Rpgr gene therapy for retinitis pigmentosa

Abstract

A method for treating a human subject suffering from X-linked retinitis pigmentosa (XLRP) or another clinically defined ophthalmic condition due to a loss-of-function mutation in a gene encoding a protein encoding the GTPase regulatory factor (RPGR) protein of retinitis pigmentosa, the method comprising administering a nucleic acid comprising an adeno-associated viral vector containing a shortened human RPGR cDNA to the subject.

Description

[0001] This application is a divisional application. The original application was filed on July 17, 2015, with application number 201580051512.3, and was entitled "RPGR gene therapy for retinitis pigmentosa".

[0002] Priority requirements This application claims priority to U.S. Patent Application Serial No. 62 / 028,638, filed July 24, 2014, pursuant to 35 USC § 119(e). The entire contents of the aforementioned patent application are incorporated herein by reference.

[0003] Federally funded research or development This invention was made with government support from grants EY10581 and 5P30EY14104 granted by the National Institutes of Health. The government holds certain rights to this invention. Technical Field

[0004] This invention relates to a method for treating a human subject suffering from X-linked retinitis pigmentosa (XLRP) or another ophthalmic condition caused by a loss-of-function mutation in a gene encoding a protein encoding the GTPase regulatory factor (RPGR) protein of retinitis pigmentosa, the method comprising administering a nucleic acid comprising an adeno-associated viral vector containing a shortened human RPGR cDNA to the subject. Background Technology

[0005] Retinitis pigmentosa (RP) is the leading form of inherited blindness in humans. Of the three common inheritance patterns (autosomal dominant, autosomal recessive, and X-linked), X-linked RP (XLRP) is associated with a severe form of disease involving rods and cones as primary targets (Berson 1993; Sandberg and others 2007). More than 70% of X-linked RP cases and 10-20% of all RP cases are caused by mutations in the gene encoding RPGR (Bader and others 2003; Branham and others 2012; Churchill and others; Pelletier and others 2007). Given that mutations in more than 100 genes are currently known to cause RP and more severe X-linked disease, RPGR is one of the most important genes associated with RP. Summary of the Invention

[0006] This invention is based on the discovery of successfully regenerating a shortened form of human RPGR with functional RPGR activity, and therefore includes a method for treating subjects suffering from RP caused by mutations in RPGR. Subjects who can be treated by the method of this invention may include those with visual function loss (e.g., impaired response to an electroretinogram (ERG) test) but who retain some photoreceptor cells (as determined by optical coherence tomography (OCT)).

[0007] Therefore, in one aspect, the present invention provides a method for treating a human subject suffering from XLRP or another clinically defined ophthalmic condition due to a loss-of-function mutation in a gene encoding a GTPase regulatory factor (RPGR) protein for retinitis pigmentosa. The method comprises administering a nucleic acid comprising an adeno-associated viral vector containing a shortened human RPGR cDNA, wherein the shortened human RPGR cDNA encodes a protein at least 80% identical in length to SEQ ID NO:2, the protein optionally having a deletion of up to 200 additional amino acids in the region surrounding the deletion region in SEQ ID NO:2 (i.e., between amino acids 861 and 862 of SEQ ID NO:2).

[0008] In some implementations, the RPGR cDNA is controlled by a human rhodopsin kinase (hRK) promoter (e.g., an hRK promoter comprising SEQ ID NO:5 or substantially consisting of SEQ ID NO:5).

[0009] In some embodiments, the adeno-associated virus vector is AAV-2, serotype-8 (AAV2 / 8), or AAV-8.

[0010] In some embodiments, the RPGR cDNA contains at least 80% the same sequence as SEQ ID NO: 1 or is substantially composed of that sequence.

[0011] In some embodiments, the human RPGR cDNA encodes a protein that is at least 95% identical in length to the full-length SEQ ID NO:2.

[0012] In some implementations, the method includes using approximately 2 × 10 10 A low dose of vg / mL, approximately 2×10 11 A moderate dose of vg / mL, or about 2×10 12The nucleic acid is administered at a high dose of vg / mL. In some embodiments, the nucleic acid is administered into the subretinal space. In some embodiments, a microinjection cannula is inserted into the subretinal space—located in the temporal region of the optic nerve and just above the major arcade vessel—to allow fluid flow toward the macula of the retina.

[0013] In another aspect, the present invention provides a nucleic acid encoding a shortened human RPGR, wherein the shortened human RPGR cDNA encodes a protein that is at least 80% identical to the full length of SEQ ID NO:2, the protein optionally having a deletion of up to 200 additional amino acids around the deletion region in SEQ ID NO:2.

[0014] In some implementations, the RPGR cDNA is controlled by a human rhodopsin kinase (hRK) promoter (e.g., an hRK promoter comprising SEQ ID NO:5 or substantially consisting of SEQ ID NO:5).

[0015] In some embodiments, the RPGR cDNA contains at least 80% the same sequence as SEQ ID NO: 1 or is substantially composed of that sequence.

[0016] In some embodiments, the human RPGR cDNA encodes a protein that is at least 95% identical in length to the full-length SEQ ID NO:2.

[0017] In some embodiments, the human RPGR cDNA is at least 80% identical to the full length of SEQ ID NO: 1, and optionally has deletions of nucleotides encoding up to 200 additional amino acids around the deletion region.

[0018] This document also provides vectors, such as adeno-associated virus vectors, such as AAV-2, serotype-8 (AAV2 / 8), or AAV-8, which contain nucleic acids encoding the shortened human RPGR described herein, and isolated cells (i.e. cells not present in live human subjects or host animals) containing nucleic acids encoding the shortened human RPGR and optionally expressing the shortened human RPGR protein.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials used in this invention are described herein; alternatively, suitable methods and materials known in the art may also be used. The materials, methods, and examples described are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0020] Other features and advantages of the invention will become apparent from the following detailed description, accompanying drawings, and claims. Attached Figure Description

[0021] Figure 1A -B: (A) Diagram of the coding region of human RPGR ORF15 and human ORF15 cDNA delivered in two shortened forms of AAV. (B) Two recombinant human ORF15 cDNAs. RPGR-ORF15 Immunoblot. AAV delivery of small deletions in human cDNA ( AAV- ORF15-L The "long" shape leads to the expression of the human RPGR-ORF15 protein, which is approximately 160 kD in size. Large deletions in human cDNA result in AAV delivery (…). AAV-ORF15-S The "short form" results in the expression of a protein approximately 130 kD in size. Both forms of human RPGR-ORF15 protein are smaller than the endogenous human RPGR ORF15 (approximately 200 kD) found in human retinal tissue.

