches the retained autofluorescence observed in D (F / H). FIGS. 4A – 4H: The c.-69 variants downregulate TMEM216 expression in vitro and in vivo. ( 4A) Characteristics of the 931-bp region upstream of the TMEM216 gene used in the luciferase assay (GRCh38: chr11:61,391,712-61,392,642). This 931-bp region contains cis-regulatory elements for known retinal transcription factors, such as CRX and OTX2 and two predicted promoter sequences (P1 and P2).37,46( 4B) Relative luciferase activity for the reference and mutated constructs containing the TMEM216 c.-69G>A, c.-69G>T and additional constructs harboring deletions of the predicted P1 and P2 promoters. Luciferase activity was normalized by a construct with no promoter sequence and the WT control. **** denotes a p-value of <0.0001. ( 4C) Real-time quantitative PCR quantification of TMEM216 and the neighboring TMEM138 gene expression in blood samples from two WT controls and two patients carrying the homozygous TMEM216 c.-69G>T variant (T-24 and T-23). ( 4D) IGV representation of CDNA reads from Oxford Nanopore sequencing of leukocyte RNA. Top lane: an affected patient homozygous for c.-69G>T, Middle lane: the heterozygous mother, Bottom lane: a healthy WT control. Red vertical line represents the variant base c.- 69G, which is not included in the RNA, sequence. The right panel shows the relative read depths for a benign coding cSNP (rs3741265, c.264G>A, p.Pro88Pro) for which the mother is heterozygous. The A allele, in cis with the c.-69G>T mutation, is represented at a significantly lower concentration than the G allele (read depth 8184 (A) versus 23255(G)). ( 4E) qRT-PCR analysis of gene expression in hTERT-RPE1 cells. Quantitative PCR analysis of TMEM216 and TMEM138 expression across all three genotypes. Expression is relative to GAPDH housekeeping gene. One-way ANOVA results are shown (* = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001). ( 4F) Functional validation of TMEM216 c.-69G>A in Atty. Dkt. No.: 114198-2110 hTERT-RPE1 cells. CRISPR-Cas9 edited hTERT-RPE‘1 cells with c. -69G>A variant in homozygous state showed loss of cilia or abnormal cilia (green) whereas the wild-type and heterozygous cells showed presence of cilia. The cilia were stained with Acetylated tubulin (green) | and the nucleus were stained with DAPI (blue). The Scale bar = 10um. Two independent clones with each genotype were analyzed. ( 4G) Reduction in percent ciliated hTERT-RPE1 cells with c.-69G>A. Percent ciliated cells is significantly low when c.-69G>A variant is present in the homozygous (3%) or heterozygous (54%) state when compared to the wild-type. Corrected p-value <0.0001 (Kruskal-Wallis non-parametric test with Dunn’s multiple comparison of the difference between all of the conditions). ( 4H) Total TMEME216 expression in the human peripheral retina compared to all GTEx tissues. Distribution of total TMEM216 expression in combined GTEx tissues (mean expression = 11.02 + / - 0.05 TPM, n=17382) and the human peripheral retinal samples (mean expression = 19.96 + / - 0.24 TPM, n=411), showing a significantly higher expression in the retina (Mann Whitney nonparametric test p-value <0.0001). FIGS. 5A – 5G: Sequence of the region encompassing the TMEM216 c.-69G>T and G>A variants. Representative electropherograms of the DNA sequence surrounding the TMEM216 -69 G>T and G>A variants in an unaffected individual ( 5A), unaffected carriers ( 5B / 5D), affected individuals ( 5C / 5E), wild-type hTERT-RPE1 cells ( 5F), hTERT-RPE cells with the heterozygous ( 5F) and homozygous ( 5G) genotypes. Shown with an asterisk is an additional variant present in homozygous cells. FIGS. 6A – 6B: Homozygosity mapping for four homozygous affected using AutoMap. ( 6A) Homozygosity mapping for two individuals homozygous for TMEM216 c.- 69G>T. ( 6B) Homozygosity mapping for two individuals homozygous for TMEM216 c.- 69G>A. The blue bars represent regions of homozygosity, and the red arrows indicate regions containing TMEM216 gene. FIGS. 7A and 7B are vector maps. 7A is a map of TMEM216-Native Promoter-Long isoform flanked by ITR sequences. 7B is a map of TMEM216-Native Promoter-Short isoform flanked by ITR sequences. 8 graphically depicts the two major isoforms of TMEM216. Atty. Dkt. No.: 114198-2110 FIGS. 9A to 9C show cilia phenotype in mutant cells before and after gene augmentation. 9A shows loss of cilia in hTERT-RPE1 cells with the TMEM216 c.-69G>A mutation. Left image shows wild type cells with Cilia. Right image shows loss of cilia in cells homozygous for the mutation. 9B is one of two images of hTERT-RPE cells with homozygous TMEM216 c.-69G>A mutation before and after treatment with hAAV-CAG- TMEM216-HA (Long isoform). Lack of cilia is evident. 9C shows mutant cells treated with AAV-CAG-TMEM216-HA full length transcript. Mutant cells develop cilia (shown with arrows), after treatment. FIGS. 10A to 10C show the impact of c.-69A mutation in patient cells. 10A shows cilia present in normal control human iPSC-RPE cells. Cilia are seen as center dots. 10B shows lack of cilia in iPSC-RPE cells derived from a patient homozygous for c.-G69>A. No center dots are seen indicating a lack of cilia. 10C shows relative expression of TMEM216 in retinal pigment epithelial cells (RPE) generated from patient iPSC. FIGS. 11A and 11B show restoration of cilia phenotype and increased levels of TMEM216 gene augmentation in edited hTERT-RPE1 with the homozygous c.-69C>A mutation. 11A shows 3 panels. Left panel shows wildtype hRPE1 cells with cilia (note arrows). Center panel shows no cilia in cells homozygous for the c.-G69>A genotype. Right panel shows restoration of cilia upon TMEM216 gene augmentation using AAV8-CAG-GFP. As understood in the art, clinical grade vectors will not possess the marker protein such as GFP or HA. 11B shows relative expression of TMEM216 in wildtype and homozygous following gene augmentation.

