Retinitis Pigmentosa GTPase Regulator Protein Research Articles

AAV-RPGR Gene Therapy Rescues Opsin Mislocalisation in a Human Retinal Organoid Model of RPGR-Associated X-Linked Retinitis Pigmentosa.

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.

Identification of a novel RPGR mutation associated with retinitis pigmentosa and primary ciliary dyskinesia in a Slovak family: a case report

BackgroundThe mutations in the RPGR (retinitis pigmentosa GTPase regulator) gene are the most common cause of X-linked retinitis pigmentosa (XLRP), a rare genetic disorder affecting the photoreceptor cells in the retina. Several reported cases identified this gene as a genetic link between retinitis pigmentosa (RP) and primary ciliary dyskinesia (PCD), characterised by impaired ciliary function predominantly in the respiratory tract. Since different mutations in the same gene can result in various clinical manifestations, it is important to describe a correlation between the gene variant and the observed phenotype.MethodsTwo young brothers from a non-consanguineous Slovak family with diagnosed retinal dystrophy and recurrent respiratory infections were examined. Suspected PCD was diagnosed based on a PICADAR questionnaire, nasal nitric oxide analysis, transmission electron microscopy, high-speed video microscopy analysis, and genetic testing.ResultsWe identified a novel frameshift RPGR mutation NM_001034853: c.309_310insA, p.Glu104Argfs*12, resulting in a complex X-linked phenotype combining PCD and RP. In our patients, this mutation was associated with normal ultrastructure of respiratory cilia, reduced ciliary epithelium, more aciliary respiratory epithelium, shorter cilia, and uncoordinated beating with a frequency at a lower limit of normal beating, explaining the clinical manifestation of PCD in our patients.ConclusionThe identified novel pathogenic mutation in the RPGR gene expands the spectrum of genetic variants associated with the X-linked PCD phenotype overlapping with RP, highlighting the diversity of mutations contributing to the disorder. The described genotype–phenotype correlation can be useful in clinical practice to recognise a broader spectrum of PCD phenotypes as well as for future research focused on the genetic basis of PCD, gene interactions, the pathways implicated in PCD pathogenesis, and the role of RPGR protein for the proper functioning of cilia in various tissues throughout the body.

Translational Read-Through Therapy of RPGR Nonsense Mutations.

X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, TranslamaTM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in RPGR and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in RPGR and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in RPGR or other genetic diseases.

Association of a Novel Intronic Variant in RPGR With Hypomorphic Phenotype of X-Linked Retinitis Pigmentosa

Pathogenic variants in retinitis pigmentosa GTPase regulator (RPGR) gene typically lead to a severe form of X-linked retinitis pigmentosa, which is associated with early severe vision loss. To investigate an X-linked retinal degeneration family with atypical preservation of visual acuity in the presence of a novel deep intronic splice site RPGR c.779-5T>G variant. In this case series, 3 members of an X-linked retinal degeneration family were studied by in-depth phenotyping and genetic screening at a single center. Data were collected and analyzed from November 2018 to March 2020. Data were collected on full ophthalmic history, examination, and retinal imaging. A full retinitis pigmentosa gene panel was analyzed by next-generation sequencing. The pathogenicity of the RPGR c.779-5T>G variant was assessed by in silico splice prediction tools and by purpose-designed in vitro splicing assay. An 84-year-old man was referred with clinical diagnosis of choroideremia and possible inclusion into a gene therapy trial. He presented with late-stage retinal degeneration and unusually preserved visual acuity (78 and 68 ETRDS letters) that clinically resembled choroideremia. His 23-year-old grandson was still in early stages of degeneration but showed a very different clinical picture, typical of retinitis pigmentosa. Next-generation sequencing identified a sole RPGR c.779-5T>G variant of undetermined pathogenicity in both cases. The daughter of the proband showed an RPGR carrier phenotype and was confirmed to carry the same variant. The molecular analysis confirmed that the RPGR c.779-5T>G variation reduced the efficiency of intron splicing compared with wild type, leading to a population of mutant and normal transcripts. The predicted consequences of the pathogenic variant are potential use of an alternative splice acceptor site or complete skipping of exon 8, resulting in truncated forms of the RPGR protein with different levels of glutamylation. These results support the importance of careful interpretation of inconsistent clinical phenotypes between family members. Using a molecular splicing assay, a new pathogenic variant in a noncoding region of RPGR was associated with a proportion of normal and hypomorphic RPGR, where cones are likely to survive longer than expected, potentially accounting for the preserved visual acuity observed in this family.