[0022] Figure 2A -D: In AAV-RPGR ORF15 After subretinal delivery, RPGR - / - RPGRORF 15 expression in mouse retina. (A) Fluorescence images of the short (ORF15-S) and long (ORF15-L) forms of human RPGR ORF15 protein superimposed on a Nomarski image to illustrate the layering of the outer retina. Unfixed frozen retinal sections were stained 3 weeks after treatment at 1–2 months of age. (B) Fluorescence images of the two forms of human RPGR ORF15 colocalizing with rootletin. Similar to wild-type, the two forms of human RPGR ORF15 are precisely located at the photoreceptor junction cilia just distal to rootletin. RPE, retinal pigment epithelium; OS, outer segment; CC (TZ), junction cilia (transition zone); IS, inner segment; ONL, outer nuclear layer. (C) For treatment with ORF15-S. Rpgr - / - Eyes (n=3), processed using ORF15-L Rpgr - / - The ratio of hRPGR fluorescent particles to fluorescent microradicin fibers at the cilia junction was calculated for both wild-type and mid-type eyes (n=3). Microradicin fibers within the inner segment and RPGRs just distal to the microradicin fibers on a 100 μm long mid-peripheral retina were counted. Values ​​are presented as mean ± standard error. (D) Rpgr - / - Expression patterns of short and long ORF15 proteins in fixed, floating retinal sections from mice. Sections were stained 4–6 weeks after treatment at 2–3 months of age to localize human RPGR ORF15 protein. In wild-type retinas, mouse RPGR ORF15 protein was observed as discrete green fluorescent signals (dots) occupying the region between the inner and outer segments of photoreceptor cells, at the level of the transition zone or connective cilia. In contrast, the fluorescence signal of short ORF15 (AAV-ORF15-S) was not limited to the level of the connective cilia of photoreceptor cells, but it was also observed as a discrete signal throughout the inner and outer segments. The fluorescence signal of long ORF15 showed very few (if any) mislocalizations and was mainly limited to connective cilia regions similar to those in wild-type mice. OS, outer segment; CC (TZ), connective cilia (transition zone); IS, inner segment; ONL, outer nuclear layer.

[0023] Figure 3 At 13 months of age (6 months post-injection), the treated (long and short ORF 15) and control RPGR were compared. - / - Immunohistochemical analysis (initially yellow) of rod and cone cells in the mouse retina. RPGR treated with short ORF 15 (AAV8-ORF15-s) - / - In the mouse retinas, the mislocalization staining patterns of rhodopsin and cone opsins (mixed S&M cones in the lower half of the retina) were virtually indistinguishable from those observed in control retinas. In both mouse retinas, mislocalization of cone opsins was noted in the inner segment and synaptic layer. Similarly, compared to age-matched wild-type retinas, the outer segments of rods and cones were shortened and disordered, with a reduced outer nuclear layer. In contrast, RPGR treated with long ORF15 (AAV8-ORF15-1) showed improved contrast. - / - In the mouse retina, rhodopsin exhibits partitioning similar to that of wild-type mouse retinas. Furthermore, compared to the control retina, the outer segments of the rods are longer and more organized in the ORF 15 long-type treated retina, and the on-row lenticule (ONL) is thicker. Compared to the control, the RPGR in the ORF 15 long-type treated retina is also higher. - / - In the mouse retina, cone opsin staining revealed more cone cells with elongated and well-organized outer segments.

[0024] Figure 4A -B: In RPGR - / - Rescue of photoreceptor cells in mice after treatment with RPGR ORF 15-1. (A) Stacked bar graphs showing ONL thickness (top) and IS / OS length (bottom) in the treated eye (initially red) and contralateral control eye (initially blue) of three 18-month-old mice. (B) From wild-type mice and from RPGR ORF 15-1 mice. - / - Representative optical micrographs of the ORF15-1 treated eye and the contralateral control eye of mice. Images were taken from the mid-periphery of the upper retina along the vertical meridian.

[0025] Figure 5A -C: (A) From 16 RPGRs aged 11-14 months - / - Amplitudes of rod a-wave, rod b-wave, and cone b-wave in mice. Compared to the lower limit of wild-type mice, the control eye (OD) showed a disproportionate loss in cone b-wave amplitude relative to rod b-wave amplitude. Except in one case, all treated eyes (OS) showed a greater response than the contralateral control eye. In particular, it was noted that more than half of the treated eyes at this age had rod ERG b-wave amplitudes equal to or greater than the wild-type lower limit. The mean values ​​of all three measurements differed significantly between eyes (P < 0.01). (B) RPGR of 22 mice aged 9 to 18 months on a logarithmic scale for dark-adapted (rod) b-wave (top panel) and light-adapted (cone) b-wave (bottom panel). - / - Scatter plot of ERG amplitude in mice. For each age group, data points were slightly shifted horizontally to minimize data overlap. Regression lines for the treated and control eyes were fitted using repeated measures longitudinal regression with SAS PROC MIXED based on all available data. (C) RPGR from a pair of 18-month-old mice treated with ORF15-1 and the contralateral control. - / - Representative dark adaptation (DA) and light adaptation (LA) ERG waveforms of the eye. Wild-type (age-matched) ERG waveforms are shown for comparison. At this age, the control eye had severely reduced or almost absent rod and cone ERGs, respectively. However, the treated eye still had significant rod and cone functions at this time point, approximately 70% and 35% of the wild-type values, respectively.

[0026] Figure 6Full-field ERG of 5 patients with XLRP due to the RPGR ORF 15 mutation was performed on a 0.5 Hz white light flash and on the same white light flash at 30 Hz. Three or more traces were superimposed to illustrate reproducibility. Dots above the traces indicate the onset of the flash. Although the response to the 0.5 Hz flash was only 6% to 65% lower than the lower limit of normal (350 μV), the response to the 30 Hz flash was not detectable as illustrated in the figure (i.e., without bandpass filtering and signal averaging). Detailed Implementation

[0027] Viral vector-mediated somatic gene therapy has shown great promise in animal models of human retinal degenerative diseases. To date, there have been numerous successful studies using adeno-associated virus (AAV)-mediated gene delivery to rescue photoreceptor degeneration in both small animal models (Ali and others 2000; Pang and others 2012; Pawlyk and others 2010; Pawlyk and others 2005; Tan and others 2009) and large animal models (Acland and others 2001; Alexander and others 2007; Beltran and others 2012; Komaromy and others 2010; Lheriteau and others 2009). In these cases, the retinal pigment epithelium (RPE), or photoreceptor cells, are the primary targets of transgene expression. In addition, some successes have been achieved in Phase I clinical trials—including gene therapy for patients with congenital Lieber's amaurosis (LCA) targeting RPE (Bainbridge and others 2008; Cideciyan and others 2008; Maguire and others 2008) and more recently, gene therapy for patients without choroid (Maclaren and others 2014). Currently, no clinical trials are using AAV-mediated gene replacement therapy to treat patients with X-linked RP.