[0002] Atty. Dkt. No.: 114198-2110 DETAILED DESCRIPTION It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology. The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. In practicing the present technologies, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Atty. Dkt. No.: 114198-2110 Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. Definitions Throughout this application, patent and technical publications are referenced by an identifying citation or an Arabic numeral that refers to a citation with the full bibliograph provided in the reference section, immediately preceding the claims. The disclosures of these technical and patent publications are provided to more clearly understand the state of the art and are incorporated by reference herein. Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting of' and / or “consisting essentially of' aspects and variations. Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of Atty. Dkt. No.: 114198-2110 the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range. The term "at least" prior to a value or series of values is understood to include the values adjacent to the term "at least," and all subsequent values (numbers, integers, or fractions) that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer, e.g., "at least 18 nucleotides of a 21- nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures). In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof. -11- Atty. Dkt. No.: 114198-2110 It is to be noted that, as used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component; for example, "a nucleic acid sequence," is understood to represent one or more nucleic acid sequences, unless stated otherwise. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. Furthermore, "and / or", where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or± 10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. The term “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation. Atty. Dkt. No.: 114198-2110 As used herein, the term “determining the genotype of a cell or tissue sample” intends to identify the genotypes of polymorphic loci of interest in the cell or tissue sample. In one aspect, a polymorphic locus is a single nucleotide polymorphic (SNP) locus. If the allelic composition of a SNP locus is heterozygous, the genotype of the SNP locus will be identified as “X / Y” wherein X and Y are two different nucleotides, e.g., A / C for the rs1042044 A / C SNP. If the allelic composition of a SNP locus is heterozygous, the genotype of the SNP locus will be identified as “X / X” wherein X identifies the nucleotide that is present at both alleles, e.g., G / G for the rs1042044 A / G SNP. The term "derived from," as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism. As used herein, the term "coding sequence" or a sequence "encoding" refers to a particular molecule which is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of RNA) into protein, in vitro or in vivo, when operably linked to an appropriate regulatory sequence, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. Although a "stop codon" (e.g., TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence. As used herein, the term "exon" refers to coding sections of a DNA molecule, or of an RNA molecule which is transcribed from a DNA molecule that are translated into protein. Exons can be separated by intervening sections of DNA that do not code for proteins, known as "introns". Therefore, the term "intron", as used herein, refers to a segment of nucleic acid that is transcribed and is present in the "pre-mRNA" but excised by the splicing machinery and therefore not present in the mature mRNA transcript. Following transcription, new, Atty. Dkt. No.: 114198-2110 immature strands of messenger RNA, called "pre-mRNA", may contain both introns and exons. These pre-mRNA molecules go through a modification process in the nucleus called splicing during which the noncoding introns are cut out and only the coding exons remain in the "mature mRNA'. Splicing produces a mature messenger RNA molecule that is then translated into a protein. The term "first exon" refers to a coding sequence or sequence of nucleic acid that encodes a polypeptide or polypeptide region and the term "second exon" refers to a different second coding sequence or sequence of nucleic acid that encodes a second polypeptide region. Where the two exons are separated by an intervening intron in the pre-mRNA, the splicing machinery operates to remove the intervening intron and join the two exons in the mature mRNA. The term "polyadenylation signal" refers to a nucleic acid sequence present in the RNA transcript that allows for the transcript, when in the presence of the enzyme polyadenyl transferase, to be polyadenylated. The term "promoter," as used herein in, refers to a sequence sufficient to direct transcription, in a cell. A promoter is intended as a DNA region to which RNA polymerases bind and that directs the enzyme to transcribe an operably linked DNA sequence. A DNA sequence is operably linked to a promoter if the promoter is capable of directing transcription of that DNA sequence. Promoters for use in the invention include prokaryotic, eukaryotic (e.g., mammalian or yeast), and viral promoters, e.g., the CMV (mammalian cytomegalovirus) promoter, the CAG promoter (also known as CBA promoter; CMV early enhancer / chicken β actin promoter), the UbC (polyubiquitin C gene) promoter, or the CBh (an engineered CBA promoter in which the SV40 intron is replaced with a hybrid intron composed of a 5′ donor splice site from the chicken β-actin 5′ UTR and a 3′ acceptor splice site from MVM). A promoter can be a "constitutive" promoter that is a promoter that, when operably linked to a polynucleotide encoding a gene product, results in the production of a gene product in the cell under most or all conditions of the cell. A promoter can be a "regulatable" promoter that is a promoter whose activity is affected by a cis or trans acting factor (e.g., an inducible promoter, such as an external signal or agent).The term "inducible" promoter means that when the promoter is operably linked to a polynucleotide encoding a specified gene product, it results in the production of a gene in the cell basically only when Atty. Dkt. No.: 114198-2110 the inducer corresponding to the promoter is present in the cell. A promoter can be a "ubiquitous" promoter that is a promoter that is active in a wide range of cells, tissues and cell cycles, or a "tissue-specific" promoter, that is a promoter that has activity only or mostly in certain cell types, i.e., drives the expression of the operably linked nucleotide sequence only or mostly in certain cell types. A promoter can be a "bidirectional" promoter, which is a promoter that is an intergenic region between two divergent genes located on complementary strands of the DNA and drives their coordinated transcription in opposite directions. As used herein, the term "regulatory sequence" refers to a nucleic acid sequence capable of regulating the expression of a nucleic acid sequence operably linked to said regulatory sequence, non-limiting examples of regulatory sequences are enhancers (a DNA sequence that increases the level of transcription of an operably linked gene), and silencers (a DNA sequence that decreases the level of transcription of an operably linked gene). The term "regulatory sequence" also refers to nucleic acid sequence in RNA transcripts capable of regulating, for example, the processing or the expression of said transcripts. Non-limiting examples of regulatory sequences that can be in RNA transcripts are nucleotide sequences that regulate localization or splicing of said RNA transcripts. The term "regulatory sequence" further refers to an amino acid sequence capable of regulating, for example, the localization (e.g., subcellular localization signals, such as nuclear localization signals), or the stability (e.g., degradation signals) of a protein. The terms "operatively linked," "operatively inserted," "operatively positioned," "under control" means, with reference to two or more nucleic acid sequences, that the nucleic acid sequences are arranged in such a way that one of the two or more nucleic acid sequences can mediate a function that is exerted upon at least one of the other two or more nucleic acid sequences. For example, a regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer) can be "operatively linked," to a coding nucleic acid sequence, that is the regulatory nucleic acid sequence is in the correct location and orientation in relation to the coding nucleic acid sequence to control expression of the coding nucleic acid sequence (e.g., via control of RNA polymerase initiation). Wherein a regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer) is "operatively linked," to a coding region, the coding Atty. Dkt. No.: 114198-2110 region is "under transcriptional control" of the regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer). The term "operably linked" means that a nucleic acid sequence and a regulatory sequence(s) are arranged in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). The term "operably inserted" means that a nucleic acid sequence of interest is positioned adjacent a regulatory nucleic acid sequence which directs transcription and translation of the nucleic acid sequence of interest (i.e., facilitates the production of, e.g., a polypeptide encoded by a DNA of interest). As used herein, the term "RNA" relates to a nucleic acid molecule that comprises ribonucleotide residues. In some aspects, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a b-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non- nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA. The term "mRNA," as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more peptide (e.g., oligopeptide, or polypeptide) or protein. The term "mRNA," as used herein includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. An mRNA molecule may also contain a 5' untranslated region (5'-UTR), and / or a 3' untranslated region (3'-UTR). In some aspects, the RNA is produced by in vitro transcription or chemical synthesis. In some aspects, the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides. Atty. Dkt. No.: 114198-2110 As used herein the term "splicing" refers to the process by which introns are removed from primary transcripts (pre-mRNA) and exons are joined to form the mature mRNA. Introns are removed by the pre-mRNA by cleavage at conserved sequences called "splice sites", or "splicing sites". These sites are located at the 5′ and 3′ ends of introns. Most commonly, the RNA sequence that is removed begins with the dinucleotide GU at its 5′ end, and ends with AG at its 3′ end. These consensus sequences are known to be critical, because changing one of the conserved nucleotides may result in the inhibition of splicing. Another important sequence occurs at what is called the branch point, located anywhere from 18 to 40 nucleotides upstream from the 3′ end of an intron. The branch point always contains an adenine, but it is otherwise loosely conserved. A typical sequence is YNYYRAY, where Y indicates a pyrimidine, N denotes any nucleotide, R denotes any purine, and A denotes adenine. Rarely, splice site sequences are found that begin with the dinucleotide AU and end with AC, these are spliced through a similar mechanism. Splicing occurs in several steps and is catalyzed by small nuclear ribonucleoproteins (snRNPs, commonly pronounced "snurps"). First, the pre-mRNA is cleaved at the 5′ end of the intron following the attachment of a snRNP called U1 to its complementary sequence within the intron. The cut end then attaches to the conserved branch point region downstream through pairing of guanine and adenine nucleotides from the 5′ end and the branch point, respectively, to form a looped structure known as a lariat. The bonding of the guanine and adenine bases takes place via a chemical reaction known as transesterification, in which a hydroxyl (OH) group on a carbon atom of the adenine attacks the bond of the guanine nucleotide at the splice site. The guanine residue is thus cleaved from the RNA strand and forms a new bond with the adenine. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked Atty. Dkt. No.: 114198-2110 coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may comprise specific functional sequences needed for insertion and / or expression of the desired DNA fragments. A "vector" can be any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. In some aspects, insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that may or not have complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes described in the literature include: genes providing resistance to neomycin, ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters described in the literature include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters. As used herein, the term "adeno-associated vector" or "AAV vector" refers to a vector comprising one or more polynucleotides of interest (e.g., transgenes, such as micro- dystrophin) that are flanked by AAV terminal repeat sequences (ITRs). AAV is a single- stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York); Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1- 61 (1974)). Such AAV vectors can be replicated and packaged into infectious viral particles Atty. Dkt. No.: 114198-2110 when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. "AAV Cap" means AAV Cap proteins, VP1, VP2, and VP3 and analogs thereof. "AAV Rep" means AAV Rep proteins and analogs thereof. "Serotype," with respect to a vector or virus capsid, is defined by a distinct immunological profile based on the capsid protein sequences and capsid structure. Non-limiting examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6P1, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12, and variants thereof. As used herein, "flanked," with respect to a sequence that is flanked by other elements, indicates the presence of one or more the elements upstream and / or downstream, i.e., 5′ and / or 3′, relative to the sequence. The term "flanked" is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element. A sequence (e.g., a transgene) that is "flanked" by two other elements (e.g., ITRs), indicates that one element is located 5′ to the sequence and the other is located 3′ to the sequence; however, there may be intervening sequences between. As used herein, the terms "AAV virion," "AAV viral particle," or "AAV particle" refer to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. The particle can comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell). An AAV particle comprising a heterologous polynucleotide can be also referred to as a "recombinant AAV particle," "recombinant AAV vector," "rAAV particle," or "rAAV vector." As used herein, the term "transfection" of a cell refers to the introduction of genetic material into a cell by means of a non-viral vector for the purpose of genetically modifying the cell. As used herein, the term "transduction" of a cell refers to the introduction of genetic material into a cell by means of a viral vector for the purpose of genetically modifying the cell. For example, the coding region of a gene or of a portion thereof (e.g., a micro- dystrophin) can be administrated / delivered to a recipient cell either in vivo or in vitro, via an Atty. Dkt. No.: 114198-2110 AAV particle resulting in the expression of the gene or portion thereof (e.g., a micro- dystrophin) in the recipient cell. As used herein, the term "cell" or "cells" or “host cell or cells” refers not only to the particular subject cell, but also to the progeny or to the potential progeny of such cell(s). The scope of the term as used herein also encompasses the progeny that may or may not in fact be identical to the parent cell because certain modifications may occur in succeeding generations due to either mutation or environmental influences. “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The host cell can be a prokaryotic or a eukaryotic cell. In some embodiments, the host cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293T cell, or an a549 cell. “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, canine, bovine, porcine, murine, rat, avian, reptilian and human. Nonlimiting examples include packaging cells lines or retinal cells or RPE cells, e.g., WERI-RB (available from ATCC HTB-169). “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells’ genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2µm in diameter and 10 µm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, Atty. Dkt. No.: 114198-2110 bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coli bacterium, and Salmonella bacterium. The term “genetic marker” refers to an allelic variant of a polymorphic region of a gene of interest and / or the expression level of a gene of interest. The term “wild-type allele” refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes. The term “polymorphism” refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a “polymorphic region of a gene.” A polymorphic region can be a single nucleotide, the identity of which differs in different alleles. A “polymorphic gene” refers to a gene having at least one polymorphic region. The term “genotype” refers to the specific allelic composition of an entire cell or a certain gene and in some aspects a specific polymorphism associated with that gene, whereas the term “phenotype” refers to the detectable outward manifestations of a specific genotype. The phrase “amplification of polynucleotides” includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR) and amplification methods. These methods are known and widely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu, D.Y. et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified. Atty. Dkt. No.: 114198-2110 Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from a particular gene region are preferably complementary to, and hybridize specifically to sequences in the target region or in its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively, the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments is known in the art. The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. The term “isolated” as used herein refers to molecules or biological or cellular materials being substantially free from other materials. In one aspect, the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues. The term “suitable for a therapy” or “suitably treated with a therapy” shall mean that the patient is likely to exhibit one or more desirable clinical outcome as compared to patients having the same disease and receiving the same therapy but possessing a different Atty. Dkt. No.: 114198-2110 characteristic that is under consideration for the purpose of the comparison. In one aspect, the characteristic under consideration is a genetic polymorphism or a somatic mutation. In another aspect, the characteristic under consideration is expression level of a gene or a polypeptide. The term “blood” refers to blood which includes all components of blood circulating in a subject including, but not limited to, red blood cells, white blood cells, plasma, clotting factors, small proteins, platelets and / or cryoprecipitate. This is typically the type of blood which is donated when a human patent gives blood. As used herein, a “biological pathway” refers to (1) a bespoke set of genes for which their protein products are known to interact in a biological pathway (e.g., complement pathway, JAK / STAT pathway, MAPK pathway, etc.), or (2) an unsupervised learning approach (e.g., principal component analysis (PCA)) that yields patterns in the data such that clusters of genes comprising a pathway may be identified. In some embodiments, approach (1) is based on canonical pathways identified by literature and external pathway databases, while approach (2) is a data-driven analysis that results in identification of pathways. As used herein, “biological pathway activity” is defined by information form the literature or external databases that provide evidence for gene or protein expression indicative of pathway activity (e.g., ‘up regulation’ or ‘down regulation’ of pathway X in disease Y). As used herein, a “drug target expression” refers to a protein expression as measured in participants of a large cohort (e.g., UK Biobank), where the protein analyzed is a known drug target or is a target that may be druggable (even if not already drugged). In some embodiments, a biomarker stratifier score (e.g., a polygenic score, a proteomics score, a transcriptomics score, or a polymorphism score) for “a drug target expression” is computed same as it would be for any other quantitative trait, where the outcome of the model is a quantitative measurement. As used herein, the phrase “genetic variant” refers to an alteration, mutation, variant or polymorphism in a nucleic acid sample or genome of a subject. Such alteration, variant or polymorphism can be with respect to a reference genome, which may be a reference genome of the species (e.g., for human, hGl9 or hG38), the subject or other individual. Variations Atty. Dkt. No.: 114198-2110 include one or more single nucleotide variations (SNVs), insertions, deletions, repeats, small insertions, small deletions, small repeats, structural variant junctions, variable length tandem repeats, and / or flanking sequences, copy number variants (CNVs), transversions, gene fusions and other rearrangements are also forms of genetic variation. A variation can be a single nucleotide variation (SNV), insertion or deletion (indel), repeat, copy number variation (CNV), transversion, or combination thereof. A sample intends a biological sample isolated from the subject and includes blood, plasma, solid tissue and can be preserved (frozen or fixed) a primary sample just isolated from the subject. The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical Atty. Dkt. No.: 114198-2110 representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. The term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins. As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. As used herein, the term “overexpress” intends a level of expression of the mRNA, the protein or the polypeptide” that is greater than or exceeds the level of expression of the mRNA, the protein or the polypeptide in a native, wild-type or cell that has not been engineered to increase expression. A “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated. In some embodiments, the gene product may refer to an mRNA or other RNA, such as an interfering RNA, generated when a gene is transcribed. The term “a regulatory sequence”, “an expression control element” or “promoter” as used herein, intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed or replicated, and facilitates the expression or replication of the target polynucleotide. A promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. Polymerase II and III are examples of promoters. In some embodiments, a regulatory sequence is bidirectional, i.e., acting as a regulatory sequence for the coding sequences on both sides of the regulatory sequence. Such bidirectional regulatory sequence may comprise, or consists essentially of, or consists of a Atty. Dkt. No.: 114198-2110 bidirectional promoter (see for example Trinklein ND, et al. (2004) An abundance of bidirectional promoters in the human genome. Genome Res. Jan;14(1):62-6). The term “protein,” “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. As used herein, the term “administer” or “administration” or “administering” intends to mean delivery of a substance to a subject such as an animal or human. Administration can be accomplished in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, as well as the age, health or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and animals, treating veterinarian. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and the target cell or tissue. Non-limiting examples of route of administration include intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation. In one aspect, the agent or vector is administered by intraocular injection. Atty. Dkt. No.: 114198-2110 As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that corresponds to "inverted terminal repeat sequences" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. An “AAV vector” as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. An “AAV virion,” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector.” Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle. Atty. Dkt. No.: 114198-2110 Multiple AAV serotypes have retinal tropism, including AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, and AAV10. See Pupo et al. 2022, doi: 10.1016 / j.ymthe.2022.09.015. In one aspect, the AAV serotype is or comprises AAV8. Additional considerations in the development of vectors can be found, for example, in Pupo A, Fernández A, Low SH, François A, Suárez-Amarán L, Samulski RJ. AAV vectors: The Rubik's cube of human gene therapy. Mol Ther. 2022 Dec 7;30(12):3515-3541. doi: 10.1016 / j.ymthe.2022.09.015. Epub 2022 Oct 5. PMID: 36203359; PMCID: PMC9734031, the contents of which are incorporated herein. A “composition” typically intends a combination of the active agent, and a naturally- occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g. sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, derivatized sugars (alditols, aldonic acids, esterified sugars), and polysaccharides, can be present alone or in combination, making up 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. As used herein, the terms "pharmaceutical formulation" or "pharmaceutical composition" refer to an admixture comprising an effective amount of a therapeutically Atty. Dkt. No.: 114198-2110 and / or prophylactic effective agent and at least one pharmaceutically acceptable excipient (e.g., carrier, diluent, stabilizer, or any combination thereof) or adjuvant. Examples of pharmaceutically acceptable excipients are, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, colorants, amino acids, stabilizers, bulking agents, surfactants, antimicrobials, preservatives, metal ions, chelators, cyclodextrin-based excipients, polyanions, polycations, salts, solubilizers, detergents, compatible solid or liquid fillers, encapsulating substances, or any combination thereof, which are suitable for administration to a subject. Specific examples of excipient include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers, or any combination thereof. Examples of pharmaceutically acceptable carriers are, but are not limited to, lipids, polymers, polysaccharides, peptides, proteins, lipidoids, and any combination thereof. In some aspects, the lipid is selected from the group consisting of: cationic lipids, non-cationic lipids, steroid lipids, ionizable lipids, PEG-conjugated lipids, and any combination thereof. In some aspects, the fusion proteins, the expression cassettes, and the vectors disclosed herein are complexed, or packaged in a liposome, a nanoliposome, a lipid nanoparticle, a lipoplex, a micell, a nanomicell, a nanoemulsion, an oil-in-water emulsions, a PEG-conjugated lipid nanoparticle, a polymeric nanoparticle, a lipid-polymer hybrid nanoparticle, a polysaccharidic nanocarrier, an RNA / DNA-peptide nanoparticle, an RNA / DNA -peptide nanocomplex, a biomimetic nanovesicle, a lipidoid-RNA / DNA complex, a virus-like particle, dendrimer nanoparticle, a nanogel, a metallic nanoparticle, a gold nanoparticle (AuPNs), a magnetic nanoparticle, a theranostic nanoparticle, or any combination thereof. In some aspects, the combination of the fusion proteins, the expression cassettes, and the vectors disclosed herein with one or more of the carriers described herein facilitates, enhances or enables administration of the fusion proteins, the expression cassettes, and the vectors disclosed herein to the subject. In some aspects, the combination of a therapeutic agent with one or more of the carriers described herein facilitates, enhances or enables the delivery of the therapeutic agent to a target cell. Atty. Dkt. No.: 114198-2110 Pharmaceutically acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Pharmaceutical excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice. As used herein, the terms "treat," "treated," and "treating" mean both therapeutic and prophylactic treatment or preventative measures wherein the object is to reverse, alleviate, ameliorate, lessen, inhibit, slow down progression, development, severity or recurrence of an undesired symptom, complication, condition, biochemical indicia of a disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. In some aspects, treatment includes eliciting a clinically significant response without excessive levels of side effects. In some aspects, treatment includes prolonging survival as compared to expected survival if not receiving treatment. As used herein, the term "amelioration" or "ameliorating" refers to a lessening of severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or forestalling the onset, development or progression of a condition or disease for a period of time, including weeks, months, or years. As used herein, the term "prophylactic" (e.g., "prophylactic agent", "prophylactic treatment", "prophylactically effective amount"), refers to any complete or partial prevention of a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect and / or symptom attributable to the disease. As used herein, the term "gene therapy" refers to the administration into an individual's cells and / or tissues of an exogenous molecule (e.g., a nucleic acid sequence (e.g., a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a gene product capable of interfering with the genomic sequence of the individual's cells and / or Atty. Dkt. No.: 114198-2110 tissues)) to treat, reduce the symptoms of, or reduce the likelihood of a disease, disorder, syndrome, or condition. An exogenous molecule or sequence is understood to be molecule or sequence not normally occurring in the cell, tissue and / or individual to be treated. Both acquired and congenital diseases are amenable to gene therapy. As used herein, the term "subject" refers to any organism to which a composition or a substance (e.g., a nucleotide molecule) can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal. A subject can seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be under care by a trained professional for a particular disease or condition. As used herein, the term "expression cassette" refers to a nucleic acid molecule, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleotide sequence in a competent host cell, such that a particular gene product (e.g., RNA or protein) is expressed. Expression of any gene product may be dependent upon presence of cellular factors or additional gene products from other expression cassettes. An expression cassette may be part of a vector, such as a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. As used herein, the terms "genetic construct" or "construct" refer to a nucleic acid molecule (RNA, DNA, or a combination thereof) made recombinanlty or syntheiticallycomprising one or more nucleotide sequences, such as a nucleotide sequence encoding (i.e., a coding sequence) a particular gene product (e.g., RNA or protein), and comprising initiation and termination signals; a regulatory element, such as a promoter; and / or a polyadenylation signal. The one or more nucleotide sequences comprised in a genetic construct can be operably linked, such that they are capable of directing expression of the coding sequence in a cell, such as a cell maintained in culture (in vitro), or a cell comprised in an individual (in vivo), to which the genetic construct is administered. A genetic construct can be, for example, an expression cassette, or a vector, e.g., a vector comprising an expression cassette. Atty. Dkt. No.: 114198-2110 As used herein, the terms “percent identity” and / or “percent identical,” as applicable to a particular nucleotide or amino acid sequence, refer to the proportion of identical residues between this particular reference sequence and another sequence, as calculated by a pairwise alignment using the Needleman-Wunsch algorithm using a generally available alignment program, e.g., the Needle (EMBOSS) program. The compositions used in accordance with the disclosure, including cells, treatments, therapies, agents, drugs and pharmaceutical formulations can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein. Modes for Carrying out the Disclosure Diagnostic Methods In some aspects, the disclosure provides a method for diagnosing or prognosing a TMEM216-related disorder in a subject, the method comprising, or consisting essentially of, or consisting of detecting a mutation at the genomic location GRCh38: chr11:g.61392563G, in the 5’ UTR region, or upstream of the start codon of the ciliopathy gene TMEM216 at a location from about -1000 to about -1, wherein a mutation at the location from about -1000 to about -1 is a positive diagnosis or prognosis for the TMEM216-related disorder in the subject. Alternatively, the region in the 5’UTR for diagnosis or prognosis is at a location Atty. Dkt. No.: 114198-2110 from about 1000 to about 1 base pair upstream of the start codon of the ciliopathy gene TMEM216, e.g., or alternatively at a location from about -1000 to about -1, or alternatively from about -1000 to about -30, or from about -1000 to about -35, or from about -750 to about -1, or from about -750 to about -35, or alternatively from about -750 to about -30, or from about -750 to about -35, or from about -500 to about -1, or from about -500 to about -35, or alternatively from about -500 to about -30, or from about -500 to about -35, or alternatively from about -500 to about -30, or from about -250 to about -1, or from about -250 to about - 35, or alternatively from about -250 to about -30, or from about -100 to about -1, or from about -150 to about -10, or alternatively from about -100 to about -20, or from about -100 to about -35, or from about -100 to about -40, or alternatively from about -75 to about -50, or about -70 to about -35, and ranges in between. Non-limiting examples of such mutations are at a genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents of SEQ ID NO: 8. In some aspects, the disclosure provides the above method, wherein the mutation is at the genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8. Alternatively, the mutation is at the genomic location selected from c.