Molecular Strategies for RPGR Gene Therapy.

Mutations affecting the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the commonest cause of X-linked and recessive retinitis pigmentosa (RP), accounting for 10%–20% of all cases of RP. The phenotype is one of the most severe amongst all causes of RP, characteristic for its early onset and rapid progression to blindness in young people. At present there is no cure for RPGR-related retinal disease. Recently, however, there have been important advances in RPGR research from bench to bedside that increased our understanding of RPGR function and led to the development of potential therapies, including the progress of adeno-associated viral (AAV)-mediated gene replacement therapy into clinical trials. This manuscript discusses the advances in molecular research, which have connected the RPGR protein with an important post-translational modification, known as glutamylation, that is essential for its optimal function as a key regulator of photoreceptor ciliary transport. In addition, we review key pre-clinical research that addressed challenges encountered during development of therapeutic vectors caused by high infidelity of the RPGR genomic sequence. Finally, we discuss the structure of three current phase I/II clinical trials based on three AAV vectors and RPGR sequences and link the rationale behind the use of the different vectors back to the bench research that led to their development.

Disease mechanisms and neuroprotection by tauroursodeoxycholic acid in Rpgr knockout mice.

Mutations in theretinitis pigmentosaGTPase regulator (RPGR)gene are the predominant cause of retinitis pigmentosa. RPGR plays a critical role as a scaffold protein in the regulation of protein trafficking from the basal body to the axoneme, where the cargoes are transported to the outer segments (OSs) of photoreceptors. This trafficking process is controlled directly by intraflagellar transport complexes and regulated by the RPGR protein complex, although the precise mechanisms have yet to be defined. We used an Rpgr conditional knockout (cko) mouse model to investigate the disease mechanisms during retinal degeneration and to evaluate the protective effects of tauroursodeoxycholic acid (TUDCA). Rhodopsin, cone opsins and transducin were mislocalized in Rpgr cko photoreceptors, while localization of NPHP4 to connecting cilia was absent, suggesting that RPGR is required for ciliary protein trafficking. Microglia were activated in advance of retinal degeneration in Rpgr cko mouse retinas. TUDCA treatment suppressed microglial activation and inflammation and prevented photoreceptor degeneration in Rpgr cko mice. Our data demonstrated that TUDCA has therapeutic potential for RPGR-associated RP patients.

Molecular mechanisms of X‐linked retinitis pigmentosa

PurposeRetinitis Pigmentosa is a class of inherited retinal degeneration that causes progressive vision impairment even blindness. The mutations of Retinitis Pigmentosa GTPase Regulator (RPGR) gene account for up to 20% of RP cases. RPGR protein is highly expressed at the connecting cilium of vertebrates’ photoreceptors. RPGR protein has the function of regulating protein trafficking from basal body to axoneme. The trafficking is controlled by the protein complex formed by RPGR, RPGRIP1 and IFT proteins, although its precise function yet to be identified. RPGR−/− mice model was employed for investigating disease mechanisms during retinal degeneration.MethodsCell death pathway and protein trafficking were detected by immunohistochemistry.ResultsThe retina of RPGR−/− mice started to undergo degeneration from 3M old. The caspase dependent cell death pathway was found to be activated during degeneration. The Apoptosis Inducing Factor existed in outer nuclear layer indicated that caspase‐independent apoptosis also contributed to cell death. The activated microglia, showed migration from outer plexiform layer to ONL in the retina of RPGR−/− mice and the downstream inflammation pathway were activated in RPGR−/− retina. Prior to the onset of retinal degeneration, the key elements involved in the phototransduction such as rhodopsin already mislocalized and accumulated in inner segment and outer nuclear layer. The mislocalization may result from the abnormal formation of the regulator of membrane trafficking Rab8 in RPGR−/− mice that controls the fusion of rhodopsin transport carrier and directs the cargo delivery across the CC.ConclusionsIn Conclusion, multi‐pathways were involved in the photoreceptor cell death and the deficiency of RPGR led to abnormal localization of the crucial proteins that participate in photoreceptor maintenance and phototransduction.