[0028] Previously, the inventors used a transgenic approach to demonstrate the functional and morphological rescue of rod and cone cells in RPGR-deficient mice using a shortened mouse RPGR ORF15 homolog lacking approximately 600 bp at the carboxyl terminus of a purine-rich region (Hong and others 2005). Variations in the length of the repeat region have been frequently found in normal individuals (Bader and others 2003; Jacobi and others 2005; Karra and others 2006). However, the function of the shortened human RPGR has not yet been described.

[0029] In this study, a shortened human RPGR ORF15 replacement gene, driven by a previously documented rhodopsin kinase (RK) promoter (Khani and others 2007; Sun and others 2010) and delivered in a rapidly acting AAV8 vector (Allocca and others 2007; Natkunarajah and others 2008), was able to rescue the photoreceptor degeneration phenotype in an RPGR knockout mouse model. The purine-rich repeat region of exon 15 is essential for the precise subcellular localization and function of RPGR, but shortening its length by up to one-third does not appear to impair its function. In future human gene therapy trials, this shortened RPGR replacement gene provides a viable alternative to the previously avoided "full-length" RPGR ORF15.

[0030] RPGR RPGR is expressed in a complex pattern, with both a default variant and an ORF 15 variant documented (Vervoort and others 2000). The default or constitutive form of RPGR spans exons 1–19, and ORF 15 terminates in a large variable exon designated as ORF 15 before beginning exons 16–19. The unique feature of the ORF 15 exon is that it contains a long, purine-rich repetitive sequence that has proven difficult to clone into cDNA and unstable in many recombinant DNA manipulation steps. Although the smaller default form of RPGR is the dominant form in tissues with motile cilia (Hong et al., 2003) and many types of procilia (data not published), the ORF 15 homolog of RPGR is essential for normal rod and cone function in the retina (Vervoort and others 2000; Vervoort and Wright 2002) and is primarily expressed in photoreceptor cells (Hong and others 2003). ORF 15 is also a common site for RPGR mutations, with mutations identified in 22–60% of X-linked RP patients (Breuer and others 2002; Vervoort and others 2000).

[0031] The inventor facilitated the development of a portable RPGR The first X-linked RP mouse model in which the level of null mutations and any RPGR homologs was undetectable (Hong and others 2000). RPGRIneffective mice exhibited slowly developing retinal degeneration characterized by early mislocalization of opsins in cone cells within the cell body and synapses, and decreased rhodopsin levels in rod cells. Thus, these mice possessed cone-rod degeneration. By 12 months of age, significant photoreceptor cell loss and diminished cone and rod function, as measured by electroretinography (ERG), became apparent. In the retina, RPGR binds to connective cilia between the inner and outer segments of the photoreceptor cells via the RPGR-interacting protein (RPGRIP1) (see, e.g., Boylan and Wright 2000; Hong and others 2001; Roepman and others 2000). These connective cilia resemble transitional zones of motile cilia or protocilia, serving as pathways to the outer segment. This subcellular localization pattern and the mutant mouse phenotype suggest that RPGR plays a role in protein transport between the inner and outer segments of the rod and cone (Hong and Li 2002; Hong and others 2000; Hong and others 2001). In order to develop a faster mutation process RPGR Mutant mouse models, and several other [models], have recently been developed. RPGR Mouse strains (Brunner, et al., 2010; Huang et al., 2012). A naturally occurring X-linked RPGR model (rd9) has also been recently reported (Thompson and others 2012). In all these cases, including... RPGR The nullipopulation mice exhibited slowly progressing photoreceptor loss, but with varying degrees of rod and cone involvement, possibly due in part to differences in strain and / or pigmentation. These findings suggest that the slow rate of degeneration in the knockout model is due to species differences rather than incomplete ablation, and confirm the nullipopulation of this mouse model in patients. RPGR Applicability in therapeutic research for mutations.

[0032] Two variants of the full-length human RPGR (A and C) (also known as CRD; RP3; COD1; PCDX; RP15; XLRP3; orfl5; and CORDX1) are described in GenBank; homolog A has accession numbers NM_000328.2 (nucleic acid) and NP_000319.1 (protein); homolog C has accession numbers NM_001034853.1 (nucleic acid) and NP 001030025.1 (protein). Compared to variant C, variant (A) uses a variable splicing site and contains multiple variable exons in the 3' coding region, and encodes homolog A (also known as homolog 1), which is shorter than homolog C and has a different C-terminus. The sequence used in the exemplary composition described herein is shown in SEQ ID NO: 1 below. The human RPGR sequence that can be used in the compositions and methods described herein may be at least 80%, for example 85%, 90%, 95% or 100% identical to the full length of SEQ ID NO: 1, with up to 50, 100, 150 or 200 additional amino acids deleted from the deletion region, the deletion being indicated by dashes in the following sequence.

[0033] The shortened form of the human RPGRORF15 sequence with a 378 bp deletion, where the deletion is indicated by dashes ("-"; the number of dashes is not related to the size of the deletion). The shortened form of the human RPGRORF15 protein sequence, with deletion regions indicated by dashes ("-"; the number of dashes is not related to the size of the deletion). Full-length human RPGRORF15 cDNA sequence; the 378 bp deleted in the shortened form is shown in bold and underlined in the following sequence. Full-length human RPGRORF15 amino acid sequence; amino acids missing in the shortened form are shown in bold and underlined in the following sequence. Mathematical algorithms can be used to perform sequence comparisons and determine the percentage of identity between two sequences. For example, the Needleman and Wunsch ((1970) J. Mol. Biol.48:444-453) algorithm (which is incorporated into the GAP program in the GCG software package (available at gcg.com)) can be used with default parameters (e.g., Blossum 62 scoring matrix, vacancy penalty 12, vacancy extension penalty 4, frameshift vacancy penalty 5) to determine the percentage of identity between two amino acid sequences.

[0034] RK promoter In some embodiments of the methods described herein, a substitution gene construct is used, wherein a shortened human RPGRcDNA is placed under the control of a human rhodopsin kinase (hRK) promoter. In some embodiments, the RK promoter is approximately 200 bp in length (a short promoter from the rhodopsin kinase (RK) gene, which has been shown to drive cell-specific expression in rod and cone cells (Khani et al., 2007; Sun et al., 2010; Young et al., 2003)). An exemplary hRK promoter sequence is -112 / +87 (Khani et al., 2007): Viral delivery vector The shortened human RPGR cDNA described above is packaged into delivery vectors, such as AAV8 or AAV2 / 8 vectors.

[0035] The replacement gene (cDNA) can be administered using any effective vector (e.g., any formulation or composition capable of efficiently delivering the component gene into cells in vivo). Methods include inserting the gene into a non-pathogenic, non-replicating viral vector (including recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1) or a recombinant bacterial or eukaryotic plasmid. The viral vector can be directly transfected into cells; the plasmid DNA can be delivered naked, or via, for example, cationic lipofectamine or derived (e.g., antibody-conjugated) poly-lysine conjugates, gramacidin S, artificial viral envelopes, or other such intracellular vectors, as well as direct injection or CaPO4 precipitation of the gene construct in vivo.

[0036] A preferred method for introducing nucleic acids into cells in vivo is by using a viral vector containing nucleic acids (e.g., cDNA). The advantage of infecting cells with a viral vector is that most target cells are able to receive the nucleic acids. Furthermore, molecules encoded within the viral vector (e.g., via cDNA contained within the viral vector) can be efficiently expressed in cells that have absorbed the viral vector's nucleic acids.

[0037] Adenoviral vectors and adeno-associated virus vectors can be used as recombinant gene delivery systems for the in vivo transfer of exogenous genes, particularly to humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the host's chromosomal DNA. The development of specific cell lines that produce only replication-deficient retroviruses (referred to as "packaging cells") has increased the practicality of retroviruses for gene therapy, and these replication-deficient retroviruses have been characterized for gene transfer for gene therapy purposes (see Miller, Blood 76:271 (1990)). Replication-deficient retroviruses can be packaged into viral particles that can be used to infect target cells using helper viruses via standard techniques. Protocols for producing recombinant retroviruses and infecting cells with such viruses in vitro or in vivo are described in Ausubel, et al., eds. Current Protocols in Molecular BiologySee Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, known to those skilled in the art. Suitable packaging virus lines for preparing tropophilic and facultative retroviral systems include ΨCrip, ΨCre, Ψ2, and ΨAm. Retroviruses are used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and / or in vivo (see, for example, Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA). 88:8377-8381; Chowdhury et al. (1991)Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci.USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; US Patent No. 4,868, 116; US Patent No. 4,980,286; PCT application WO 89 / 07136; PCT application WO 89 / 02468; PCT application WO 89 / 05345; and PCT application WO 92 / 07573).

[0038] Another viral gene delivery system that can be used in the methods of this invention utilizes adenovirus-derived vectors. The genome of the adenovirus can be manipulated to encode and express the target gene product, but it loses its ability to replicate in the normal lysing viral life cycle. See, for example, Berkner et al, BioTechniques 6:616 (1988); Rosenfeld et al, Science 252:431-434 (1991); and Rosenfeld et al, Cell 68: 143-155 (1992). Suitable adenovirus vectors derived from adenovirus strain Ad 5 dl324 or other adenovirus strains (e.g., Ad2, Ad3, or Ad7) are known to those skilled in the art. Recombinant adenoviruses have the advantage in some cases that they cannot infect non-dividing cells and can be used to infect a variety of cell types, including epithelial cells (Rosenfeld et al, (1992), see above). Furthermore, viral particles are relatively stable and easily purified and concentrated, and as mentioned above, they can be modified to affect the range of infectivity. Additionally, the introduced adenovirus DNA (and the exogenous DNA it contains) is not integrated into the host cell's genome but remains free, thus avoiding potential problems arising from in situ insertional mutagenesis (the location where the introduced DNA integrates into the host genome, such as retroviral DNA). Moreover, the adenovirus genome has a greater capacity to carry exogenous DNA compared to other gene delivery vectors (up to 8 kilobases) (Berkner et al, see above; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).

[0039] Another viral vector system that can be used to deliver nucleic acids is adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires another virus (such as adenovirus or herpesvirus) as a helper virus to achieve efficient replication and a productive life cycle (see Muzyczka et al, Curr. Topics in Micro, and Immunol. l58:97-129 (1992)). It is also one of the few viruses that can integrate its DNA into non-dividing cells and exhibits a high frequency of stable integration (see, for example, Flotte et al, Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al, J. Virol. 63:3822-3828 (1989); and McLaughlinet et al, J. Virol. 62: 1963-1973 (1989)). AAV vectors containing as few as 300 base pairs can be packaged and integrated. The space of exogenous DNA is confined to about 4.5 kb. AAV vectors (such as those described in Tratschin et al, Mol. Cell. Biol. 5:3251-3260 (1985)) can be used to introduce DNA into cells. AAV vectors have been used to introduce many nucleic acids into different cell types (see, for example, Hermonat et al, Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al, Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al, Mol. Endocrinol. 2:32-39 (1988); Tratschin et al, J. Virol. 51 :611-619 (1984); and Flotte et al, J. Biol. Chem. 268:3781-3790 (1993)).

[0040] In a preferred embodiment, the viral delivery vector is a recombinant AAV2 / 8 virus.

[0041] Before administration, the final product will undergo a series of ultrapurification steps to meet clinical-grade standards.

[0042] Subject selection Subjects who are candidates for the treatment methods of the present invention include those diagnosed with RP caused by a mutation in the gene encoding RPGR. The methods described herein can also be used to treat subjects with other clinically determined ophthalmic conditions (e.g., X-linked cone-rod dystrophy) caused by mutations in the gene encoding RPGR. Methods known in the art can be used to diagnose XLRP or another ophthalmic condition caused by mutations in the gene encoding RPGR.

[0043] The methods described herein may include identifying subjects (e.g., children, adolescents, or young adults) who have XLRP or another ophthalmic condition caused by a mutation in a gene encoding RPGR, or who are suspected of having XLRP or another ophthalmic condition caused by a mutation in a gene encoding RPGR (e.g., based on the presence of symptoms of said condition and the absence of other obvious causes); obtaining samples containing genomic DNA from said subjects; detecting the presence of mutations in RPGR using known molecular biology methods; and selecting patients with mutations in RPGR that cause XLRP or another condition. Detection of mutations in RPGR may include detecting mutations in ORF15, as described in, for example, Sandberg et al, (2007). InvestOphthalmol Vis Sci 48, 1298-304; Dror et al, Am J Hum Genet. Nov 2003; 73(5):1131-1146.

[0044] Mutations in RPGR ORF15 include frameshift mutations, nonsense mutations, splice site mutations, and missense mutations. Exemplary mutations include... ORF15Glu446 (1-bp-del), ORF15Glu447 (2-bp-del) and ORF15GLys521(l-bp- ins) .

[0045] Detection of mutations in RPGR may also include sequencing the entire or a portion (e.g., the ORF15 region) of the subject's RPGR gene, and comparing the sequence with a reference sequence (e.g., GenBank accession number NG_009553.1) to detect mutations. Frameshift mutations, truncation mutations, mutations that alter conserved amino acids, or mutations affecting regulatory regions (e.g., promoters) can be considered mutations leading to XLRP as described herein or another ophthalmic condition; functional alterations can be confirmed by expressing the mutant in vitro (e.g., in cultured cells) or in vivo (e.g., in transgenic animals) and determining, for example, function or subcellular localization.

[0046] Patients with XLRP or another ophthalmic condition caused by an RPGR mutation who can be treated using the methods described herein preferably retain some photoreceptor cells and visual function, as measured, for example, by standard visual function or visual field tests and / or optical coherence tomography (OTC, e.g., spectral domain OTC (SD-OCT)); see, for example, Sandberg et al, Invest Ophthalmol Vis Sci. 2007;48: 1298-1304. The methods described herein may include identifying subjects diagnosed with XLRP or another ophthalmic condition caused by an RPGR mutation, having a confirmed mutation in the RPGR that causes their condition; and detecting their visual abilities and the presence of remaining central photoreceptor cells. Subjects who can be treated using the methods of the present invention (e.g., children, adolescents, young adults, or adult subjects) preferably have a visual acuity of at least 20 / 200 (methods for measuring visual acuity are well known in the art; see, for example, Johnson, Deafness and Vision Disorders: Anatomy and Physiology, Assessment Procedures, Ocular Anomalies, and Educational Implications , Charles C. Thomas Publisher;1999; Carlson, N; Kurtz, D.; Heath, D.; Hines, C. Clinical Procedures for Ocular Examination. Appleton & Lange; Norwalk, CT. 1990) and a detectable outer nuclear layer in the fovea (e.g., at least 75%, 80%, 90%, 95%, or 99% of normal thickness).

[0047] Example The invention is further described in the following embodiments, which do not limit the scope of the invention as defined in the claims.

[0048] Materials and methods The following materials and methods are used in the embodiments described below.

[0049] animal Previously recorded RPGR - / - Mice production and analysis (Hong and others 2000). Mice used in this study. RPGR - / - The mice are azygotic mice bred in our institutional animal facility. RPGR Male and homozygous ( RPGR - / -The mice were bred through sibling mating between females. Wild-type mice used in the study were C57BL mice from the Charles River Laboratory (Wilmington, MA). Mice were housed in a 12hr light / 12hr dark cycle. The study was conducted in accordance with the ARVO statement for the use of animals in ophthalmological and visual studies and was approved by the IACUC of the Massachusetts Eye and Ear Hospital.

[0050] Plasmid construction and generation of recombinant AAV8 Human RPGR ORF 15 cDNA was amplified from human retinal cDNA by PCR using primers designed to include the entire coding region of the RPGR ORF 15 homolog. Despite repeated attempts using various methods, full-length ORF 15 cDNA was not obtained, consistent with the experience of other researchers and our own (Hong and others 2005). Instead, we obtained a shortened ORF 15 cDNA (remaining 2517 bp) containing a large 314-codon (942 bp) frame of deletion within the ORF 15 exon, where purine-rich repeat regions were removed (codons 696–1010 del, “short”). Figure 1A A second ORF15 cDNA was constructed using recombinant DNA manipulation, containing a 126-codon (378 bp) in-frame deletion in the highly repetitive region of exon 15 (leaving 3081 bp in exon 15) (codons 862-988del, “long”). These ORF15 cDNAs were sequenced to verify fidelity. To construct AAV vectors, RPGR cDNA was inserted into the multiple cloning site of the parental pAAV-RK-zsGreen vector. The resulting pAAV-RK-mRPGR and pAAV-RK-hRPGR vectors were packaged into AAVs. AAV2 / 8 pseudotype vectors were generated by three-part transfection: (1) an AAV vector plasmid encoding the target gene; (2) an AAV helper plasmid pLT-RC03 encoding the AAV Rep protein from serotype 2 and the Cap protein from serotype 8; and (3) an adenovirus helper plasmid pHGTI-Adeno1 that entered 293A cells. Transfection was performed using a protocol developed by Xiao et al. (Xiao, et al., 1998). Two days post-transfection, cells were lysed using repeated cold-thaw cycles. After initial removal of cell debris, nucleic acid components from virus-producing cells were removed by benzonase treatment. Recombinant AAV vector particles were purified using an iodixanol density gradient. The purified vector particles were dialyzed extensively with PBS and titrated by dot blot hybridization.

[0051] Subretinal injection Mice were generally anesthetized by intraperitoneal injection of ketamine (90 mg / kg) / toluidine (9 mg / kg). 0.5% propylcaine solution was administered as a local anesthetic to the cornea. The pupils were dilated by local application of cephalexin and phenylephrine hydrochloride. Under an ophthalmic surgical microscope, a small incision was made in the cornea adjacent to the limbus using an 18-gauge needle. A 33-gauge blunt needle fitted into a Hamilton syringe was inserted through the incision, posterior to the lens, and pushed past the retina. The entire injection was administered subretinally within the nasal quadrant of the retina. Each eye received 2 × 10-10 injections. 9 Vector genome ( AAV-ORF15-L ) / μl or 5×10 9 Vector genome ( AAV- ORF15-S ) / μl. The RPGR-ORF15 vector was administered to the left eye (OS, left eye), and the control vector ( AAV8-RK-EGFP The drugs were administered to the right eye (OD, right eye). These were referred to herein as “treated” or “control”, respectively. Visualization during injection was aided by adding 0.1% v / v fluorescein (100 mg / ml AK-FLUOR, Alcon, Inc.) to the carrier suspension. Fundus examination after injection revealed >30% retinal separation in most cases, confirming successful subretinal delivery. A cohort of mice (n=50 total) was injected at 1 month of age for protein expression studies, and injections were given at 3–7 months of age prior to loss of major photoreceptor cells (as ERG remained normal during this period) for functional (ERG) and histological studies.

[0052] Histology and immunofluorescence For both optical and transmission electron microscopy (TEM) examinations, the excised eye was fixed in 1% formaldehyde and 2.5% glutaraldehyde in 0.1 M dimethylarsine buffer (pH 7.5) for 10 minutes. After removing the anterior segment and lens, the eyecup was left in the same fixative overnight at 4°C. The eyecup was rinsed with buffer, then post-fixed in osmium tetroxide, dehydrated by a series of fractionated alcohols, and embedded in Epon. Semi-thin sections (1 μm) were cut for optical microscopy. For EM, ultrathin sections were stained in uranium acetate and lead citrate and then examined on a JEOL 100CX electron microscope.

[0053] For immunofluorescence staining of ciliates, the eyes were removed, flash-frozen, and sectioned into 10 μm sections in a cryostat. Unfixed frozen sections were then collected on slides and stained. For immunostaining of all other proteins, floating retinal sections were collected and stained. For this procedure, the eyes were placed in a fixative (2% formaldehyde, 0.25% glutaraldehyde / PBS) and their anterior segments and lenses were removed. Fixation typically lasted 20 minutes. The fixed tissue was immersed in 30% sucrose / PBS for at least 2 hours, flash-frozen, and sectioned similarly to the unfixed tissue. The sections were then collected in PBS buffer and allowed to remain free-floating for the duration of the immunostaining process. The stained sections were observed and photographed on a laser scanning confocal microscope (model TCS SP2; Leica). The antibodies used were mouse RPGR (SI), human RPGRC100, anti-rootletin, 1D4 (anti-rhodopsin), mixed blue / green cone anti-opsin, and Hoechst 33342, stained with nuclear dyes.

[0054] Immunoblotting analysis Retinal tissue was homogenized in RIPA buffer, boiled in Laemmli buffer, and loaded onto a 5% SDS-PAGE gel at 15 μg / lane. After gel separation, proteins were electrotransferred onto a PVDF membrane. The membrane was blocked with 5% skim milk and incubated overnight at room temperature with a primary antibody. After rinsing, the membrane was incubated with a peroxidase-conjugated secondary antibody. SuperSignal® West Pico chemiluminescent substrate (Pierce) was used for detection. For normalization, protein samples were separated on SDS-PAGE and detected with a transductionin α antibody (a gift from Dr. Heidi Hamm of Vanderbilt University).

[0055] ERG Record Mice were acclimatized to darkness overnight and anesthetized by intraperitoneal injection of sodium pentobarbital prior to the experiment. Phenylephrine hydrochloride and cyclopentolate hydrochloride were used to locally dilate both pupils of each animal, and the mice were then placed on a heated platform. A 10-μs flash of white light (1.37 × 10⁻⁶) at 1-minute intervals was used in the Ganzfeld dome. 5 cd / m 2 In darkness, this elicits a rod-dominated response. At 41 cd / m 2 Against a white light background where the visual poles were desensitized, the same flashes (1.37 × 10⁻⁶) occurring at 1 Hz intervals were used. 5 cd / m 2Emissions of light-adapted cone responses were induced. ERG was monitored simultaneously from both eyes using silver wire loop electrodes in contact with each cornea (which was locally anesthetized with propacaine hydrochloride and moistened with hydroxypropyl methylcellulose eye drops (Goniosol)), with a subcutaneous electrode in the neck used as a reference; an electrically shielded chamber was used as the ground.

[0056] All responses were differentially amplified at 1000 gain (-3dB at 2 Hz and 300 Hz; AM502, Tektronix Instruments, Beaverton, OR), digitized at 16-bit resolution using adjustable peak-to-peak input amplitude (PCI-6251, National Instruments, Austin, TX), and displayed on a PC using custom software (Labview, version 8.2, National Instruments). Individually for each eye, the cone responses were tuned through a 60 Hz notch filter and an adjustable artifact-reject window, summed (n=4–20), and then fitted to a cubic spline function with variable stiffness to improve the signal-to-noise ratio without affecting the temporal characteristics; in this way, we were able to resolve the b-wave response as small as 2 μV.

[0057] Statistical analysis JMP, version 6 (SAS Institute, Cary, NC) was used to compare transverse ERG amplitudes and implied frequencies. Repeated measures analysis using PROC MIXED OF SAS, version 9.3 (SAS Institute) was used for histological comparisons and to compare longitudinal ERG data of treated eyes relative to untreated eyes.

[0058] patient We examined full-field electroretinography (ERG) data from 111 patients with XLRP due to the ORF15RPGR mutation, obtained from the data set described in Sharon et al. (2003), to compare b-wave amplitudes to 0.5 Hz white light, reflecting the remaining rod + cone function, and b-wave amplitudes to a 30 Hz flash of the same white light, reflecting the remaining cone function alone. To determine whether they had rod-cone or cone-rod disease, we calculated the ratio of their amplitude to the 0.5 Hz flash for OD and their amplitude to the 30 Hz flash for OS; the same ratio at the lower limit of normal in our system was 350 μV / 50 μV = 7. To more precisely quantify the response amplitude to the 0.5 Hz flash and minimize the possible secondary effects of major photoreceptor degeneration, we focused on those patients (n=14) whose amplitude to the 0.5 Hz flash was >50 μV (reflecting early or milder disease).

[0059] ERG of patients with ORF15 mutation For the 14 patients who exhibited the strongest response to a 0.5 Hz white light flash (reflecting remaining rod + cone function), the amplitude range for this condition was 53 μV to 329 μV OD and 59 μV to 282 μV OS. Their amplitudes for a 30 Hz flash of the same white light (reflecting isolated cone function and monitored using bandpass filtering and signal averaging with amplitudes < 10 μV) ranged from 0.98 μV to 23.5 μV OD and 0.95 μV to 20 μV OS. The mean ± standard error of the ratio of the response amplitude to the 0.5 Hz flash to the response amplitude to the 30 Hz flash was 47.0 ± 12.7 OD and 48.7 ± 13.0 OS. These means differed significantly from the 7.0 – ratio based on the lower limit of normal (nonparametric signed-rank test, p = 0.0004 OD and p = 0.001 OS). In other words, these patients with the ORF15 mutation have a significantly disproportionate loss of cone function. Examples of these ERGs are shown in... Figure 6 .