-69G>A, c.-69G>T, c.- 69G>C, or c.-95G>C; or selected from c.-69G>A, c.-69G>T, c.-69G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof of SEQ ID NO: 8.; or the mutation is selected from the genomic location selected from c.-69G>A, c.-69G>T, or c.-69G>C, c.- 95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8.; or the mutation is selected from the genomic location selected from the genomic location selected from c.-95G.C of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8. In one aspect, the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2. In some aspects, the disclosure provides a method, wherein the mutation is at the genomic location selected from c.-95G>C or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8. In one aspect, the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2. Atty. Dkt. No.: 114198-2110 In one aspect the subject being diagnosed is an animal, e.g. a mammal such as a human patient. It is to be understood that information obtained using the diagnostic and prognostic assays described herein can be used alone or in combination with other information, such as, but not limited to, genotypes or expression levels of other genes, clinical chemical parameters, histopathological parameters, or age, gender and weight of the subject. When used alone, the information obtained using the diagnostic assays described herein is useful in determining or identifying the clinical outcome of a treatment, selecting a patient for a treatment, or treating a patient, etc. When used in combination with other information, on the other hand, the information obtained using the assays described herein is useful in aiding in the determination or identification of clinical outcome of a treatment, aiding in the selection of a patient for a treatment, or aiding in the treatment of a patient and etc. In a particular aspect, the genotypes or expression levels of one or more genes as disclosed herein are used in a panel of genes, each of which contributes to the final diagnosis, prognosis or treatment. The methods are useful in the assistance of an animal, a mammal or yet further a human patient. For the purpose of illustration only, a mammal includes but is not limited to a human, a simian, a murine, a bovine, an equine, a porcine or an ovine subject. The disclosure further provides diagnostic, prognostic and therapeutic methods, which are based, at least in part, on determination of the identify of a genotype of interest identified herein. For example, information obtained using the diagnostic assays described herein is useful for determining if a subject is suitable for treatment of a given type. Based on the prognostic information, a doctor can recommend a therapeutic protocol such as the therapy described herein, useful for disease symptoms or disease progression. The methods are useful in the assistance of an animal, a mammal or yet further a human patient. For the purpose of illustration only, a mammal includes but is not limited to a human, a simian, a murine, a bovine, an equine, a porcine or an ovine subject. Biological Sample Collection and Preparation Atty. Dkt. No.: 114198-2110 The methods and compositions disclosed herein can be used to detect nucleic acids using a biological sample obtained from a patient. Biological samples can be obtained by standard procedures and can be used immediately or stored, under conditions appropriate for the type of biological sample, for later use. Any liquid or solid biological material obtained from the patient believed to contain nucleic acids comprising the region containing the genetic region of interest can be a suitable sample. Methods of obtaining test samples are known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, swabs, drawing of blood or other fluids, surgical or needle biopsies. In some aspects, the biological sample is a tissue or a cell sample. Suitable patient samples in the methods include, but are not limited to, blood, plasma, serum, a biopsy tissue, fine needle biopsy sample, amniotic fluid, plasma, pleural fluid, saliva, semen, serum, tissue or tissue homogenates, frozen or paraffin sections of tissue or combinations thereof. In some aspects, the biological sample is an original sample recently isolated from the patient, a fixed tissue, a frozen tissue, a resection tissue, or a microdissected tissue. In some aspects, the biological samples are processed, such as by sectioning of tissues, fractionation, purification, nucleic acid isolation, or cellular organelle separation. In some embodiments, nucleic acid (DNA or RNA) is isolated from the sample according to any methods known to those of skill in the art. In some aspects, genomic DNA is isolated from the biological sample. In some aspects, RNA is isolated from the biological sample. In some aspects, cDNA is generated from mRNA in the sample. In some embodiments, the nucleic acid is not isolated from the biological sample. In some embodiments, the amplification includes a labeled primer or probe, thereby allowing detection of the amplification products corresponding to that primer or probe. In particular embodiments, the amplification can include a multiplicity of labeled primers or probes; such primers can be distinguishably labeled, allowing the simultaneous detection of multiple amplification products. Atty. Dkt. No.: 114198-2110 In some embodiments, the amplification products are detected by any of a number of methods such as gel electrophoresis, column chromatography, hybridization with a nucleic acid probe, or sequencing the amplicon. Detectable labels can be used to identify the primer or probe hybridized to a genomic nucleic acid or amplicon. Detectable labels include but are not limited to fluorophores, isotopes (e.g.,32P,33P, .35S,3H,14C,125I,131I) electron-dense reagents (e.g., gold, silver), nanoparticles, enzymes commonly used in an ELISA (e.g., horseradish peroxidase, beta- galactosidase, luciferase, alkaline phosphatase), chemiluminiscent compounds, colorimetric labels (e.g., colloidal gold), magnetic labels (e.g., Dynabeads®), biotin, digoxigenin, haptens, proteins for which antisera or monoclonal antibodies are available, ligands, hormones, oligonucleotides capable of forming a complex with the corresponding oligonucleotide complement. In one embodiment, a primer or probe is labeled with a fluorophore that emits a detectable signal. The term "fluorophore" as used herein refers to a molecule that absorbs light at a particular wavelength (excitation frequency) and subsequently emits light of a longer wavelength (emission frequency). While a suitable reporter dye is a fluorescent dye, any reporter dye that can be attached to a detection reagent such as an oligonucleotide probe or primer is suitable for use in the methods described. Suitable fluorescent moieties include, but are not limited to, the following fluorophores working individually or in combination: 4- acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives, e,g, acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino- N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino- 1-naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5''- dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- Atty. Dkt. No.: 114198-2110 isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6- dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6- carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6- carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescein (TET); fluorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; and tetramethyl rhodamine isothiocyanate (TRITC). In some aspects, the primer or probe is further labeled with a quencher dye such as Tamra, Dabcyl, or Black Hole Quencher®(BHQ), especially when the reagent is used as a self-quenching probe such as a TaqMan®(U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemless or linear beacon probe (Livak et al., 1995, PCR Method Appl., 4:357-362; Tyagi et al, 1996, Nature Biotechnology, 14:303-308; Nazarenko et al., 1997, Nucl. Acids Res., 25:2516-2521; U.S. Pat. Nos. 5,866,336 and 6,117,635). Atty. Dkt. No.: 114198-2110 In some aspects, methods for real time PCR use fluorescent primers / probes, such as the TaqMan® primers / probes (Heid, et al., Genome Res 6: 986-994, 1996), molecular beacons, and Scorpion™ primers / probes. Real-time PCR quantifies the initial amount of the template with more specificity, sensitivity and reproducibility, than other forms of quantitative PCR, which detect the amount of final amplified product. Real-time PCR does not detect the size of the amplicon. The probes employed in Scorpion®™ and TaqMan® technologies are based on the principle of fluorescence quenching and involve a donor fluorophore and a quenching moiety. The term "donor fluorophore" as used herein means a fluorophore that, when in close proximity to a quencher moiety, donates or transfers emission energy to the quencher. As a result of donating energy to the quencher moiety, the donor fluorophore will itself emit less light at a particular emission frequency that it would have in the absence of a closely positioned quencher moiety. The term "quencher moiety" as used herein means a molecule that, in close proximity to a donor fluorophore, takes up emission energy generated by the donor and either dissipates the energy as heat or emits light of a longer wavelength than the emission wavelength of the donor. In the latter case, the quencher is considered to be an acceptor fluorophore. The quenching moiety can act via proximal (i.e., collisional) quenching or by Forster or fluorescence resonance energy transfer ("FRET"). Quenching by FRET is generally used in TaqMan® primers / probes while proximal quenching is used in molecular beacon and Scorpion™ type primers / probes. The detectable label can be incorporated into, associated with or conjugated to a nucleic acid primer or probe. Labels can be attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties. See, e.g., Mansfield, Mol. Cell. Probes (1995), 9:145-156. Detectable labels can be incorporated into nucleic acid probes by covalent or non- covalent means, e.g., by transcription, such as by random-primer labeling using Klenow polymerase, or nick translation, or, amplification, or equivalent as is known in the art. For example, a nucleotide base is conjugated to a detectable moiety, such as a fluorescent dye, e.g., Cy3™ or Cy5™ and then incorporated into nucleic acid probes during nucleic acid synthesis or amplification. Nucleic acid probes can thereby be labeled when synthesized using Cy3™- or Cy5™-dCTP conjugates mixed with unlabeled dCTP. Atty. Dkt. No.: 114198-2110 Nucleic acid probes can be labeled by using PCR or nick translation in the presence of labeled precursor nucleotides, for example, modified nucleotides synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptens (such as biotin or digoxigenin) can be used; this method allows custom preparation of most common fluorescent nucleotides, see, e.g., Henegariu et al., Nat. Biotechnol. (2000), 18:345- 348. Nucleic acid probes can be labeled by non-covalent means known in the art. For example, Kreatech Biotechnology's Universal Linkage System® (ULS®) provides a non- enzymatic labeling technology, wherein a platinum group forms a co-ordinative bond with DNA, RNA or nucleotides by binding to the N7 position of guanosine. This technology can also be used to label proteins by binding to nitrogen and sulfur containing side chains of amino acids. See, e.g., U.S. Pat. Nos. 5,580,990; 5,714,327; and 5,985,566; and European Patent No. 0539466. Labeling with a detectable label also can include a nucleic acid attached to another biological molecule, such as a nucleic acid, e.g., an oligonucleotide, or a nucleic acid in the form of a stem-loop structure as a "molecular beacon" or an "aptamer beacon". Molecular beacons as detectable moieties are described; for example, Sokol (Proc. Natl. Acad. Sci. USA (1998), 95:11538-11543) synthesized "molecular beacon" reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends. In the absence of a complementary nucleic acid strand, the molecular beacon remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. See also, e.g., Antony (Biochemistry (2001), 40:9387-9395), describing a molecular beacon consist of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. See also U.S. Pat. Nos. 6,277,581 and 6,235,504. Aptamer beacons are similar to molecular beacons; see, e.g., Hamaguchi, Anal. Biochem. (2001), 294:126-131; Poddar, Mol. Cell. Probes (2001), 15:161-167; Kaboev, Atty. Dkt. No.: 114198-2110 Nucleic Acids Res. (2000), 28:E94. Aptamer beacons can adopt two or more conformations, one of which allows ligand binding. A fluorescence-quenching pair is used to report changes in conformation induced by ligand binding. See also, e.g., Yamamoto et al., Genes Cells (2000), 5:389-396; Smimov et al., Biochemistry (2000), 39:1462-1468. The nucleic acid primer or probe can be indirectly detectably labeled via a peptide. A peptide can be made detectable by incorporating predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, transcriptional activator polypeptide, metal binding domains, epitope tags). A label can also be attached via a second peptide that interacts with the first peptide (e.g., S--S association). As readily recognized by one of skill in the art, detection of the complex containing the nucleic acid from a sample hybridized to a labeled probe can be achieved through use of a labeled antibody against the label of the probe. In one example, the probe is labeled with digoxigenin and is detected with a fluorescent labeled anti-digoxigenin antibody. In another example, the probe is labeled with FITC, and detected with fluorescent labeled anti-FITC antibody. These antibodies are readily available commercially. In another example, the probe is labeled with FITC, and detected with anti-FITC antibody primary antibody and a labeled anti-anti FITC secondary antibody. Nucleic acids can be amplified prior to detection or can be detected directly during an amplification step (i.e., "real-time" methods, such as in TaqMan® and Scorpion™ methods). In some embodiments, the target sequence is amplified using a labeled primer such that the resulting amplicon is detectably labeled. In some embodiments, the primer is fluorescently labeled. In some embodiments, the target sequence is amplified and the resulting amplicon is detected by electrophoresis. With regard to the exemplary primers and probes, those skilled in the art will readily recognize that nucleic acid molecules can be double-stranded molecules and that reference to a particular site on one strand refers, as well, to the corresponding site on a complementary strand. In defining a variant position, allele, or nucleotide sequence, reference to an adenine, a thymine (uridine), a cytosine, or a guanine at a particular site on one strand of a nucleic acid Atty. Dkt. No.: 114198-2110 molecule also defines the thymine (uridine), adenine, guanine, or cytosine (respectively) at the corresponding site on a complementary strand of the nucleic acid molecule. Thus, reference can be made to either strand in order to refer to a particular variant position, allele, or nucleotide sequence. Probes and primers, can be designed to hybridize to either strand and detection methods disclosed herein can generally target either strand. Methods of Treatment In some aspects, disclosed herein are polynucleotides encoding such one or more therapeutic polypeptides; vectors comprising such polynucleotides (e.g., AAV vectors comprising such expression cassettes); methods of making those vectors; recombinant AAV (rAAV) particles comprising such vectors; pharmaceutical compositions comprising the polynucleotides, the vectors, and / or the rAAV particles disclosed herein; and methods of using the polynucleotides, the vectors, the rAAV particles, and / or the pharmaceutical compositions disclosed herein. The polynucleotides can further comprise regulatory regions necessary for replication and / or expression in vitro or in vivo, e.g., promoters, polyA tails and enhancer elements. These compositions are useful for making and delivering the therapeutic TMEM216 polypeptides and fragments for use in the disclosed methods. In some respects, the disclosure provides a method for treating, preventing or delaying the progression of a TMEM216-related disorder in a subject in need thereof, the method comprising, or consisting essentially of, or consisting of administering to the subject an effective amount of at least one gene that supports or restores functional TMEM216 in the patient. Non-limiting examples of TMEM216-related disorder are retinitis pigmentosa (RP) and Joubert syndrome 2. The at least one gene can be administered in a gene delivery vehicle, e.g. a vector or plasmid as described herein, that are optionally linked to one or more regulatory elements, e.g., a promoter and / or enhancer element. In some aspects, the subject in need of treatment harbors a mutation at the genomic location GRCh38: chr11:g.61392563G, in the 5’UTR or alternatively at a location from about 1000 to 1 base pairs upstream of the start codon of the ciliopathy gene TMEM216, e.g., alternatively at a location from about -1000 to about -1, or alternatively from about -1000 to about -30, or from about -1000 to about -35, or from about -750 to about -1, or from about - Atty. Dkt. No.: 114198-2110 750 to about -35, or alternatively from about -750 to about -30, or from about -750 to about - 35, or from about -500 to about -1, or from about -500 to about -35, or alternatively from about -500 to about -30, or from about -500 to about -35, or alternatively from about -500 to about -30, or from about -250 to about -1, or from about -250 to about -35, or alternatively from about -250 to about -30, or from about -100 to about -1, or from about -150 to about - 10, or alternatively from about -100 to about -20, or from about -100 to about -35, or from about -100 to about -40, or alternatively from about -75 to about -50, or from about -70 to about -35, and ranges in between. Non-limiting examples of such mutations are at a genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents of SEQ ID NO: 8. In some aspects, the disclosure provides the above treatment method, wherein the mutation is at the genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.- 95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8. Alternatively, the mutation is at the genomic location selected from c.-69G>A, c.- 69G>T, c.-69G>C, or c.-95G>C; or selected from c.-69G>A, c.-69G>T, c.-69G>C, or c.- 41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof of SEQ ID NO: 8.; or the mutation is selected from the genomic location selected from c.-69G>A, c.-69G>T, or c.- 69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8.; or the mutation is selected from the genomic location selected from the genomic location selected from c.-95G.C of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8. In one aspect, the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2. In some aspects, the disclosure provides a treatment method, wherein the mutation is at the genomic location selected from c.-95G>C or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof or SEQ ID NO: 8. In one aspect, the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2. In one aspect the subject being diagnosed is an animal, e.g. a mammal such as a human patient. Atty. Dkt. No.: 114198-2110 The method comprises administering to the subject in need thereof an effective amount of the wildtype TMEM216 gene, or a functional fragment thereof, examples of such are provided herein. In one aspect the TMEM216 gene is the long form. In another aspect the TMEM216 gene is the short form of the gene. Examples of such are provided herein. To perform the therapy, the at least one gene is administered in construct designated TMEM216_WT herein, or an equivalent thereof. In some aspects, the at least one gene is TMEM216 as designated herein or a functional equivalent thereof. Alternative embodiments include the TMEM216 isoform, optionally the long or the short isoform. In some aspects, an effective amount of the gene is administered in a gene delivery vehicle, e.g., a viral particle, wherein the viral particle is or is optionally derived from a herpes simplex virus, a retrovirus, an adenovirus, or an adeno-associated virus. For example, the effective amount of the gene is administered in an adeno-associated viral (AAV) particle or a derivative or variant thereof. Non-limiting examples include an AAV is selected from an AAV serotype selected from the group of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, or AAV-11. In one particular aspect, the AAV is AAV-8. Administration can be by any appropriate route, as known in the art or as described herein. Non-limiting examples include a route such as intravitreal injection, sub-retinal injection or sub-RPE injection. In another aspect, the one or more gene is administered in a pharmaceutical composition, and is administered in a dose that is selected by the treating physician. One or more doses can be administered as determined by the treating physician. Also provided herein are nucleic acids for use in the above methods. One example is the TMEM216_Native promoter-Short Isoform-AAV genome as shown in 7, optionally without the HA tag or a marker sequence. Another embodiment includes a nucleic acid including the TMEM216_Native promoter-Long Isoform-AAV genome shown in 7, optionally without the HA tag, a marker sequence, e.g., the AmpR gene and promoter. In a further aspect, a nucleic acid including the TMEM216_Short Isoform-AAV genome as shown in 7, optionally without the HA tag or marker sequence is provided. Atty. Dkt. No.: 114198-2110 In some aspects, the nucleic acids disclosed herein further comprise additional nucleotide sequences. In some aspects, the nucleic acids disclosed herein can be expression cassettes encoding the TMEM216 polynucleotide. In some aspects, the nucleic acids disclosed herein further comprise a promoter, a Kozak sequence, an enhancer, a silencer, a polyA tail, a polyadenylation signal, a 3'-UTR, a 5'-UTR, an intronic sequence, a nucleotide sequence encoding a molecular tag, a nucleotide sequence encoding a selectable marker, a linker, or any other nucleotide sequence that may facilitate the expression of the nucleotide sequences encoding the polypeptides disclosed herein. Any further nucleotide sequences present in the polynucleotide are in the appropriate position required to exert its function. For example, the nucleotide sequence of a promoter is operably linked to the nucleotide sequence encoding the polypeptides disclosed herein. In some aspects, the nucleic acid of the disclosure further comprises: a promoter, e.g., the TMEM216 wildtype promoter or the CAG promoter; a nucleotide sequence encoding a TMEM216 gene or functional fragment thereof, and optionally a polyadenylation signal, e.g., an SV40 polyadenylation signal. In further aspects, the nucleic acid of the disclosure comprises a first ITR at the 5’ of the polynucleotide and a second ITR at the 3’ of the polynucleotide. Examples of such are shown in 7. In one aspect, the AmpR and AMP promoter elements or polynucleotides and optionally the HA tag are excluded from the nucleic acids as shown in 7. The promoter for the nucleic acids can be a constitutive, a regulatable, an inducible, an ubiquitous, or a tissue-specific promoter. In some aspects, the promoter is a constitutive promoter. Non-limiting example of constitutive promoters are a CBA promoter, a CMV promoter, an EF1α promoter, or a CAG promoter. In some aspects, the promoter is a tissue-specific promoter. In some aspects, the promoter drives expression of the therapeutic protein in a cell of the eye, e.g., an RPE. Inverted Terminal Repeats (ITRs) Atty. Dkt. No.: 114198-2110 In some aspects, the nucleic acids further comprise a nucleotide sequence encoding a first inverted terminal repeat (ITR) and a second ITR. In some aspects, the first inverted terminal repeat (ITR) and the second ITR are derived from the same serotype. In some aspects, the first inverted terminal repeat (ITR) and the second ITR are derived from different serotypes. In some aspects, the serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 serotype. In some aspects, the serotype is AAV8 serotype. In some aspects, a nucleic acid including the TMEM216_Long Isoform-AAV genome as shown in 7 is provided, optionally without the HA tag, antibiotic resistance gene, or marker sequence. The nucleic acid can be DNA, RNA or mixtures thereof. They may be included with an expression or delivery vector, such as for example a plasmid or an AAV vector, e.g., an AAV8 vector. The nucleic acids and / or vectors can also be combined with a carrier, such as a pharmaceutically acceptable carrier. In one aspect, the short and long form of the TMEM216 gene is delivered or provided in the same composition, and delivered as a form of gene therapy, as described herein. Alternatively, the short and long form are separately delivered sequentially or concurrently. In a further aspect, provided herein is a cell, e.g., an isolated eukaryotic or prokaryotic cell or host cell comprising the one or more nucleic acids or vectors as described herein for use in replication and packaging of the nucleic acid molecules into viral particles, e.g., AAV particles. In one aspect, the cells are packaging cell lines for the incorporation of the nucleic acid into an AAV capsid particle. Non-limiting packaging cell lines include for example, HEK 293 cells, MRC-5 cells, WI-38 cells, Vero cells, or FrhL-2 cells. Thus, also provided is an AAV particle comprising the nucleic acid and / or vector, wherein the AAV is a serotype selected from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 or AAV-12. In one aspect, the AAV is a serotype selected from AAV8, AAV-10 or AAV-11. In a further aspect, the AAV is AAV8. In some aspects, the rAAV particle comprises, or alternatively consists essentially of, or yet further consists of a capsid protein and the nucleic acid encoding a functional Atty. Dkt. No.: 114198-2110 TMEM216, wherein in one aspect, the TMEM216 is the short or long form, as described herein, wherein the capsid protein is derived from a AAV8 capsid protein. The nucleic acid molecules and the vectors (e.g., plasmids or AAV vectors) of the disclosure may be produced by any of the suitable means disclosed in the published literature. For example, nucleic acid molecules may be produced by chemical synthesis or by recombinant DNA technology. Methods for producing recombinant AAV vectors are disclosed in the published literature (e.g., Kimura, T., Ferran, B., Tsukahara, Y. et al. Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci Rep 9, 13601 (2019); Viral Vectors for Gene Therapy: Methods and Protocols, Manfredsson F.P, Benskey M.J., Springer Link (2019)). The disclosure further provides methods of treating a patient selected by any method of the above embodiments or identified as likely to experience a more favorable clinical outcome by any of the above methods, following the therapy. In some embodiments, the methods entail administering to the patients such a therapy. In some respects, the patient is selected by a method comprising screening a tissue or cell sample isolated from the patient for the polymorphism. Exemplary methods for screening are described in the diagnostic methods provided above and throughout the present disclosure. Any such diagnostic methods disclosed for the detection of a polymorphism can be combined with the treatment methods provided herein. The methods are useful in the assistance of an animal, a mammal or yet further a human patient. For the purpose of illustration only, a mammal includes but is not limited to a human, a simian, a murine, a bovine, an equine, a porcine or an ovine subject. Accordingly, a formulation comprising the necessary therapy or equivalent thereof is further provided herein. The formulation can further comprise one or more preservatives or stabilizers. The agents or drugs can be administered as a composition. A “composition” typically intends a combination of the active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include Atty. Dkt. No.: 114198-2110 pharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates. Various delivery systems are known and can be used to administer a gene of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis. See e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432 for construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of delivery include but are not limited to intra-arterial, intra- muscular, intravenous, intranasal and oral routes. In a specific embodiment, it can be desirable to administer the pharmaceutical compositions of the disclosure locally to the area in need of treatment; this can be achieved by, for example, and not by way of limitation, local injection into the RPE. The agents identified herein as effective for their intended purpose can be administered to subjects or individuals identified by the methods herein as suitable for the therapy. Therapeutic amounts can be empirically determined and will vary with the pathology being treated and the subject being treated. Also provided is a therapy or a medicament comprising an effective amount of a gene as described herein for treatment of a patient having a polymorphism. Methods of administering pharmaceutical compositions are well known to those of ordinary skill in the art and include, but are not limited to, oral, microinjection, intravenous or parenteral administration. The compositions are intended for topical, oral, or local administration as well as intravenously, subcutaneously, or intramuscularly. Administration can be continuously or intermittently throughout the course of the treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the disease being treated and the patient and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Kits In some embodiments, a kit comprises at least one reagent necessary to perform the assay. For example, the kit can comprise an enzyme, a buffer or any other necessary reagent Atty. Dkt. No.: 114198-2110 (e.g. PCR reagents and buffers). For example, in some respects, a kit contains, in an amount sufficient for at least one assay, any of the hybridization assay probes, amplification primers, and / or antibodies suitable for detection in a packaging material. In some embodiments, the kit or panel comprises primer and / or probes suitable for screening for the polymorphism(s). The various components of the kit can be provided in a variety of forms. For example, in some respects, the required enzymes, the nucleotide triphosphates, the probes, primers, and / or antibodies are provided as a lyophilized reagent. These lyophilized reagents can be pre-mixed before lyophilization so that when reconstituted they form a complete mixture with the proper ratio of each of the components ready for use in the assay. In addition, the kits can contain a reconstitution reagent for reconstituting the lyophilized reagents of the kit. In some respects, the kits further comprise a solid support for anchoring the nucleic acid of interest on the solid support. The target nucleic acid can be anchored to the solid support directly or indirectly through a capture probe anchored to the solid support and capable of hybridizing to the nucleic acid of interest. Examples of such solid support include but are not limited to beads, microparticles (for example, gold and other nano particles), microarray, microwells, multiwell plates. The solid surfaces can comprise a first member of a binding pair and the capture probe or the target nucleic acid can comprise a second member of the binding pair. Binding of the binding pair members will anchor the capture probe or the target nucleic acid to the solid surface. Examples of such binding pairs include but are not limited to biotin / streptavidin, hormone / receptor, ligand / receptor, and antigen / antibody. In one aspect, the kit further comprises an effective amount of a therapy. The kit can comprise at least one probe or primer which is capable of specifically hybridizing to the gene of interest and instructions for use. For example, in some aspects, the kits comprise at least one of the above described nucleic acids. The test samples used in the diagnostic kits include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are known in the art and Atty. Dkt. No.: 114198-2110 can be readily adapted in order to obtain a sample which is compatible with the system utilized. The kits can include all or some of the positive controls, negative controls, reagents, primers, sequencing markers, probes and antibodies described herein for determining the subject’s genotype in the genes of interest or target region. As amenable, these suggested kit components can be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components can be provided in solution or as a liquid dispersion or the like. Typical packaging materials would include solid matrices such as glass, plastic, paper, foil, micro-particles and the like, capable of holding within fixed limits hybridization assay probes, and / or amplification primers. Thus, for example, the packaging materials can include glass vials used to contain sub-milligram (e.g., picogram or nanogram) quantities of a contemplated probe, primer, or antibodies or they can be microtiter plate wells to which probes, primers, or antibodies have been operatively affixed, i.e., linked so as to be capable of participating in an amplification and / or detection methods. The instructions will typically indicate the reagents and / or concentrations of reagents and at least one assay method parameter which might be, for example, the relative amounts of reagents to use per amount of sample. In addition, such specifics as maintenance, time periods, temperature, and buffer conditions can also be included. The diagnostic systems contemplate kits having any of the hybridization assay probes, amplification primers, or antibodies described herein, whether provided individually or in one of the combinations described above, for use in determining the presence or amount of the polymorphism(s) in a test sample. The disclosure now being generally described, it will be more readily understood by reference to the following example which is included merely for purposes of illustration of certain aspects and embodiments of the present disclosure and are not intended to limit the disclosure. Experiment No. 1 Atty. Dkt. No.: 114198-2110 Introduction Retinitis pigmentosa (RP [MIM: 613731]), the largest clinical subset of inherited retinal disorders (IRDs), is characterized by progressive degeneration of rod photoreceptor cells. RP is estimated to affect approximately 1 in 2,500 to 4,000 individuals worldwide, typically presenting with night blindness, followed by the gradual constriction of the visual field, often leading to legal blindness.1–3Fundus abnormalities include bone spicule-like pigment deposits in the peripheral retina, attenuation of retinal vessels, and a pale optic nerve head. For individuals who undergo genetic testing, there is a significantly lower rate of molecular diagnosis in ethnicities that are under-represented in aggregated genome datasets.1,4,5Missing molecular diagnoses may be attributed to non-coding variants, structural rearrangements, variants in unreported RP genes, and incorrect clinical diagnosis.1,4,6–8Pathogenic variants in over 280 IRD genes have been identified across all Mendelian inheritance patterns (RetNet database, https: / / sph.uth.edu / RetNet / ). Variants impacting protein-coding genes involved in various pathways including phototransduction, photoreceptor structure, ciliogenesis, and RNA splicing have been identified as causes of syndromic and non-syndromic IRDs.7,9–11Many IRDs are classified as ciliopathies, with variants in genes that are involved with cilia biogenesis and transport.12–17Variants in ciliary genes can cause a spectrum of phenotypes ranging from non-syndromic retinal degenerations to syndromic ciliopathies.18–23This study identified two candidate pathogenic variants on chromosome 11 at the genomic position g.61392563 (GRCh38) upstream of TMEM216 (c.–69G>T, c.–69G>A, GenBank: NM_001173991.3; MIM: 613277) and 23 kb downstream of TMEM138 (MIM: 614459). These variants were identified in a total of 74 individuals affected with RP of African (c.–69G>T) and South Asian (c.–69G>A) ancestry. Variants in both TMEM216 and TMEM138 have previously been implicated in Joubert (MIM: 608091) and Meckel (MIM: 603194) syndromes, often exhibiting retinal findings.24–26In a separate study, using methods as described herein, two additional variants were discovered, c.-95G>C in patients of South Asian and European ancestry as well as c.-41C>T The expression of these genes is regulated Atty. Dkt. No.: 114198-2110 by the conserved regulatory elements located in the intergenic region.25Variants in TMEM216 and TMEM138 identified to date in Joubert or Meckel syndrome are limited to the coding region and splice sites with a likely loss, or reduced, function as the disease mechanism.24,27–29Subjects and Methods Ethical statement All studies were performed in accordance with the Declaration of Helsinki and the approval of the institutional review boards (IRB) of University of California San Diego, La Jolla, CA, USA; Johns Hopkins University, Baltimore, MD, USA; the CNS IRB at the National Institutes of Health, Bethesda, MD, USA; Moorfields Eye Hospital, London UK (Genetic Study of Inherited Eye Disease Research Ethics Committee [REC] ref. 12 / LO / 0141) or Genomics England 100,000 Genomes project (REC ref. 14 / EE / 1112) or NIHR BioResource for Rare Disease (REC ref. 13 / EE / 0325); the Ethikkommission Nordwest-und Zentralschweiz; the Commission Cantonale d’Étique de la Recherche sur l’Être Humain du Canton de Vaud; the Comissão de Ética para a Saúde do Instituto de Oftalmologia Dr. Gama Pinto; the Health Research Institute-Fundación Jiménez Díaz University Hospital; Universidad Autónoma de Madrid; Massachusetts General Brigham IRB; and the University of Punjab, Lahore, Pakistan. Blood samples were collected from affected individuals and available family members after obtaining their written informed consent to participate in Applicant’s study. Study cohorts and analysis Whole-genome sequence (WGS) data of individuals from a large UK cohort (Genomics England 100,000 Genomes Project4 [UK100k]1 were analyzed to uncover the underlying cause of retinal degeneration in previously unresolved cases. In the UK100k main cohort, 2,316 participants (from 2,038 families) with inherited retinal degeneration (IRD) were recruited, with 35% of cases categorized as solved through the initial Genomics England variant analysis pipeline. This pipeline involved tiering of variants within relevant gene panels but did not initially include non-coding and structural variants. Scrutiny of genomic data from the unsolved UK100k IRD cohort included independent analysis of Atty. Dkt. No.: 114198-2110 homozygous coding and noncoding rare variants (MAF < 0.01) across a panel comprising 216 retinal genes, known to be associated with either syndromic or non-syndromic retinal dystrophy (https: / / panelapp.genomicsengland.co.uk / panels / 307 / ). The aim was to detect apparent homozygosity caused by heterozygous large deletions missed through the initial standard pipeline and to allow further scrutiny of variants of interest. Conducting case- specific analysis within the Genomics England’s Interactive Variant Analysis (IVA) tool enabled each variant to be viewed across the whole UK100k rare disease cohort, alongside zygosity and key phenotypic information for each participant listed. Independently, while the UK cohort screen was underway, a homozygosity mapping approach was used to study two consanguineous RP-affected families of Pakistani origin (A- 4 and A-5) (FIGS. 1R and 1S). Prior to this analysis, these two families were unsolved as no likely causal / pathogenic coding sequence variants in known IRD genes of MAF 0.1% had been identified after WGS analysis.6To identify additional RP-affected individuals with the two novel candidate causative TMEM216 variants detected in the UK100k cohort and the two Pakistani families (A-4 and A-5), 5,930 molecularly uncharacterized RP-affected individuals at five additional centers were tested either by reviewing their NGS data or by targeted DNA sequencing: Ocular Genomics Institute, Massachusetts Eye and Ear, Boston, MA, USA (800 probands from 800 families); the University of Punjab, Lahore, Pakistan (215 affected individuals from 194 families); the Institute of Molecular and Clinical Ophthalmology Basel, Basel, Basel-Stadt, Switzerland (2,738 affected individuals from 2,703 families); the Health Research Institute- Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (2,013 probands from 2,013 families); and the NIHR Bioresource for Rare Disease1 at Moorfields Eye Hospital, London (NIHRRD) and the UK Inherited Retinal Disease Consortium (IRDC), London, UK (164 probands from 164 families). Homozygosity mapping Genome-wide homozygosity mapping of families A-4 and A-5 (FIGS. 1R and 1S) was performed as previously described using WGS data.30,31Briefly, homozygosity was calculated from common SNPs and indels with MAF >1%. For each individual, a data frame Atty. Dkt. No.: 114198-2110 with the genomic position of all genotyped SNVs and a binary variable representing all heterozygous and homozygous alternative SNVs was constructed. The smooth.spline function with default parameters in R was used to calculate the frequency of homozygous and heterozygous SNPs. A stretch of homozygosity was defined if the smoothed frequency was <0.1% in at least 10,000 consecutive SNVs. The homozygosity mapping for families T-24, T-23, A-6, and A-7 (FIGS. 1E, 1F, 1T, and 1U) was performed using AutoMap with default parameters on whole-exome sequencing (WES) data ( 6).32Haplotype analysis Haplotypes of affected individuals carrying TMEM216 (GenBank: NM_001173991.3) c.–69G>T or c.–69G>A alleles were constructed from SNPs at 50 kb intervals 1 Mb upstream and downstream of the variant on chromosome 11 (g.60392563– 62392563 [GRCh38]). The shared haplotype was identified by inspection in each group and was used to estimate the frequency of that haplotype in the corresponding population based on the 1000 Genomes database for the Punjabis in Lahore (PJL) chromosome 11 dataset for the Pakistani individuals and the combined Esan in Nigeria (ESN), Gambian in Western Division (GWD), Luhya in Webuye, Kenya (LWK), and Yoruba in Ibadan, Nigeria (YRI) datasets for the African individuals. Haplotype estimations were done by the EM (expectation maximization) and CHM (composite haplotype method) algorithms as incorporated in the Golden Helix SVS (SNP & Variation Suite) using default variables except that the minimum haplotype frequency was set to 0.0001 and a maximum of 100 EM iterations were allowed in order to predict accurate frequencies for rare haplotypes such as the risk haplotype conserved among all individuals homozygous for the causative g.61392563G>A and G>T alleles on chromosome 11. rs572262418 and rs760001653 were not found in the Pakistani and African 1000 Genomes datasets, respectively, probably due to a combination of low frequency of the rs572262418 minor allele in the Pakistani population and different frequencies of rs760001653 alleles in the various African populations included in the 1000 Genomes datasets. Because of this they were excluded from the haplotype analysis. This should not affect the haplotype frequency estimation as they are in complete Atty. Dkt. No.: 114198-2110 linkage disequilibrium with the remaining SNPs in the risk haplotype in all affected individuals. Clinical evaluation of affected individuals All available participants underwent a full ophthalmic examination with a detailed clinical and family history. Retrospective clinical data were also gathered where available. Ophthalmic examination included visual acuity (Snellen and / or logMAR) and slit-lamp examination. Imaging included spectral domain optical coherence tomography (Spectralis, Heidelberg Engineering Ltd), ultra-widefield (UWF) color fundus photography (200°, Optos plc), and fundus autofluorescence (FAF) imaging, performed with 55° Spectralis or UWF Optos. Electrophysiological testing, when performed, included full-field and pattern electroretinography (ERG), which incorporated the International Society for Clinical Electrophysiology of Vision (ISCEV) standards.33The centers where the clinical evaluation of pedigrees was performed are listed in Table 5. Table 5: Internal pedigree identifiers and site of clinical evaluation for all families. Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Note: De-identified IDs were used for all pedigrees. Lowercase letters added to family IDs represent members of the same family. * = Heterozygous deletion of GRCh38 chr11:61382891-61393975 found in trans with TMEM216 c.-69G>T. HOM = Homozygous. HET = Heterozygous. N / A = Not available. NS = Not shown in 1. Impact of TMEM216 c.–69 G>A and G>T variants on transcription factor binding The Find Individual Motif Occurrences (FIMO) tool (MEME Suite 5.5.4) was used to scan a set of sequences for individual matches to motifs provided from JASPAR and HOCOMOCO databases.34–36Transcription factor (TF) positional weight matrices (PWMs) are used as input, and motif occurrences with a p value less than 0.001 were retained and analyzed. Luciferase reporter gene assay A genomic fragment spanning the TMEM2165' untranslated region (5' UTR) and upstream non-coding sequence on chromosome 11 (g.61391712–61392642 [GRCh38]) was Atty. Dkt. No.: 114198-2110 chosen for dual reporter luciferase assays. The fragment was chosen because of overlap with known retinal cis-regulatory elements (CREs) found in previous studies.37Six constructs were designed: wild-type, two versions harboring the variants c.–69G>A or c.–69G>T, and two constructs that removed predicted promoter 1 or 2, and a promoterless sequence (Tables 1 and 7). The gene fragments were generated commercially (Twist Bioscience) and cloned (Gibson Assembly cloning Master Mix, New England Biolabs) into a modified version of the psiCHECK2 luciferase vector (Promega) lacking the SV40 promoter. The resulting plasmids were validated by Sanger sequencing and transfected into WERI-Rb1 cells (ATCCHTB-169) using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer’s guidelines. Each experimental condition was tested in 10 biological replicates. Forty-eight hours post-transfection, luciferase assay was performed (Dual-Glo Luciferase assay, Promega) with luminescence reading performed by a plate spectrophotometer (SpectraMaxM3, Molecular Devices). Raw data were processed by subtracting the background luminescence of the un-transfected cell samples, normalization of the test renilla luciferase activity with the background firefly luciferase activity, and normalization with the wild-type construct. A promoterless construct served as a negative control. Statistical significance was assessed using Brown-Forsythe and Welch ANOVA and Dunnett’s multiple comparison test (compared all groups against control group). Table 1: Sequence of the seven genomic fragments cloned into the psiCHECK™2 luciferase vector. Each fragment was flanked by an upstream (5’- GAAATAACCTCTGAAAGAGGAACTTGGTTAGGTACC-3’) and a downstream (5’- AGGCCTAGGCTTTTGCAAAAAGCTTGATTCTTCTGAC-3’) recombination sequence arm. The reference nucleotide at position c.-69 in TMEM216_WT is bolded and italicized, while variants TMEM216 c.-69 G>T and G>A are bolded. The 20-bp sequence between double parentheses corresponds to GRChg38 chr11:61392544-61392563 and it was deleted in fragment TMEM216_Δ20bp. Two 60-bp promoter sequences (P2 in brackets and underlined) and P1 (in single parenthesis), corresponding to GRChg38 chr11:61392331- 61392390 and chr11:61392583-61392642, respectively, were deleted in fragments TMEM216_ΔP2 and TMEM216_ΔP1. The sequence of the TMEM2165’UTR (in bold italics) is located within promoter 1. Fragment TMEM216_ ΔP2+P1 contains the largest deletion, Atty. Dkt. No.: 114198-2110 spanning from GRChg38 chr11:61392331-61392642. The dashes (-) indicate the sequence of P2, P2, and GRChg38 chr11:61392544-61392563 in the constructs. Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Atty. Dkt. No.: 114198-2110 Expression of TMEM216 and TMEM138 in blood cells of individuals with TMEM216 c.–69G>T RNA was isolated from individuals homozygous for TMEM216 c.–69G>T (T-23 and T-24) and unaffected control subjects from blood samples collected in Tempus Blood RNA tube (Thermo Fisher) and extracted using the Tempus Spin RNA isolation kit (Thermo Fisher). The extracted RNA was then purified using RNA cleanup and concentration kit (Qiagen) and converted into cDNA using the MultiScribe, High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed on 4 ng of cDNA in 20 µL reactions with Fast-SYBR Green (4385612, Roche). Cycle threshold (CT) values were normalized using GAPDH (MIM: 138400) expression, and relative expression was calculated using 2-ΔΔCT method.38The experiment was repeated in triplicate using the same cDNA samples on the same day (experimental replicates). The primers used are listed in Table 5. For statistical analysis, two-tailed t test was performed. Sequencing of TMEM216 transcript from blood samples of individuals with TMEM216 c.–69G>T PaxGene blood samples were collected from an affected individual homozygous for c.–69G>T, unaffected carrier parent, and an unaffected control. Total RNA was purified using the PaxGene extraction kit (Qiagen) followed by reverse transcription to cDNA using random hexamers and SuperScript IV reverse transcriptase (Invitrogen). PCR was performed using primers designed to capture the canonical transcript (GenBank: NM_001173991.3) Atty. Dkt. No.: 114198-2110 from exon 1 to –30 UTR (Table 5). PCR product was purified (Ampure XP, Beckman Coulter Inc.). Up to 30 fmol of amplicon (Qubit High Sensitivity dsDNA quantification) was used for end preparation (Ultra II end-prep kit; NEB) and native barcoding (Oxford Nanopore Technologies; ONT: EXP-NBD104). Barcoded samples were pooled for library preparation (ONT ligation sequencing kit SQK-LSK110 Flongle protocol). Libraries were sequenced for 12 h using the Flongle flowcell and MinION sequencer. Base calling was performed using the high-accuracy base calling mode in Minknow. Adaptor sequences were removed using Porechop v.0.2.4,39 and reads were filtered using NanoFilt v.2.8.040 for reads 800 to 1,800 bp with a quality score ≥ Q10. Resulting FastQ data were aligned to the human reference genome (build GRCh38) using minimap2 v.2.22.41 BAM files were generated using SAMtools v.1.9.42 The Integrative Genome Viewer43 v.2.7.2 and v.2.14.1 were used to visualize aligned long reads. The heterozygous coding SNV present in the carrier parent (c.264G>A [p.Pro88Pro]) was used to visualize the relative amplification of the c.–69G vs. c.–69T alleles. Generation of cell lines with TMEM216 c.–69G>A The hTERT-RPE1 cells (CRL-4000, ATCC) were used to generate the cell lines harboring the TMEM216 c.–69G>A variant using two sets of sgRNAs and donor oligo sequences (Table 6) performed by Synthego. The cells were screened for the TMEM216 c.– 69G>A variant using PCR amplification and sequencing with the primers listed in Table 6. Table 6: Sequence of primers, gRNA, and donor sequences used in this study. Atty. Dkt. No.: 114198-2110 Analysis of gene expression in hTERT-RPE1 cells with TMEM216 c.–69G>A Atty. Dkt. No.: 114198-2110 The relative expression of TMEM216 and TMEM138 transcripts was studied in the WT and genome-edited hTERT-RPE1 cells with the TMEM216 c.–69G>A variant in the homozygous or heterozygous state using primers listed inTable 6. Total mRNA was isolated from these cells, treated with RNase-free DNase, and purified using Qiagen RNeasy Mini Kit (Qiagen). Preparation of cDNA and RT-qPCR analyses were performed as described earlier.44The relative quantity was normalized to the expression levels of housekeeping genes GAPDH and ACTB (MIM: 102630) presented on an arbitrary scale to represent the relative levels of expression. The statistical significance (p value) was calculated using Student’s t test as described previously.44Evaluation of cilia in hTERT-RPE cells with TMEM216 c.–69G>A Genome-edited hTERT-RPE1 cells carrying TMEM216 c.–69G>A variant in the heterozygous and homozygous states along with wild-type hTERT-RPE1 cells were studied using two independent clones of each genotype. These hTERT-RPE1 cells were cultured under serum-starved conditions and stained with acetylated α-tubulin antibodies (sc-23950, 1:200, Santa Cruz Biotechnologies) after 24 h of plating. Images were captured using Nikon confocal microscope system (A1R STORM). The ciliary phenotype was compared relative to the ciliary marker acetylated-α-tubulin. ImageJ64 software was used for measuring the number of the cilia in >200 cells for all genotypes in all clones. Materials and Methods Identification of TMEM216 c.–69G>T in individuals with retinitis pigmentosa that lack molecular diagnosis One individual with simplex, non-syndromic retinitis pigmentosa, unsolved through the UK100k WGS analysis pipeline, was queried for apparent homozygous rare variants (MAF < 0.001) across a panel of 216 retinal dystrophy genes. 24 autosomal rare homozygous variants were found, none of which were homozygous in other individuals with retinal dystrophy across the entire UK100k cohort, with the exception of TMEM216 c.– 69G>T on chromosome 11 (g.61392563G>T [GRCh38], GenBank: NM_001173991.3). Analysis of the entire UK100k dataset identified 48 heterozygous and 18 apparent homozygous variant calls for this variant (Table 2). All 18 homozygotic individuals from 14 Atty. Dkt. No.: 114198-2110 unrelated families had a clinical diagnosis of RP and remained undiagnosed through prior analysis. Zygosity of the apparent homozygotes was checked by inspection of trio genome segregation data and individual read data using IGV. Two affected siblings from one additional family (M-2) showed hemizygosity for the variant and a deletion in trans, encompassing exons 1–3 of TMEM216 and much of the intergenic region distal to TMEM138 upstream of the gene on chromosome 11 (g.61,382,891–61,393,975 [GRCh38]) ( 1Q). An additional 24 individuals homozygous for TMEM216 c.–69G>T were identified across 24 families following genetic analysis of 5,874 families with 5,930 unsolved IRD cases from additional cohorts. One further individual (M-1) was a compound heterozygote for this and TMEM216 c.35–2A>G on chromosome 11 (g.61393229A>G [GRCh38]), a previously reported pathogenic variant ( 1P).2All 45 individuals from 40 families were of either African (Zimbabwe, Ghana, Nigeria, Angola), Caribbean (Jamaica, Barbados, Haiti), or African American descent (FIGS. 1A–1Q; Table 1). The variant has an allele frequency (AF) of 0.0002739 (407 / 1,485,934 alleles) in the gnomAD v4 dataset with enrichment in the African / African American genetic ancestry group (362 / 71,782 alleles, AF: 0.005043) including one homozygote, but was completely absent in non-Finnish European individuals (1,105,112 alleles).45Identification of TMEM216 c.–69G>A in individuals with RP In 2021, Biswas et al. performed whole-genome sequencing in 108 unrelated pedigrees from three different ethnic populations.6The WGS data from nine members of two consanguineous Pakistani families with RP that were not solved by the latter study (A-4 and A-5, FIGS. 1R and 1S) were subjected to a careful search for homozygous regions of the genome that segregated with the disease phenotype. This analysis revealed a ~8.25 Mb homozygous region on chromosome 11 (g.55,000,000–63,258,298) shared by the three affected individuals of A-4 (IV:2, IV:4, and IV:5 in 1R; 2A). This homozygous region is also partially shared with one affected individual (V:4 in 5) from the second family, A-5. Among all four affected individuals, the stretch of homozygosity overlapped, and the size of the shared region was 1.57 Mb on chromosome 11 (g.61,145,940–62,715,657). Atty. Dkt. No.: 114198-2110 Analysis of all rare variants within this shared homozygous region and segregation analysis of additional family members revealed a rare non-coding homozygous variant, TMEM216 c.–69G>A on chromosome 11 (g.61392563G>A [GRCh38], GenBank: NM_001173991.3). This variant segregated with RP in both pedigrees (FIGS. 1R and 1S) and was found in the gnomAD v4 dataset with a frequency of 0.00005855 (87 / 1,485,932 alleles), with enrichment in the South Asian genetic ancestry group (79 / 82,738 alleles, AF: 0.0009548).45Table 2. Summary of demographic information and clinical phenotype of selected affected individuals from African and South Asian families with TMEM216 variants

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[0005] Atty. Dkt. No.: 114198-2110 Demographics, pedigree, and phenotype information obtained where available. Lowercase in family ID represent members of the same family. “Age of onset” as reported by subject of earliest signs of nyctalopia or vision loss. Ethnicity data gathered as reported by subject. Visual acuities converted to imperial Snellen scale. *Heterozygous deletion of g.61,382,891–61,393,975 (GRCh38) on chromosome 11 found in trans with TMEM216 c.– 69G>T. HOM, homozygous; HET, heterozygous; YOB, year of birth; F, female; M, male; BE, both eyes; RE, right eye; LE, left eye; RP, retinitis pigmentosa; VA, visual acuity; CF, counting fingers; NPL, no perception of light; LP, light perception; N / A, not available; NS, not shown in 1. Atty. Dkt. No.: 114198-2110 Analysis of the genomic data of 315 additional pedigrees with affected individuals that remained without molecular diagnosis after WGS or WES analysis out of a total of ~716 unrelated IRD-affected pedigrees recruited from Pakistan resulted in the identification of TMEM216 c.–69G>A in the homozygous state in affected individuals from two additional families: A-6 and A-7. Examination of the homozygous regions identified in individuals from these two families narrowed the shared homozygous interval to 330 kb ( 2B). Further, targeted variant analysis of affected individuals from additional Pakistani pedigrees by Sanger sequencing revealed the presence of the TMEM216 c.–69G>A variant in the homozygous state in affected individuals from two pedigrees, A-8 and A-9 (FIGS. 1V and 1W). This variant segregated in the homozygous state with RP in one family (A-8) while in the second family (A-9), one affected member had the variant in the homozygous state and two other affected members carried this variant in the heterozygous state (FIGS. 1V and 1W). The underlying cause of disease in these heterozygous carriers that lacked potentially pathogenic variants in other genes including TMEM216 remains unknown and may be independently inherited. Subsequent screening of the datasets of UK100k and NIHRRD cohorts identified four additional RP-affected individuals from three families (A-1, A-2, and A-3) of South Asian origin, homozygous for TMEM216 c.–69G>A (Table 2). In summary, the homozygous TMEM216 c.–69G>A variant was observed in affected individuals from 9 families of South Asian origin with a clinical diagnosis of RP and no molecular diagnosis (FIGS. 1R–1W; Table 2). Haplotype mapping Haplotypes formed by SNPs selected for having a minimum fraction of heterozygotes in the gnomAD database for that particular population (African and South Asian) of 0.3 in the TMEM216 region on chromosome 11 of affected individuals were assembled in the Pakistani and African individuals and extended from TMEM216 c.–69G (g.61392563) until at least one haplotype in each group diverged (Table 3). In the African affected individual group, the shared haplotype extends 177 kb from rs61895905 on chromosome 11 (g.61340630) to rs3019201 (g.61517826). In the Pakistani individuals, it spans 330 kb from rs373641413 -77- Atty. Dkt. No.: 114198-2110 (g.61264929) to rs79136768 (g.61594967) on chromosome 11 (Table 4). These two conserved haplotypes strongly suggest a single founder mutation unique to each group. The conserved haplotypes were not found in the predicted haplotype sets for either the Pakistani or African population groups, so the 1000 Genomes datasets were seeded with a single homozygous carrier for the shared founder haplotype to give a conservative upper limit for the frequency. The estimated population frequencies for the shared haplotypes when the single carriers are included are 0.01 for the Pakistani population and 0.0023 for the African population giving a p < 1.3 ×10-18and p < 3.6310-53for a single founder mutation for the Pakistani and African population, respectively. All individuals with the c.–69G>A variant were from consanguineous unions while all with the c.–69G>T variant did not report consanguinity (Table 2). This is exemplified by high total size of regions of homozygosity (ROHs) in the four families with c.–69G>A and low total size of ROHs in the two families with c.–69G>T (FIGS. 2 and 6). Individuals reporting consanguinity typically have more than 100 Mb of total size of ROHs. Table 3. Conserved region of homozygosity across TMEM216 c.–69G>T in African families

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[0007] Atty. Dkt. No.: 114198-2110 Table 4. Conserved region of homozygosity across TMEM216 c.–69G>A in South Asian families Clinical phenotype of subjects with TMEM216 c.–69G>A and c.–69G>T Of the bi-allelic TMEM216 individuals, detailed clinical data were available for 31 seen at Moorfields Eye Hospital, London, UK (aged 9–73 years, 16 females / 15 males) (Table 12). All had night blindness in the first decade of life, with progressive loss of peripheral field over subsequent decades. The childhood-onset night blindness resembled the clinical profile typically seen in individuals with RP consequent upon bi-allelic variants of genes involved in rod phototransduction such as CNGB1 (MIM: 600724), CNGA1 (MIM: 123825), PDE6B (MIM: 180072), and PDE6A (MIM: 180071). Foveal structure and function were initially preserved, with loss of acuity from the third decade. OCT and en face imaging were of typical RP in which there is primary loss of rod photoreceptor structure with secondary loss of the cone-rich central macula and fovea. Representative images are shown in 3. Electrophysiology showed abrogated responses to both rod and cone stimuli using ISCEV standard conditions. Even at an early age (3 individuals were tested prior to 10 years of age), there were barely detectable cone-driven and undetectable rod-driven responses. There were no evident differences in the age of onset and progression of those with homozygosity for TMEM216 c.–69G>T compared to TMEM216 c.–69G>A, nor in the two Atty. Dkt. No.: 114198-2110 mixed heterozygotes for TMEM216 c.–69G>T. No evident systemic features suggestive of generalized ciliopathy were noted in affected persons (Table 2). Evaluation of affected members of other families with these non-coding variants showed a clinical course consistent with that described above. c.–69G>A and c.–69G>T downregulate TMEM216 expression in vitro A dual reporter luciferase assay was performed to test whether the noncoding variants c.–69G>A and c.–69G>T alter TMEM216 gene expression. The 931 bp test sequence contained the genomic sequence spanning the 5' untranslated region (UTR) of TMEM216 and upstream non-coding sequence on chromosome 11 (g.61,391,712–61,392,642 [GRCh38]) ( 4A). Two promoters were predicted in this region according to the Eukaryotic Promoter Database46: promoter 1 (P1, g.61,392,583–61,392,642 [GRCh38] on chromosome 11) overlapping with the TMEM2165' UTR (GenBank: NM_001173991.3) and an upstream promoter 2 (P2, g.61,392,331–61,392,390 [GRCh38] on chromosome 11). The region contains known retinal transcription factor binding sites (CRX and OTX2) and lies within an ATAC peak ( 4A).37,46FIMO transcription factor analysis using JASPAR TF motif PWMs predicted differential binding of transcription factors between wild-type and the sequence containing both variants. Five constructs were tested: a reference and two variant constructs containing either the c.–69G>A or c.–69G>T variant and two deletion constructs with deletions involving individual predicted promoter sequences P1(ΔP1) and P2 (ΔP2) ( 4B). Both TMEM216 c.– 69G>A and c.–69G>T variants resulted in a significant downregulation of luciferase activity (26% and 36% of reference expression respectively, p value < 0.0001), suggesting a hypomorphic nature of these variants. A more pronounced downregulation was observed when the proximal promoter (P1) was missing (p value < 0.0001). Deletion of the distal promoter (P2) did not show significant differences when compared to the reference construct ( 4B). Reduced expression of TMEM216 in leukocytes of individuals with TMEM216 c.– 69G>T Atty. Dkt. No.: 114198-2110 RT-qPCR was performed to analyze the expression levels of TMEM216 and TMEM138 in blood samples from the WT control subjects and affected individuals from pedigrees T-23 and T-24 homozygous for TMEM216 c.–69G>T. The results revealed a significant reduction of 51% of TMEM216 expression in homozygous compared to wild-type individuals (p value = 0.0075) ( 4C). However, no significant difference was observed in the expression of TMEM138 in affected individuals compared to control subjects ( 4C). Skewed allele balance in a c.–69G>T carrier IGV read depth analysis of blood-derived TMEM216 RT-chPCR transcripts using ONT single-molecule sequencing yielded 26,922 reads from a homozygous individual, 32,854 from a carrier individual, and 20,963 from a control subject ( 4D). The reads were phased in the carrier parent sample using the heterozygous SNV c.264G>A (A allele) in cis with the c.–69T allele. Reads covering this nucleotide position showed an allele balance of 74% / 26% (23,255 reads / 8,184 reads) of the c.–69G; c.264G allele to the c.–69T; c.264A allele, suggesting a relative reduction in transcription from the c.–69T allele. The generation of two distinct isoforms from differential usage of the exon 1 donor site is shown in each of the samples with a similar ratio of long to short exon 13' ends across samples ( 4D). Moreover, when examined separately, the mutant and wild-type alleles from the heterozygous parent (2nd row) showed exon 1 usage short:-long as follows: mutant, 5,106:962 (16% long); wild-type, 18,892:5,970 (24% long). Overall, these data would support the model that the c.–69G>T variant causes reduced but not abrogated expression. Reduced expression of TMEM216 in hTERT-RPE1 cells with TMEM216 c.–69 G>A hTERT-RPE1 cells were generated by introducing this variant using CRISPR-Cas9, which resulted in the generation of cell lines heterozygous and homozygous for this TMEM216 c.–69G>A variant. Unfortunately, the cell line homozygous for the TMEM216 c.–69G>A variant also had an additional variant TMEM216 c.–71C>T (g.61392561C>T on chromosome 11) introduced during the gene editing process ( 5). Compared to the control wild-type, the relative expression of TMEM216 was reduced by 86.3% (p value < 0.001) in the genome-edited cells homozygous for c.–69G>A / c.–71C>T Atty. Dkt. No.: 114198-2110 and by 62.1% (p value < 0.05) in cells heterozygous for c.–69G>A. These results indicate that TMEM216 c.–69G>A lowers the expression of TMEM216 ( 4E). However, the relative expression of TMEM138 which is known to share a regulatory region with TMEM216 was not altered based on the presence of the variant ( 4E). The expression of three other IRD genes was also evaluated in these cells, due to their proximity to the TMEM216 c.–69G>A variant. The BEST1 (MIM: 607854), ASRGL1 (MIM: 609212), and ROM1 (MIM: 180721) genes located on chromosome 11 (55 kb, 95 kb, and 1.2 Mb away, respectively, from c.–69G>A) showed no significant difference in their expression between mutant and control hTERT-RPE1 cells (data not shown). Abnormal ciliogenesis in hTERT-RPE1 cells with TMEM216 c.–69 G>A The ciliary morphology of hTERT-RPE1 cells carrying the c.–69G>A variant was evaluated as TMEM216 deficiency is known to affect primary ciliogenesis.47Immunocytochemistry of hTERT-RPE1 cells with the ciliary marker acetylated tubulin antibodies revealed reduced ciliogenesis with apparent normal gross morphology of cilia in cells with the heterozygous c.–69G>A genotype compared to the wild-type hTERT-RPE1 cells while a majority of cells homozygous for the c.–69G>A / c.–71C>T genotype lacked cilia ( 4F). The percentage of ciliated cells are significantly low among cells with the heterozygous and homozygous genotype compared to the wild-type (54% and 3% of wild- type, respectively, with adjusted p values of <0.0001) ( 4G). These findings suggested abnormal ciliogenesis in hTERT-RPE1 cells with c.–69G>A variant and reduced levels of TMEM216 transcript. The impact of the additional variant c.–71C>T in cells with the homozygous genotype is unknown. Analysis of GTEx data revealed significantly higher levels of TMEM216 transcript in the human peripheral retina compared to those recorded from additional tissues in the GTEx database ( 4H).48,49Discussion Forty-five individuals with recessive RP from 40 pedigrees have been found so far to be homozygous or compound heterozygous for the G>T change and 29 individuals from nine families have been found to be homozygous for the G>A variant. Two affected siblings were compound heterozygotes for the G>T variant and a deletion encompassing exons 1–3 of Atty. Dkt. No.: 114198-2110 TMEM216 and the upstream region. One affected individual was compound heterozygous for the G>T variant and a splice site variant c.35-2A>G. The c.35-2A>G substitution in TMEM216 observed in one of the compound heterozygotes would only be expected to affect TMEM216 specifically, consistent with the pathology being consequent on reduced TMEM216 expression rather than an effect on other nearby genes. The population frequencies reported for both variants are consistent with carrier frequencies in recessive IRD genes. Haplotype analysis is consistent with a single ancestral mutation event giving rise to all extant disease-associated alleles for each of the two variants. The relatively high prevalence of the G>T allele in the African population (0.005 in gnomAD) is noteworthy. This prevalence is higher, for instance, than the most common alleles causing recessive RP in the European population, e.g., USH2A (MIM: 608400; GenBank: NM_206933.4) (c.2299del [p.Glu767SerfsTer21] and c.2276G>T [p.Cys75 9Phe], with allele frequencies of 0.001 and 0.002, respectively).45,50Based on the allelic frequency, Applicant can estimate 1 in 40,000 in this population would be homozygous for this variant. The sum of allelic frequencies of pathogenic and likely pathogenic variants from ClinVar as well as clear loss-of-function variants from gnomAD (v.4.0.0)45is 0.00033 for African / African American population (for nine variants). Therefore, Applicant expect 1:300,000 individuals to carry the c.–69G>T variant in trans with a pathogenic variant. This makes the expected frequency of individuals carrying c.–69G>T homozygous or in trans with another pathogenic variant to ~1:35,000 in that population. This would be expected to be a significant proportion of non-syndromic RP in this population. The nature and location of the sequence encompassing the TMEM216 c.–69G site and the in silico analysis suggest a critical role for this genomic region in transcriptional regulation of TMEM216. Four independent assays presented in Applicant’s study (dual reporter Luciferase assay, RT-qPCR, and nanopore sequencing of affected individuals’ leukocytes and RT-qPCR of edited cell lines) demonstrated that the c.–69 G>A or G>T noncoding variants downregulate TMEM216 expression. Applicant’s experimental validations indicate that the TMEM216 expression is reduced but not abrogated by these variants without affecting nearby IRD genes. Atty. Dkt. No.: 114198-2110 It is of interest that all individuals presented here (age range at exam 5–72 years) did not show any symptoms suggestive of the systemic pathology that occurs in affected individuals reported with bi-allelic variants in TMEM216 which cause Joubert, Meckel, and related disorders.24None of the subjects with TMEM216 c.–69G>A or G>T variants exhibited neurological symptoms. However, because neuroimaging was not clinically indicated, Applicant cannot exclude asymptomatic minor structural changes in the cerebellum. One possible explanation for the specificity of involvement of the rod photoreceptors in these individuals might be a need for higher TMEM216 gene expression in these cells. This is supported by the presence of significantly higher levels of TMEM216 transcript in the human peripheral retina compared to the levels listed in GTEx.48,51It is likely that the photoreceptor cells are more sensitive to the reduction of gene expression, and therefore the TMEM216 c.–69G>A or G>T variants leading to such gene expression reduction, rather than complete loss of expression, cause specific dysfunction of photoreceptors. This phenomenon has been observed in other ciliopathy genes such as CEP290 (MIM: 610142) which is associated with a spectrum of phenotypes ranging from Leber congenital amaurosis (LCA [MIM: 611755]), to the lethal Meckel syndrome (MKS).18–23However, cases have been reported with a lack of apparent retinal involvement in the individuals with homozygous c.218G>T (p.Arg73Leu) reported by Valente et al.24It is possible that the mechanism of disease of this mutation is different than the reduced expression / function caused by TMEM216 c.–69G>A and G>T, which lead to differences in the retinal phenotype. It is noted that there are at least two major transcripts generated from the human TMEM216 gene. The longer transcript (GenBank: NM_001330285) has a shorter open reading frame (–87 amino acids compared with 148 from the shorter transcript [GenBank: NM_001173991.3]) due to the use of a downstream start codon in the longer transcript. The presence of both isoforms is observed in the majority of tissues.49Applicant’s analysis of TMEM216 transcripts in affected individuals’ leukocytes to study the impact of TMEM216 c.–69G>T in individuals with the c.–69G>T genotype in the homozygous and heterozygous states appeared to show similar reduction in both transcripts equally in the homo- and heterozygote on read-counting of nanopore-generated sequencing. It remains possible that Atty. Dkt. No.: 114198-2110 one of the two transcripts is critical for rod photoreceptor function and maintenance and that the c.–69 variants have a specific effect on this specific retinal transcript. Additional studies are needed to understand the mechanism underlying non-syndromic RP phenotype due to the non-coding variants c.–69G>A and c.–69G>T. Notably, all TMEM216 variants reported so far in individuals affected with Joubert syndrome involve only the coding sequence and would be expected to affect both isoforms.24The TMEM216 protein is part of the “MKS module” that localizes to the cilia transition zone and plays a key role in ciliogenesis.17,52,53This complex includes additional proteins involved in ciliopathies.54The phenotypes observed in affected individuals with variants in TMEM216 are Joubert and Meckel syndrome, which are severe ciliopathies that often involve retinal degeneration.24,55,56In previous studies, fibroblasts derived from individuals with TMEM216 variants show impaired ciliogenesis.24,25It is well established that the integrity of cellular ciliary machinery is critical in the formation and maintenance of photoreceptors and as a result, variants in TMEM216 are predicted to cause retinal degeneration along with other ciliary phenotypes.53Further, TMEM216 knockdown zebra- fish show abnormal outer segment formation compared to wild-type, while complete loss of TMEM216 leads to embryonic and postnatal lethality in mice.47,53Consistent with the above observations, the hTERT-RPE1 cells with c.–69G>A genotype lacked cilia, indicating abnormal ciliogenesis.24,47These observations suggest the involvement of TMEM216 in retinal pathology observed in individuals with the c.–69G>T and c.–69G>A variants upstream of this gene and abnormal ciliogenesis as a possible mechanism underlying retinal pathology. Identification of noncoding variants with a high impact on gene expression that lead to IRD is an emerging phenomenon; for example, causative variants upstream of PRDM13 (MIM: 616741) have been shown to cause North Carolina macular dystrophy (MCDR1 [MIM: 136550]).57–59Applicant anticipate that such variants will be a major cause in the remaining genetically unsolved IRD cases. The identification of TMEM216 c.–69 G>T and G>A described herein significantly improves the molecular diagnosis for IRD-affected individuals, particularly in those of African ethnicity who are historically understudied.60–64 Atty. Dkt. No.: 114198-2110 Despite their genetic diversity, there is a scarcity of available genetic data, which makes the interpretation of pathogenicity of variants more challenging.45,65,66Example 2 - TMEM216 Constructs Methods Available individuals from seven genetically unresolved Pakistani pedigrees underwent ophthalmic evaluation including fundoscopy and ERG. Homozygosity mapping and segregation of rare non-coding variants with disease was performed. CRISPR-Cas9 was utilized to generate hTERT-RPE1 cells with the candidate non-coding variant for functional assessment. Luciferase assay, cell morphology evaluation, gene expression via qRT-PCR, immunostaining, bulk RNA-seq, and CUT&RUN using cells with A / A and G / A genotypes were performed. A genomic fragment spanning the TMEM2165’ untranslated region (5’ UTR) and upstream non-coding sequence (GRCh38 chr11: g.61391712-61392642) was chosen for dual reporter luciferase assays (931bp). The 931 bp region contains ATAC-seq, CRX, and OTX2, peaks (PMID: 35303433) and two predicted promoter sequences (P1 and P2) (PMID: 27899657) The fragment was chosen because of overlap with known retinal cis-regulatory elements (CREs) found in previous studies.37Six constructs were designed: wild-type, two versions harboring the variants c.-69G>A or c.-69G>T, and three constructs that removed predicted promoter 1, predicted promoter 2 and a promoterless sequence. (Table 1). Results Analysis of WGS identified a rare variant (GRChg38 chr11-61392563G>A; MAF: 0.00095 in South Asians) in a homozygous intergenic region of TMEM216 and TMEM138 segregating with arRD in 7 families of Pakistani origin with 3-8 affected members. This variant is located upstream of theTMEM216 (c.-69G>A) gene transcription start site and in an ATAC-seq peak in RPE and photoreceptor cells suggesting a regulatory role. Based on FIMO transcription factor (TF) motif analysis, the variant is predicted to impact binding of CHIP-seq validated TFs. Genome editing of hTERT-RPE1 cells resulted in cells homozygous (A / A) for the variant (with an additional C>T off target edit two bases distal) and heterozygous (G / A) without off target changes. qRT-PCR analysis of TMEM216 and Atty. Dkt. No.: 114198-2110 TMEM138 transcripts in these cells revealed significantly reduced expression of both transcripts. Further, the morphology of cilia in these cells was abnormal compared to wild type control cells. Luciferase assay, bulk RNA-sequencing data, and CUT&RUN sequencing results of candidate TF are being analyzed to further examine the impact of G>A change on TF binding and gene expression. Conclusions Genome analysis identified a rare non-coding variant GRChg38chr11:61,392,563G>A segregating with early-onset non-syndromic arRD in 7 Pakistani families. hTERT-RPE1 cells with this variant showed significantly lower levels of TMEM216 and TMEM138 transcripts and abnormal ciliary morphology. These results suggested that reduced expression of TMEM216 and TMEM138 leads to abnormal photoreceptor ciliogenesis and in non-syndromic RD in Pakistani patients. Example 3 - Development of an AAV vector This study investigates whether Adeno-associated virus (AAV)-delivered TMEM216 can restore cilia formation as a biomarker for restored TMEM216 expression and function in human retinal pigment epithelial (hTERT-RPE1) cells with mutations in the TMEM216 gene. Mutations in TMEM216, specifically the c.-69G>A and c.-69G>T variants in the 5'-UTR, have been associated with retinitis pigmentosa (RP), a degenerative retinal disease causing progressive vision loss. Two AAV vectors were produced, one carrying the full-length TMEM216 gene and another carrying a shorter isoform. Both vectors were diluted to various concentrations (1:100, 1:500, and 1:1000) for testing. See FIGS. 7 and 8 for vector maps and sequences provided herein. Two different homozygous mutant hTERT-RPE1 clones (D4 and F6) were generated using CRISPR / Cas9 to introduce the c.-69G>A mutation. CRISPR / Cas9 editing targeted the c.-69G>A mutation in TMEM216, introducing this mutation in the homozygous state in hTERT-RPE1 cells. Details of this procedure are described in PMID: 39191256. Atty. Dkt. No.: 114198-2110 Approximately 50% of the mutant cells showed cilia formation at a 1:500 dilution of the AAV vectors, indicating partial rescue of the cilia defect due to TMEM216 mutations. This suggests that the delivery of functional TMEM216 via AAV vectors can restore some level of cilia formation. Data shown in 9 is for the TMEM216-long isoform. Immunohistochemistry (IHC) showed that the HA-tagged TMEM216 protein was expressed in transduced cells ( 9C). Without wishing to be bound by theory, Applicant hypothesizes that the expression of wild-type TMEM216 is required for restoring the cilia formation. Example 4 – Gene Therapy to Restore Vision in Patients The restoration of cilia function is emerging as a vital biomarker for vision restoration in retinal diseases, given their essential role in maintaining retinal health. Cilia, sensory organelles found on various retinal cells, are crucial for phototransduction and the transport of vital proteins. Disruptions in ciliary function can lead to retinal degeneration and vision loss, as seen in conditions like retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Recent advancements in gene therapy have demonstrated the potential to restore ciliary function and improve vision. For example, researchers at the National Eye Institute developed a gene therapy targeting mutations in the NPHP5 gene associated with LCA. By introducing a functional version of NPHP5 into patient-derived retinal organoids, they observed significant restoration of ciliary structure and function, leading to improved localization of phototransduction proteins and enhanced photoreceptor activity. Applicant’s work focuses on the role of TMEM216 in maintaining ciliary structure and function. Mutations in this gene, particularly within the 5'-UTR are now linked to RP. Results show that AAV-mediated delivery of TMEM216 can partially restore cilia formation in mutant human retinal pigment epithelial (hTERT-RPE1) cells. This finding, combined with evidence from other studies, suggests that cilia restoration may serve as a valuable biomarker for the therapeutic efficacy of gene augmentation in retinal ciliopathies. Furthermore, studies have shown a strong correlation between cilia length and visual function in animal models of retinal degeneration. Restoration of cilia length and structure Atty. Dkt. No.: 114198-2110 has been associated with improved visual acuity and retinal function, supporting the notion that cilia restoration is a key indicator of therapeutic efficacy in gene therapy for retinal ciliopathies. These findings underscore the importance of cilia restoration as a biomarker for vision restoration. By monitoring ciliary health and function, clinicians can assess the effectiveness of therapeutic interventions and gain insights into the underlying mechanisms of retinal diseases, paving the way for more targeted and effective treatments. Applicant’s research aims to further validate the use of cilia restoration as a biomarker for vision restoration in the context of TMEM216-associated RP. By utilizing patient-derived iPSCs, retinal organoids, and in vivo models, Applicant can assess the correlation between cilia rescue, increase in the levels of TMEM216 expression and functional improvements in visual responses. This research advances the understanding of the disease mechanisms but also guide the development of effective gene therapies for RP and other related retinal ciliopathies. Applicant thus provides herein exemplary vectors with various promoter constructs, including a native promoter and cell-type-specific promoters, to maximize TMEM216 expression in RPE cells. These vectors are further optimized for targeted TMEM216 expression in RPE and photoreceptor cells, increasing efficacy and safety of gene therapy. Applicant also generated patient iPSCs, RPE cells and retinal organoids, and optimize AAV-mediated TMEM216 expression in these models. Methodology for patient iPSC-RPE generation, phenotype characterization and imaging are the same as described in https: / / pubmed.ncbi,nlm.nih.gov / 3325167 / , last accessed on June 26, 2025. Briefly, patient- derived iPSCs (induced pluripotent stem cells) were generated from individuals with c.- 69G>A mutation and no mutation (control) in TMEM216 and stained for cilia. Cilia are shown as “dots” in the center of the cells. 10A shows normal control cells. 10B shows iPSC derived RPE. The iPSC derived RPE cells do not show cilia (center dots), indicating the absence of cilia. In sum, RPE cell were generated from iPSCs of two patients (c.-69>A homozygous 1 and 2) and wildtype (wt) control. The levels of expression of TMEM216 were measured in these cells ( 10C). This study showed reduced TMEM216 expression in iPSC with the homozygous mutation compared to controls. Low levels of TMEM216 expression Atty. Dkt. No.: 114198-2110 and lack of cilia observed in patient iPSC-RPE is similar to the observations on edited hTERT-RPE cells with c.-69G>A mutation. This data shows that TMEM2165’ UTR mutations cause reduced levels of TMEM216 expression, leading to loss of cilia and retinal pathology. TMEM216 gene therapy can restore normal phenotype or reduce disease symptoms. Applicant also restored cilia phenotypes and increased levels of TMEM216 upon TMEM216 gene augmentation in edited hTERT-RPE1 with c.-69G>A. Cells, media, and antibodies were modified from the methods disclosed in https: / / pubmed.ncbi,nlm.nih.govand / 39191256 / , last accessed on June 26, 2025. Vector AAV8 with CAG-TMEM216-GFP, was inserted into cells TMEM expression increased following gene augmentation (FIGS. 11A and 11B). Experiment No. 5 - Evaluate Optimized Vector in RPE and Photoreceptor cell Conditional Knockout Mice as a Model System Due to the lack of conservation of the human TMEM216 regulatory sequence containing the c.-69G>A and c.-69G>T mutations in mice, a precise mouse model harboring these mutations cannot be generated. Applicant employs conditional knockout mice to study the delivery platform and assess its impact on photoreceptor and RPE cell function. Knockout mice can help understand the in vivo delivery efficiency and potential functional effects of TMEM216 augmentation. These methods and models can be used to refine and optimize TMEM216 gene therapy for treating RP, potentially leading to a viable therapeutic approach for restoring vision in patients with TMEM216 mutations. Clauses Clause 1. A method for treating or preventing a TMEM216-related disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one gene that supports or restores functional TMEM216 in the patient. Clause 2. The method of clause 1, wherein the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2. Atty. Dkt. No.: 114198-2110 Clause 3. The method of clause 1 or 2, wherein the subject in need harbors a mutation at the genomic location in the 5' UTR of GRCh38: chr11:g.61392563G, or from about 1000 to 1base pairs upstream of the start codon of the ciliopathy gene TMEM216, or at a location from about -1000 to about -1. Clause 4. The method of any of clauses 1-3, wherein the mutation is at the genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents of SEQ ID NO: 8. Clause 5. The method of any of clauses 1-4, wherein the at least one gene is administered in construct TMEM216_WT, or an equivalent thereof. Clause 6. The method of any of clauses 1-4, wherein the at least one gene is TMEM216, or a functional fragment thereof. Clause 7. The method of any of clauses 1-4, wherein the at least one gene is a TMEM216 isoform, optionally the long or the short isoform. Clause 8. The method of any of clauses 1-7, wherein the subject is a mammal, optionally a human patient. Clause 9. The method of any of clauses 1-8, wherein the effective amount of the gene is administered in a viral particle, wherein the viral particle is optionally derived from a herpes simplex virus, a retrovirus, an adenovirus, or an adeno-associated virus. Clause 10. The method of any of clauses 1-9, wherein the effective amount of the gene is administered in an adeno-associated viral (AAV) particle or a derivative or variant thereof. Clause 11. The method of clause 12, wherein the AAV is selected from an AAV serotype selected from the group of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, or AAV-11. Clause 12. The method of clause 11, wherein the AAV is AAV-8. Clause 13. A nucleic acid comprising the TMEM216_Native promoter-Short Isoform- AAV genome shown in 7, optionally without the HA tag or a marker sequence. Atty. Dkt. No.: 114198-2110 Clause 14. A nucleic acid comprising the TMEM216_Native promoter-Long Isoform- AAV genomeshown in 7, optionally without the HA tag or a marker sequence. Clause 15. A nucleic acid comprising the TMEM216_Short Isoform-AAV genome shown in 7, optionally without one or more of the AmpR gene, the Amp promoter, the HA tag or a marker sequence. Clause 16. A nucleic acid comprising the TMEM216_Long Isoform-AAV genome of shown in 7, optionally without one or more of the AmpR gene, the Amp promoter, the HA tag or a marker sequence. Claus 17.An adeno-associated virus particle (AAV) comprising the nucleic acid of any one of clauses 13-16. Clause 18. The AAV particle of clause 17, wherein the AAV is a serotype selected from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, or AAV-11. Clause 19. The AAV particle of clause 18, wherein the AAV is a serotype selected from AAV8, AAV-10, or AAV-11. Clause 20. The AAV particle of clause 18, wherein the AAV is AAV-8. Clause 21. A method for diagnosing or prognosing a TMEM216-related disorder in a subject, the method comprising detecting a mutation at the genomic location GRCh38: chr11:g.61392563G, 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 at a location from about -1000 to about -1 base pairs from the start codon, wherein a mutation at the location from about -1000 to about -1 base pairs from the start codon is a positive diagnosis or prognosis for the TMEM216-related disorder in the subject. Clause 22. The method of clause 21, wherein the mutation is at the genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof of SEQ ID NO: 8. Clause 23. The method of clause 22, wherein the mutation is at the genomic location selected from c.-95G>C or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof of SEQ ID NO: 8. Atty. Dkt. No.: 114198-2110 Clause 24. The method of any one of clauses 21-23, the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2. Clause 25. The method of any one of clauses 20-24, wherein the sample comprises a blood cell. Clause 26. The method of any one of clauses 21-25, further comprising administering an AAV particle of any one of claims 17-20. Clause 27. The method of any one of clauses 21 to 26, wherein the subject is a mammal, optionally a human patient. Partial Sequence Listing NM_001173991 1062 bp mRNA linear PRI 30-APR-2025 SOURCE Homo sapiens (human) REFERENCE 1 (bases 1 to 1062) FEATURES Location / Qualifiers source 1..1062 / organism="Homo sapiens" / mol_type="mRNA" / db_xref="taxon:9606" / chromosome="11" / map="11q12.2" gene 1..1062 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / note="transmembrane protein 216" Atty. Dkt. No.: 114198-2110 / db_xref="GeneID:51259" / db_xref="HGNC:HGNC:25018" / db_xref="MIM:613277" exon 1..79 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / inference="alignment:Splign:2.1.0" CDS 46..492 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / note="isoform 3 is encoded by transcript variant 3" / codon_start=1 / product="transmembrane protein 216 isoform 3" / protein_id="NP_001167462.1" / db_xref="CCDS:CCDS86205.1" / db_xref="GeneID:51259" / db_xref="HGNC:HGNC:25018" / db_xref="MIM:613277" SEQ ID NO: 7 Atty. Dkt. No.: 114198-2110 / translation="MLPRGLKMAPRGKRLSSTPLEILFFLNGWYNATYFLLELFIFLYKGVLL PYPTANLVLDVVMLLLYLGIEVIRLFFGTKGNLCQRKMPLSISVALTFPSAMMASYYLLLQTY VLRLEAIMNGILLFFCGSELLLEVLTLAAFSSMDRI misc_feature 109..171 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / note="propagated from UniProtKB / Swiss-Prot (Q9P0N5.3); transmembrane region" misc_feature 211..273 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / note="propagated from UniProtKB / Swiss-Prot (Q9P0N5.3); transmembrane region" misc_feature 310..372 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / note="propagated from UniProtKB / Swiss-Prot (Q9P0N5.3); transmembrane region" misc_feature 409..471 / gene="TMEM216" / gene_synonym="HSPC244; RP98" Atty. Dkt. No.: 114198-2110 / note="propagated from UniProtKB / Swiss-Prot (Q9P0N5.3); transmembrane region" exon 80..181 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / inference="alignment:Splign:2.1.0" exon 182..274 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / inference="alignment:Splign:2.1.0" exon 275..476 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / inference="alignment:Splign:2.1.0" exon 477..1062 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / inference="alignment:Splign:2.1.0" regulatory 1041..1046 / regulatory_class="polyA_signal_sequence" Atty. Dkt. No.: 114198-2110 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / note="hexamer: AATAAA" polyA_site 1062 / gene="TMEM216" / gene_synonym="HSPC244; RP98" / note="major polyA site" ORIGIN SEQ ID NO: 8 1 gtttccggca gcgccgcgct gctccgggag ccgctgtggc agcgtatgct gccacgggga 61 ctgaagatgg cgccgcgagg taaacggttg tcctccaccc cgctggaaat cctgttcttt 121 ctgaacgggt ggtataatgc tacctatttc ctgctggaac ttttcatatt tctgtataaa 181 ggtgtcctgc taccatatcc aacagctaac ctagtactgg atgtggtgat gctcctcctt 241 tatcttggaa ttgaagtaat tcgcctgttt tttggtacaa agggaaacct ctgccagcga 301 aagatgccgc tcagtattag cgtggccttg accttcccat ctgccatgat ggcctcctat 361 tacctgctgc tgcagaccta cgtactccgc ctggaagcca tcatgaatgg catcttgctc 421 ttcttctgtg gctcagagct tttacttgag gtgctcacct tggctgcttt ctccagtatg 481 gacaggattt gaagtacaga atttcagcca gcagcccatc aggctgacac cacacatatt 541 gcttctggta ctttagccac accagtgaga attggtgggg caagttgtcc tgagaaaggc 601 tgtgtggctt ttcttcagca cagacatttg ggcaagcaac tcagcataag gccagtgggt 661 accatcttct aaaccaggac catcagccca agagactctt ctacactcca gtatagggag 721 gggcaaggtt attcccatcc tgccccttct cagaaccagt cccctgctga cctcaagttc 781 tcctccttga tcaccgtggc cagagcatct cgtgtggacc atctaggctc cttgggcttc 841 aagcaggacc tgagccacat gctccctgta cgagctgtgc tatacctgtc ccacatgagc 901 acggagagcc tcatgttggt gggtttccag agtgatgtga aagcctctca ccccaatcct 961 cggagactga gttccacaac ttttttagta gctcatagtg ttatttttct actctcttca 1021 tgaaactaac tttattttat aataaatatg tattttctgt tg / / Atty. Dkt. No.: 114198-2110 TMEM216 Long and short isoform sequences and the maps of vectors containing these isoforms for delivery to retinal cells under the Native^promoter. TMEM216‐Native^promoter‐Long^isoform^DNA^sequence^in^a^Plasmid^flanked^by^ ITR^–^SEQ^ID^NO:^9^ TMEM216 Long Isoform (4708 bp) 1. ITR: 405..534 2. TMEM216-Native promoter: 590..1509 3. TMEM216-Native promoter- Long isoform ORF with a linker and HA tag: ^ -69G: 1441 ^ Start Codon: 1510..1512 ^ TMEM216 NM_016499.6 Long isoform: 1510..1953 ^ Linker: 1954..1968 ^ HA tag: 1969..1995 ^ Stop codon: 1996..1998 4. ITR: 2113..2442 5. Ori: 2889..3477 6. AmpR: complement (3648..4508) 7. AMP Promoter: complement (4509..4613) ORIGIN 601 TCACTGTCGC CCAGGCTGGA GTGCGGTGGC GCGATCTCAG CTCACTGCAA CCTCCGCCTC 661 CCGGGTTCGA GCGATTCTCC TACCTCAGCC TCCCAAGTAG CTGTAACTAC TGGCGCGCGC 721 CAGTACGCCC GGCTAATTTT TGTATTTTTA GTAGAGACGG GGTTTCGCCA TGTTGGCCAG 781 GCTGATTTCG AACTCCTGAC CTCAGGTGAT CCATCACCTC GGCCTCCCAA AGTGCTGGGA 841 TTACAGGCGT GAGCCACTAA GCCCGGCCAG CAACACGCTT TTACCGCTGC AAATCCGGAT 901 CAGGCAGGCT TTGAAGCCCA GCTCACAGGC CGTCTCTTGG AAACTAACTT AGATTTCCAC 961 CTGGGAGCAC TTTGTCCTCT CAGCTCCCAC TGCACCACTT TATGGATCGC GTGGTGACTC 1021 GTTTCATTTT ATTTATGTGT GTTCCTGGAA AGGACGCTCT ATTTCCTTCG TGTCTCTACC 1081 TGGTAGGTAA ATCGGCCGCA GAAAAGACCA ATGCTAACTC CACCCTGCAT TTGCAGAGAT 1141 CCGTCGCTCA CACCTTTTCT TGTCTTCCGT TGAAAAAAGA GAATCGTGGG AGTAGGACCT Atty. Dkt. No.: 114198-2110 1201 ATTTGCCAAT CTCCTGGTAA CACTATCACA ACTGGCCACT TCCGCCCAAC GCCCGGTAGT 1261 CTGAGAGCCT CCAAGGTCCT CCCCCGCACC CCTGAGGTCC TGCAGACGAC GGCGTCGTGG 1321 GTGGTCACCG TTATCCCTTA GGTCTGGAGA GGGGACATCC GAGCGAGGGC CACTTGCGGC 1381 CAGGCCCGAG CTCGTCCAGC TCCGGGTGAC CACAGAGTGC CGCGGGCGGG CAGAGGGGCC 1441 GGAAACCCAG GCCGCTTCGT CCCTGTTTCC GGCAGCGCCG CGCTGCTCCG GGAGCCGCTG Atty. Dkt. No.: 114198-2110 3961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 4021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 4081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 4141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 4201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 4261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 4321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 4381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 4441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 4501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 4561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 4621 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 4681 aataggcgta tcacgaggcc ctttcgtc TMEM216‐Long^isoform^protein^sequence^(with^a^linker^and^HA^tag)^–^SEQ^ID^NO:^ 10^ MLPRGLKMAPRGKRLSSTPLEILFFLNGWYNATYFLLELFIFLYKGVLLPYPTANLVL DVVMLLLYLGIEVIRLFFGTKGNLCQRKMPLSISVALTFPSAMMASYYLLLQTYVLR LEAIMNGILLFFCGSELLLEVLTLAAFSSMDRIGGGGSYPYDVPDYA ^ TMEM216‐Native^promoter‐Short^isoform^DNA^sequence^in^a^Plasmid^flanked^by^ ITR^–^SEQ^ID^NO:^11^ TMEM216 -Native promoter-short Isoform (4525 bp) 8. ITR: 405..534 9. TMEM216-Native promoter: 590..1509 10. TMEM216-Native promoter- Short isoform ORF with a linker and HA tag: ^ -69G: 1441 ^ Start Codon: 1510..1512 ^ TMEM216 NM_016499.6 Long isoform: 1510..1770 ^ Linker: 1771..1785 ^ HA tag: 1786..1812 ^ Stop codon: 1813-1815 11. ITR: 2130..2259 12. Ori: 2706..3294 13. AmpR: complement (3465..4325) 14. AMP Promoter: complement (4326..4430) Atty. Dkt. No.: 114198-2110 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccagctgcgc gctcgctcgc 421 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag 481 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt 541 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagatcG AGACAGAGTC 601 TCACTGTCGC CCAGGCTGGA GTGCGGTGGC GCGATCTCAG CTCACTGCAA CCTCCGCCTC 661 CCGGGTTCGA GCGATTCTCC TACCTCAGCC TCCCAAGTAG CTGTAACTAC TGGCGCGCGC 721 CAGTACGCCC GGCTAATTTT TGTATTTTTA GTAGAGACGG GGTTTCGCCA TGTTGGCCAG 781 GCTGATTTCG AACTCCTGAC CTCAGGTGAT CCATCACCTC GGCCTCCCAA AGTGCTGGGA 841 TTACAGGCGT GAGCCACTAA GCCCGGCCAG CAACACGCTT TTACCGCTGC AAATCCGGAT 901 CAGGCAGGCT TTGAAGCCCA GCTCACAGGC CGTCTCTTGG AAACTAACTT AGATTTCCAC 961 CTGGGAGCAC TTTGTCCTCT CAGCTCCCAC TGCACCACTT TATGGATCGC GTGGTGACTC 1021 GTTTCATTTT ATTTATGTGT GTTCCTGGAA AGGACGCTCT ATTTCCTTCG TGTCTCTACC 1081 TGGTAGGTAA ATCGGCCGCA GAAAAGACCA ATGCTAACTC CACCCTGCAT TTGCAGAGAT 1141 CCGTCGCTCA CACCTTTTCT TGTCTTCCGT TGAAAAAAGA GAATCGTGGG AGTAGGACCT 1201 ATTTGCCAAT CTCCTGGTAA CACTATCACA ACTGGCCACT TCCGCCCAAC GCCCGGTAGT 1261 CTGAGAGCCT CCAAGGTCCT CCCCCGCACC CCTGAGGTCC TGCAGACGAC GGCGTCGTGG 1321 GTGGTCACCG TTATCCCTTA GGTCTGGAGA GGGGACATCC GAGCGAGGGC CACTTGCGGC 1381 CAGGCCCGAG CTCGTCCAGC TCCGGGTGAC CACAGAGTGC CGCGGGCGGG CAGAGGGGCC 1441 GGAAACCCAG GCCGCTTCGT CCCTGTTTCC GGCAGCGCCG CGCTGCTCCG GGAGCCGCTG 1501 TGGCAGCGTa tgctcctcct ttatcttgga attgaagtaa ttcgcctgtt ttttggtaca 1561 aagggaaacc tctgccagcg aaagatgccg ctcagtatta gcgtggcctt gaccttccca 1621 tctgccatga tggcctccta ttacctgctg ctgcagacct acgtactccg cctggaagcc 1681 atcatgaatg gcatcttgct cttcttctgt ggctcagagc ttttacttga ggtgctcacc 1741 ttggctgctt tctccagtat ggacaggatt gggggtggag gctcttatcc ttacgacgtg 1801 cctgactacg cctaagccct agagctcgct gatcagcctc gactgtgcct tctagttgcc 1861 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 1921 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 1981 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc 2041 atgctgggga cctagatatt cctaatcgag gtcctttcta gagcatggct acgtagataa 2101 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc 2161 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 2221 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tggtcgactg cagaggcctg 2281 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 2341 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 2401 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 2461 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 2521 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 2581 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 2641 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg Atty. Dkt. No.: 114198-2110 2701 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 2761 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 2821 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 2881 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 2941 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3001 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3061 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 3121 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 3181 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 3241 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 3301 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 3361 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 3421 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 3481 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 3541 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 3601 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 3661 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 3721 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 3781 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 3841 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 3901 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 3961 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 4021 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 4081 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 4141 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 4201 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 4261 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 4321 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 4381 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 4441 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 4501 aggcgtatca cgaggccctt tcgtc TMEM216‐Short^isoform^protein^sequence^(with^a^linker^and^HA^tag)^–^SEQ^ID^NO:^ 12^ ^ MLLLYLGIEVIRLFFGTKGNLCQRKMPLSISVALTFPSAMMASYYLLLQTYVLRLEAI MNGILLFFCGSELLLEVLTLAAFSSMDRIGGGGSYPYDVPDYA The below are details of the TMEM216 Long and short isoform sequences and the maps of vectors containing these isoforms for delivery to retinal cells. CAG^promoter^was used. Atty. Dkt. No.: 114198-2110 TMEM216‐Long^isoform^DNA^sequence^in^a^Plasmid^flanked^by^ITR^‐^CAG^ promoter^–^SEQ^ID^NO:^13^ TMEM216 Long Isoform (5513 bp) 15. ITR: 405..534 16. CAG promoter / other sequences: 590..2255 17. TMEM216 Long isoform ORF with a linker and HA tag: ^ Start Codon: 2315..2317 ^ TMEM216 NM_016499.6 Long isoform: 2315..2758 ^ Linker: 2759..2773 ^ HA tag: 2774..2800 ^ Stop codon: 2801..2803 18. ITR: 3118..3247 19. Ori: 3694..4282 20. AmpR: complement (4453..5313) 21. AMP Promoter: complement (5314..5418) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccagctgcgc gctcgctcgc 421 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag 481 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt 541 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagatcg acattgatta 601 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 661 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 721 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 781 cgtcaatggg tggactattt acggtaaact gcccacttgg cagtacatca agtgtatcat 841 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 901 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 961 attaccatgg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc 1021 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg 1081 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 1141 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 1201 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgt 1261 tgccttcgcc ccgtgccccg ctccgcgccg cctcgcgccg cccgccccgg ctctgactga 1321 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc 1381 gcttggttta atgacggctc gtttcttttc tgtggctgcg tgaaagcctt aaagggctcc 1441 gggagggccc tttgtgcggg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg 1501 gggagcgccg cgtgcggccc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg 1561 ggctttgtgc gctccgcgtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg 1621 cgggggggct gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca Atty. Dkt. No.: 114198-2110 1681 gggggtgtgg gcgcggcggt cgggctgtaa cccccccctg cacccccctc cccgagttgc 1741 tgagcacggc ccggcttcgg gtgcggggct ccgtgcgggg cgtggcgcgg ggctcgccgt 1801 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg 1861 ggagggctcg ggggaggggc gcggcggccc cggagcgccg gcggctgtcg aggcgcggcg 1921 agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc 1981 caaatctggc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggc 2041 gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg 2101 ccgccgtccc cttctccatc tccagcctcg gggctgccgc agggggacgg ctgccttcgg 2161 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct 2221 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt 2281 gtgctgtctc atcattttgg caaagaattg caccatgctg ccacggggac tgaagatggc 2341 gccgcgaggt aaacggttgt cctccacccc gctggaaatc ctgttctttc tgaacgggtg 2401 gtataatgct acctatttcc tgctggaact tttcatattt ctgtataaag gtgtcctgct 2461 accatatcca acagctaacc tagtactgga tgtggtgatg ctcctccttt atcttggaat 2521 tgaagtaatt cgcctgtttt ttggtacaaa gggaaacctc tgccagcgaa agatgccgct 2581 cagtattagc gtggccttga ccttcccatc tgccatgatg gcctcctatt acctgctgct 2641 gcagacctac gtactccgcc tggaagccat catgaatggc atcttgctct tcttctgtgg 2701 ctcagagctt ttacttgagg tgctcacctt ggctgctttc tccagtatgg acaggattgg 2761 gggtggaggc tcttatcctt acgacgtgcc tgactacgcc taagccctag agctcgctga 2821 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 2881 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 2941 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 3001 ggggaggatt gggaagagaa tagcaggcat gctggggacc tagatattcc taatcgaggt 3061 cctttctaga gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg 3121 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 3181 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 3241 cgcgcagctg gtcgactgca gaggcctgca tgcaagcttg gcgtaatcat ggtcatagct 3301 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 3361 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 3421 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 3481 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 3541 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 3601 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 3661 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 3721 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 3781 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 3841 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 3901 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 3961 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 4021 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 4081 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 4141 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 4201 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 4261 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 4321 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 4381 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac Atty. Dkt. No.: 114198-2110 4441 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 4501 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 4561 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 4621 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 4681 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 4741 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 4801 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 4861 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 4921 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 4981 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 5041 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 5101 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 5161 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 5221 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 5281 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 5341 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 5401 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 5461 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc ^ TMEM216‐Long^isoform^protein^sequence^(with^a^linker^and^HA^tag)^–^SEQ^ID^NO:^ 14^ MLPRGLKMAPRGKRLSSTPLEILFFLNGWYNATYFLLELFIFLYKGVLLPYPTANLVL DVVMLLLYLGIEVIRLFFGTKGNLCQRKMPLSISVALTFPSAMMASYYLLLQTYVLR LEAIMNGILLFFCGSELLLEVLTLAAFSSMDRIGGGGSYPYDVPDYA TMEM216‐Short^isoform^DNA^sequence^in^a^Plasmid^flanked^by^ITR^‐^CAG^ promoter^–^SEQ^ID^NO:^15^ TMEM216 Short Isoform (5330 bp) 1. ITR: 405..534 2. CAG promoter / other sequences: 590..2255 3. TMEM216 Short isoform ORF with a linker and HA tag: ^ Start codon: 2315..2317 ^ TMEM216 NM_016499.6 short isoform: 2315..2575 ^ Linker: 2576..2590 ^ HA tag: 2591..2617 ^ Stop codon: 2618..2620 4. ITR: 2935..3064 5. Ori: 3511..4099 Atty. Dkt. No.: 114198-2110 6. AmpR: complement (4270..5130) 7. AMP PROMOTER: complement (5131..5235) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccagctgcgc gctcgctcgc 421 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag 481 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt 541 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagatcg acattgatta 601 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 661 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 721 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 781 cgtcaatggg tggactattt acggtaaact gcccacttgg cagtacatca agtgtatcat 841 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 901 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 961 attaccatgg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc 1021 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg 1081 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 1141 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 1201 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgt 1261 tgccttcgcc ccgtgccccg ctccgcgccg cctcgcgccg cccgccccgg ctctgactga 1321 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc 1381 gcttggttta atgacggctc gtttcttttc tgtggctgcg tgaaagcctt aaagggctcc 1441 gggagggccc tttgtgcggg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg 1501 gggagcgccg cgtgcggccc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg 1561 ggctttgtgc gctccgcgtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg 1621 cgggggggct gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca 1681 gggggtgtgg gcgcggcggt cgggctgtaa cccccccctg cacccccctc cccgagttgc 1741 tgagcacggc ccggcttcgg gtgcggggct ccgtgcgggg cgtggcgcgg ggctcgccgt 1801 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg 1861 ggagggctcg ggggaggggc gcggcggccc cggagcgccg gcggctgtcg aggcgcggcg 1921 agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc 1981 caaatctggc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggc 2041 gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg 2101 ccgccgtccc cttctccatc tccagcctcg gggctgccgc agggggacgg ctgccttcgg 2161 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct 2221 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt 2281 gtgctgtctc atcattttgg caaagaattg caccatgctc ctcctttatc ttggaattga 2341 agtaattcgc ctgttttttg gtacaaaggg aaacctctgc cagcgaaaga tgccgctcag 2401 tattagcgtg gccttgacct tcccatctgc catgatggcc tcctattacc tgctgctgca 2461 gacctacgta ctccgcctgg aagccatcat gaatggcatc ttgctcttct tctgtggctc 2521 agagctttta cttgaggtgc tcaccttggc tgctttctcc agtatggaca ggattggggg 2581 tggaggctct tatccttacg acgtgcctga ctacgcctaa gccctagagc tcgctgatca Atty. Dkt. No.: 114198-2110 2641 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2701 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2761 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2821 gaggattggg aagagaatag caggcatgct ggggacctag atattcctaa tcgaggtcct 2881 ttctagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac 2941 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 3001 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 3061 gcagctggtc gactgcagag gcctgcatgc aagcttggcg taatcatggt catagctgtt 3121 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 3181 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 3241 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 3301 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 3361 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 3421 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 3481 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3541 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 3601 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 3661 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 3721 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3781 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3841 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3901 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 3961 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 4021 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4081 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4141 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4201 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4261 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4321 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 4381 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 4441 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 4501 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 4561 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 4621 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 4681 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 4741 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 4801 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 4861 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 4921 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 4981 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 5041 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 5101 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 5161 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 5221 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 5281 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc Atty. Dkt. No.: 114198-2110 TMEM216‐Short^isoform^protein^sequence^(with^a^linker^and^HA^tag)^–^SEQ^ID^NO:^ 16^ MLLLYLGIEVIRLFFGTKGNLCQRKMPLSISVALTFPSAMMASYYLLLQTYVLRLEAI MNGILLFFCGSELLLEVLTLAAFSSMDRIGGGGSYPYDVPDYA Equivalents Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosure embodied therein herein disclosed can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. Several references are identified by an Arabic number, and the full bibliographic citation or these references are provided below. In case of conflict, the present specification, including definitions, will control. Atty. Dkt. No.: 114198-2110 References 1. 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Claims

Atty. Dkt. No.: 114198-2110 WHAT IS CLAIMED IS:

1. A method for treating or preventing a TMEM216-related disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one gene that supports or restores functional TMEM216 in the patient.

2. The method of claim 1, wherein the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2.

3. The method of claim 1 or 2, wherein the subject in need harbors a mutation at the genomic location in the 5’ UTR of GRCh38: chr11:g.61392563G, or from about 1000 to 1base pairs upstream of the start codon of the ciliopathy gene TMEM216, or at a location from about -1000 to about -1.

4. The method of any of claims 1-3, wherein the mutation is at the genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents of SEQ ID NO:

8.

5. The method of any of claims 1-4, wherein the at least one gene is administered in construct TMEM216_WT, or an equivalent thereof.

6. The method of any of claims 1-4, wherein the at least one gene is TMEM216, or a functional fragment thereof.

7. The method of any of claims 1-4, wherein the at least one gene is a TMEM216 isoform, optionally the long or the short isoform.

8. The method of any of claims 1-7, wherein the subject is a mammal, optionally a human patient.

9. The method of any of claims 1-8, wherein the effective amount of the gene is administered in a viral particle, wherein the viral particle is optionally derived from a herpes simplex virus, a retrovirus, an adenovirus, or an adeno-associated virus.Atty. Dkt. No.: 114198-2110 10. The method of any of claims 1-9, wherein the effective amount of the gene is administered in an adeno-associated viral (AAV) particle or a derivative or variant thereof.

11. The method of claim 12, wherein the AAV is selected from an AAV serotype selected from the group of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, or AAV-11.

12. The method of claim 11, wherein the AAV is AAV-8.

13. A nucleic acid comprising the TMEM216_Native promoter-Short Isoform-AAV genome shown in 7, optionally without the HA tag or a marker sequence.

14. A nucleic acid comprising the TMEM216_Native promoter-Long Isoform-AAV genomeshown in 7, optionally without the HA tag or a marker sequence.

15. A nucleic acid comprising the TMEM216_Short Isoform-AAV genome shown in 7, optionally without one or more of the AmpR gene, the Amp promoter, the HA tag or a marker sequence.

16. A nucleic acid comprising the TMEM216_Long Isoform-AAV genome of shown in 7, optionally without one or more of the AmpR gene, the Amp promoter, the HA tag or a marker sequence.

17. An adeno-associated virus particle (AAV) comprising the nucleic acid of any one of claims 13-16.

18. The AAV particle of claim 17, wherein the AAV is a serotype selected from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, or AAV-11.

19. The AAV particle of claim 18, wherein the AAV is a serotype selected from AAV8, AAV-10, or AAV-11.

20. The AAV particle of claim 18, wherein the AAV is AAV-8.Atty. Dkt. No.: 114198-2110 21. A method for diagnosing or prognosing a TMEM216-related disorder in a subject, the method comprising detecting a mutation at the genomic location GRCh38: chr11:g.61392563G, 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 at a location from about -1000 to about -1 base pairs from the start codon, wherein a mutation at the location from about -1000 to about -1 base pairs from the start codon is a positive diagnosis or prognosis for the TMEM216-related disorder in the subject.

22. The method of claim 21, wherein the mutation is at the genomic location selected from c.-69G>A, c.-69G>T, c.-69G>C, c.-95G>C, or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof of SEQ ID NO:

8.

23. The method of claim 21, wherein the mutation is at the genomic location selected from c.-95G>C or c.-41C>T of the TMEM216 gene (SEQ ID NO: 8) or equivalents thereof of SEQ ID NO:

8.

24. The method of any one of claims 21-23, the TMEM216-related disorder is retinitis pigmentosa (RP) or Joubert syndrome 2.

25. The method of any one of claims 21-24, wherein the sample comprises a blood cell.

26. The method of any one of claims 21-24, further comprising administering an AAV particle of any one of claims 17-20.

27. The method of any one of claims 21 to 26, wherein the subject is a mammal, optionally a human patient.

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