101. Rational Design and Cloning of a Stable RPGR ORF15 cDNA Encoding the Full-Length Native RPGR Protein

Introduction: The retinitis pigmentosa GTPase regulator (RPGR) cDNA contains a long purine-rich repetitive sequence in ORF15 exon that is unstable during recombinant DNA manipulation. This complicates efforts to develop AAV-based vectors for gene therapy of X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene. We reasoned that the stability of RPGR-ORF15 cDNA could be significantly improved by rational design of the cDNA sequence through codon modification without changing the amino acid sequence. Methods: A human RPGR cDNA was designed based on Genbank reference mRNA sequence NM_001034853 that encodes hRPGR isoform C. The 3459 bp coding sequence was codon-optimized based on human codon usage and further modified to reduce tandem repeats, and adjust G/C content, where possible. The codon-modified cDNA (hRPGRco), which encodes a full-length hRPGR protein of 1152 amino acids that is 100% identical to the published sequence, was synthesized and cloned into conventional cloning vector pUC57 and sub-cloned into various vectors, including an AAV plasmid (used for AAV production by transfection) and an HSV shuttle plasmid (used to make an HSV helper for AAV production using AGTC's proprietary HSV-based AAV manufacturing system). Stability of the hRPGRco cDNA sequence was verified by DNA sequencing at multiple steps: after sub-cloning, large scale plasmid production, production of recombinant HSV helper virus clones that carry the hRPGRco expression cassette and production of AAV vector. Stability of plasmids carrying the hRPGRco expression cassette was also analyzed after serial passage of bacterial clones cultured at various temperatures and during multiple passages. Results: Plasmids carrying the redesigned hRPGRco cDNA were grown stably in bacteria at 37°C. The hRPGRco cDNA was 100% correct in AAVplasmids, in shuttle vectors used to produce HSV helpers, and in the rHSV helper virus passed multiple rounds in mammalian cells. The sequence was also confirmed 100% correct in AAV vectors produced by plasmid transfection for animal efficacy studies. Conclusions: A stable RPGR cDNA that encodes full-length, native RPGR protein was obtained and will allow large scale production of an AAV vector for treatment of patients with XLRP.

Localizing the RPGR protein along the cilium: a new method to determine efficacies to treat RPGR mutations.

Retinal dystrophies constitute a group of clinically and genetically heterogeneous diseases that cause visual impairment. As treatments are not readily available, readout assays performed in patient-derived cells can aid in the development and comparative analysis of therapeutic approaches. We describe a new method with which the localization of the retinitis pigmentosa GTPase regulator (RPGR) protein along the cilium can be used as a measure for treatment efficacy. In a patient-derived fibroblast cell line, we found that the RPGR protein is mislocalized along the ciliary axoneme. The patient carried a point mutation that leads to skipping of RPGR exon 10. We confirmed that this skipping is causative for the impaired localization of RPGR using a U7 small nuclear RNA (U7snRNA)-based antisense approach in control cells. Treatment of the patient-derived fibroblasts with therapeutic U1snRNA significantly corrected the proteins' mislocalization. In this proof of principle study, we show that detecting the RPGR protein along the cilium provides a reliable and quantifiable readout assay to evaluate the efficacy of therapies intended to correct or silence RPGR gene mutations. This method opens the possibility to compare different therapeutic agents, and thus facilitate the identification of treatment options for the clinically and molecularly complex RPGR-associated diseases.