[0060] Example 1. AAV-mediated expression of human RPGR ORF15 We constructed two human RPGR ORF15 substitution genes: one with a 126-codon in-frame deletion (long type, ORF 15-L) and the other with a 314-codon in-frame deletion (short type, ORF 15-S). These two genes were inserted into the AAV8 vector, which is responsive to the human rhodopsin kinase promoter (…). Figure 1AThe control of ORF15 (Khani and others 2007; Sun and others 2010). Subretinal delivery (left eye) of the two human RPGR ORF15 substitution genes resulted in the production of recombinant RPGR protein. By Western blotting, two weeks after administration of the AAV vector, the long ORF15 produced approximately 160 kD of protein, while the short ORF15 produced approximately 125 kD of protein. Both protein products are smaller than native ORF15 in human retinal tissue (approximately 200 kD). Figure 1B When detected using an antibody targeting the C-terminus of human RPGR, both alternative ORF15 forms presented as a single band. Under our experimental conditions and at the administered doses, the expression levels of ORF15-S and ORF15-L were comparable. The control eye (right eye) received AAV-GFP.

[0061] Immunofluorescence staining of unfixed frozen sections can be used to... RPGR - / - Two forms of ORF 15 were observed in the mouse retina (3 weeks after subretinal injection), precisely located in the layer between the inner and outer segments connecting the cilia. However, the short form (AAV8-ORF15-s) produced a much weaker signal than the long form (AAV8-ORF15-l). Figure 2A In well-transmitted retinal areas, signals from the elongated retina appear indistinguishable from wild-type signals. Figure 2A B). Dual labeling with antibodies against ciliated root filaments—which originate from the proximal end of the basal body and extend into the cell, and are therefore used as excellent markers for the ciliated region (Hong and others 2003; Yang and others 2002)—confirmed the precise subcellular localization of recombinant RPGR to the cilia. Figure 2B Unlike the similarity of protein expression levels determined by Western blotting, only the long ORF 15 appeared to have a strong signal in each CC matching the number of root filaments, while in the retina of the short-type treatment, many root filaments did not have RPGR signal at their distal ends. Figure 2C The Rpgr value is shown relative to that processed with long or short human ORF 15. - / - The bar chart represents the RPGR marker counts in the mouse retina and in the retina of untreated wild-type mice. The mean ratio (RPGR signal count divided by the number of microradicals) of the ORF15 long type was not different from that of the wild type (Dunnett's method, p = .24), but the mean ratio of the ORF15 short type was significantly lower than that of the wild type (p = .0019).

[0062] Given the similar expression levels determined by immunoblotting, the difference in protein localization at the cilia junction suggests that some short ORF 15 may be mislocalized to other locations within the photoreceptor cell. Further analysis of fixed retinal sections by immunostaining (which better preserve tissue at the cost of signal intensity loss) revealed patterns of mislocalization of short ORF 15 into both intracellular and extracellular segments of the photoreceptor cell. Figure 2D No mislocalization was observed for the long ORF15, which exhibits a similar staining pattern to the wild-type. Therefore, the lack of staining at CC by the short RPGR is due to a reduced ability to localize to or be confined to this subcellular compartment, rather than a lower overall expression level.

[0063] Example 2. Human ORF15-1 (long form) expression promotes rod and cone cell survival in RPGR-ineffective mice. live To investigate the therapeutic efficacy of the two gene substitutions, we evaluated them using immunostaining. RPGR - / - Mouse photoreceptor cells were used to look for signs of improved rod and cone cell morphology. At 13 months of age (6 months post-treatment), no significant differences in rod or cone cell morphology were observed using short-type human ORF15. Figure 3 At this age, both the control and short ORF15-treated eyes exhibited typical degenerative features. Compared to wild-type eyes, the outer segments of the rods and cones were shortened and disordered, with visible mislocalization of rod opsins throughout the outer nuclear layer and additional mislocalization of cone opsins in the synaptic layer. The thickness of the outer nuclear layer was also reduced in both the control and short ORF15-treated eyes.

[0064] In contrast, eyes treated with the long human ORF 15 showed correctly distributed opsin expression in the outer segment of rod cells, without significant signs of mislocalization. Similarly, mislocalization of cone opsin was rare in these eyes treated with the longer ORF 15 construct. Furthermore, eyes treated with ORF 15-l were found to have more rods and cones (with nearly normal outer segments) than control or ORF 15-S treated eyes.

[0065] Based on these findings, a longitudinal study was conducted in mice treated with long ORF 15. To quantify the rescue effect of ORF 15-l-treated eyes relative to control eyes, we measured the rescue effect of three Rpgr mice. - / -The thickness of the outer nuclear layer (ONL) and the length of the inner / outer segment of photoreceptors in the contralateral eyes of mice were measured. These were performed in three zones in the superior hemisphere and three zones in the inferior hemisphere, each separated by 600 μm and starting 600 μm from either side of the optic nerve head along a vertical meridian; repeated measures full factorial regression was used at 11 and 18 months of age to identify differences in the eye, hemisphere, and zone as main effects, as well as their cross-products, to determine whether the therapeutic effect was altered at location. At 11 months of age, ONL thickness was normally distributed, but the inner / outer segment length was not (Shapiro-Wilk W goodness-of-fit test, p = .016); at 18 months of age, neither ONL thickness nor the inner / outer segment length was normally distributed (p = .0011 and p = .0002, respectively). At 11 months of age, the mean ONL thickness of the treated eyes (48.0 μm) was significantly greater than that of the control eyes (38.0 μm, p = .0015); the mean medial / lateral segment length (IS / OS) of the treated eyes (45.1 μm) was also significantly greater than that of the control eyes (29.5 μm, p < .0001, p < .0001 for normalized rank). At this age, the therapeutic benefits regarding ONL thickness and IS / OS length were comparable for both the superior and inferior hemispheres. At 18 months of age, the differences in retinal morphology between the contralateral eyes were more significant: the mean ONL thickness was 22.8 μm in the treated eye and 13.7 μm in the control eye (p < .0001, p < .0001 for normalized rank); while the mean medial / lateral segment length was 19.8 μm in the treated eye and 7.3 μm in the control eye (p < .0001, p < .0001 for normalized rank). At this age, we initially observed a significantly greater therapeutic benefit in the superior retina than in the inferior retina for IS / OS length at 18 months of age (p = .0036), but this no longer held true after converting the lengths to normalized rank (p = .17). Figure 4A The study demonstrated the ONL thickness and IS / OS length in the treated and control eyes of three 18-month-old mice based on region.

[0066] Figure 4B Representative light micrographs of an ORF 15-L treated eye and a contralateral control eye taken at 18 months of age are shown. In the control retina, the best-preserved area contains only about 2-3 rows of loosely arranged photoreceptor nuclei with shortened and disordered inner / outer segments. Note that the edges of the inner and outer segments are no longer distinct. On the other hand, the treated retina consistently contains about 5-6 rows of photoreceptor cells with longer, more ordered, and distinct inner and outer segments.

[0067] Example 3. Long expression of human RPGR ORF15 improves rod and cone cell function. RPGR in 9-18 months - / - Retinal function was assessed in a mouse population (n=22) using full-field rod and cone ERGs. Mice were treated between 3 and 7 months of age, and subsequent ERGs were recorded immediately 6 months after injection. Figure 5A Rod and cone ERG amplitudes are shown in the eyes of 16 mice tested between 11 and 14 months of age. Compared to the lower limit in wild-type mice, the control eyes (OD) showed a disproportionate loss in cone b-wave amplitude relative to rod b-wave amplitude, as previously shown in RPGR. - / - The results observed in mouse models, as well as those shown in cone-rod degeneration, indicate that in every mouse (except one), the treated eye (OS) had larger ERGa and b-wave amplitudes compared to the contralateral control eye (OD), suggesting improved rod and cone photoreceptor function. In fact, more than half of the treated eyes (9 / 16) had rod b-wave amplitudes equal to or greater than the lower limit of the age-matched wild-type values ​​(dashed line). The geometric mean of rod ERG a-wave amplitudes was 121 μV OS and 65 μV OD, and the geometric mean of rod ERG b-wave amplitudes was 482 μV OS and 267 μV OD. The mean cone ERG b-wave amplitudes were 22 μV OS and 11 μV OD. These data suggest that, for this age range, AAV-ORF15 treatment improves rod function by 81–86% and cone function by 100%.

[0068] In all 22 mouse groups, we used repeated measures longitudinal regression to compare the rate of change of rod and cone b-wave amplitudes in the eye. Figure 5B For the rod b-wave amplitude in the control eye, the estimated mean rate of change was -8.6% / month, and for the treated eye, it was -3.8% / month; the difference between these two means was significant (p = 0.0001). For the cone b-wave amplitude in the control eye, the estimated mean rate of change was -5.8% / month, and for the treated eye, it was -0.8% / month; the difference between these two means was also significant (p < 0.0001). Furthermore, the decay of the cone b-wave amplitude in the treated eye was not significantly different from zero (p = 0.54), indicating the stability of cone function and the absence of observable progression.

[0069] Figure 5CRepresentative rod and cone ERGs are shown to illustrate waveforms in treated and control eyes (including wild-type eyes at 18 months of age, the final time point). At this age, rod function in control eyes was severely reduced (average reduction of 75%), while cone function was minimal and virtually undetectable in some cases. In contrast, treated eyes at this time point still exhibited significant rod and cone function, although at a lower level than those seen in wild-type eyes.

[0070] References Other implementation plans It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to explain, and not limit, the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A nucleic acid encoding a shortened human RPGR protein, wherein the shortened human RPGR protein is at least 95% identical in length to the full-length SEQ ID NO:

2.

2. The nucleic acid of claim 1, wherein: (a) The nucleic acid encoding the shortened human RPGR protein is controlled by the human rhodopsin kinase (hRK) promoter; and / or (b) The hRK promoter comprises SEQ ID NO: 5; and / or (c) The hRK promoter is essentially composed of SEQ ID NO:

5.

3. The nucleic acid of claim 1 or 2, wherein the nucleic acid encodes the shortened human RPGR protein comprising SEQ ID NO:

2.

4. The nucleic acid of any one of claims 1 to 3, wherein the nucleic acid encodes a shortened human RPGR protein constituted by SEQ ID NO:

2.

5. The nucleic acid of any one of claims 1 to 4, wherein the nucleic acid encoding the shortened human RPGR protein is at least 95% identical in length to the full-length SEQ ID NO:

1.

6. Use of the nucleic acid of any one of claims 1-5 for preparing a medicament for treating a human subject suffering from X-linked retinitis pigmentosa (XLRP) or another ophthalmic condition caused by a loss-of-function mutation in a gene encoding a protein encoding the GTPase regulatory factor (RPGR) protein of retinitis pigmentosa.

7. A viral vector comprising the nucleic acid of any one of claims 1-5.

8. The viral vector of claim 7, wherein the adeno-associated virus vector is optionally AAV-2 or AAV-8.

9. The viral vector of claim 8, wherein the adeno-associated viral vector is AAV2 / 8.

10. Use of the viral vector of any one of claims 7 to 9 for preparing a medicament for treating a human subject suffering from X-linked retinitis pigmentosa (XLRP) or another ophthalmic condition caused by a loss-of-function mutation in a gene encoding a protein encoding the GTPase regulatory factor (RPGR) protein of retinitis pigmentosa.

11. An isolated host cell comprising the nucleic acid of any one of claims 1-5 or the viral vector of any one of claims 7 to 9.

12. The isolated host cell of claim 11, wherein the cell expresses the shortened human RPGR protein.

13. Use of an adeno-associated viral vector for the preparation of a medicament for treating human subjects suffering from X-linked retinitis pigmentosa (XLRP) or another ophthalmic condition due to a loss-of-function mutation in a gene encoding a GTPase regulatory factor (RPGR) protein for retinitis pigmentosa, said adeno-associated viral vector comprising nucleic acid encoding a shortened human RPGR protein, wherein said shortened human RPGR protein is at least 95% identical in length to the full-length of SEQ ID NO:

2.

14. The use of claim 13, wherein the nucleic acid encoding the shortened human RPGR protein is controlled by the human rhodopsin kinase (hRK) promoter.

15. The use of claim 13 or 14, wherein: (a) The hRK promoter comprises or substantially consists of SEQ ID NO: 5; and / or (b) The nucleic acid encoding the shortened human RPGR protein comprises or is substantially composed of a sequence that is at least 95% identical to that of SEQ ID NO:

1.

16. The use of any one of claims 13 to 15, wherein the adeno-associated virus vector is AAV2 or AAV8.

17. Use according to any one of claims 13 to 15, wherein the adeno-associated virus vector is AAV2 / 8.

18. The use of any one of claims 13 to 17, wherein the shortened human RPGR protein comprises SEQ ID NO:

2.

19. Use according to any one of claims 13 to 18, wherein the shortened human RPGR protein is composed of SEQ ID NO:

2.

20. The use of any one of claims 13-19: (a) wherein the use comprises administering the nucleic acid at a low dose of about 2 x 10 10 vg / mL, a medium dose of about 2 x 10 11 vg / mL, or a high dose of 2 x 10 12 vg / mL; and / or (b) wherein the nucleic acid is delivered into the subretinal space; Optionally, a microinjection cannula is inserted into the subretinal space—located in the temporal region of the optic nerve and just above the major arcade vessel—to allow fluid flow toward the macula of the retina.

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