The functional role of RPGR protein complex in retinitis pigmentosa

AbstractPurpose Retinitis pigmentosa (RP) is a progressive outer retinal dystrophy, affecting 1/3500 individuals in most populations. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the most common single cause of RP, accounting for up to 20% of all cases. RPGR forms a protein complex by interacting with the RPGR‐interacting protein 1 (RPGRIP1) and/or RPGRIP1 like protein (RPGRIP1L). We aim to investigate the functional role of RPGR protein complex in the pathogenesis of RP.Methods Small interfering RNA (siRNA) was used to knock‐down RPGR, RPGRIP1 or RPGRIP1L in human retinal pigment epithelium (RPE) 1 cell line. The efficiency of siRNA knock‐down was assessed by qualitative RT‐PCR and western blotting. The knock‐down effects of RPGR, RPGRIP1, or RPGRIP1L were characterized by immnocytochemistry, enzyme linked immunosorbent assay (ELISA), and western blotting.Results Knock‐down of RPGR, RPGRIP1 or RPGRIP1L caused defects in ciliogenesis, remodelling of the actin cytoskeleton, and centrosome positioning. Since planar cell polarity (PCP) pathway regulates actin cytoskeleton rearrangement through activation of small RhoA GTPases. So, we performed ELISA and found that RhoA‐GTPase activity were upregulated in the absence of RPGR, RPGRIP1, or RPGRIP1L protein. We also found knock‐down of RPGR, RPGRIP1 or RPGRIP1L decreased the stability of DVl3 proteins, key components of polar cell polarity (PCP) pathway.Conclusion We provide compelling evidence that the RPGR protein complex is required for the stability of DVl3, and regulates RhoA for apical centrosome positioning required for cilia formation. We show a novel functional role of RPGR complex in cilia formation by regulating actin cytoskeleton through alteration of PCP pathway.

Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa

Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

Replacement Gene Therapy with a HumanRPGRIP1Sequence Slows Photoreceptor Degeneration in a Murine Model of Leber Congenital Amaurosis

RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.

The retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) links RPGR to the nephronophthisis protein network

Nephronophthisis is a heterogenetic autosomal recessive disorder associated with multiple developmental abnormalities, including cystic kidney disease and retinal degeneration. Retinal dystrophies, in particular the X-linked forms, are believed to represent a distinct group of hereditary diseases; however, their genetic complexity and overlap with other syndromic diseases is increasingly apparent. In this study, we report that depletion of retinitis pigmentosa GTPase regulator (RPGR) during zebrafish embryogenesis causes developmental changes indistinguishable from the abnormalities caused by the depletion of nephrocystin-5 or nephrocystin-6. However, RPGR did not directly interact with either gene product. RPGR-interacting protein 1 was found to act as an adaptor connecting RPGR to nephrocystin-6, thereby linking it to the nephronophthisis protein network. This interaction was abolished by truncating mutations (c.1107delA) of the interacting protein. Our findings underline the importance of the interplay between the two protein networks, suggesting a phenotypic modulation in both retinitis pigmentosa and nephronophthisis.

Mutation analysis of the RPGR gene reveals novel mutations in south European patients with X-linked retinitis pigmentosa.

The RPGR (retinitis pigmentosa GTPase regulator) gene has been shown to be mutated in 10-20% of patients with X-linked retinitis pigmentosa (XLRP), a severe form of inherited progressive retinal degeneration. A total of 29 different RPGR mutations have been identified in northern European and United States patients. We have performed mutation analysis of the RPGR gene in a cohort of 49 southern European males affected with XLRP. By multiplex SSCA and automatic direct sequencing of all 19 RPGR exons, seven different and novel mutations were identified in eight of the 49 families; these include three splice site mutations, two microdeletions, and two missense mutations. RNA analysis showed that the three splice site defects resulted in the generation of aberrant RPGR transcripts. Six of these mutations were detected in the conserved amino-terminal region of RPGR protein, containing tandem repeats homologous to the RCC1 protein, a guanine nucleotide-exchange factor for Ran-GTPase. Several exonic and intronic sequence variations were also detected. None of the RPGR mutations reported in other populations were identified in our series. Our results are consistent with the notions of heterogeneity and minority causation of XLRP by mutations in RPGR in Caucasian populations.

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

The RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%-15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis.