Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.

Evaluation of CRISPR/Cas9 exon-skipping vector for choroideremia using human induced pluripotent stem cell-derived RPE.

BACKGROUND: Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene. METHODS: We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting. RESULTS: Artificial intelligence-based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM. CONCLUSIONS: CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.

Choroideremia: molecular mechanisms and therapies.

Choroideremia (CHM) is a monogenic X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE), and choroid; it is caused by mutations involving the CHM gene. CHM is characterized by night blindness in early childhood, progressing to peripheral visual field loss and eventually to complete blindness from middle age. CHM encodes the ubiquitously expressed Rab escort protein 1 (REP1), which is responsible for prenylation of Rab proteins and is essential for intracellular trafficking of vesicles. In this review we explore the role of REP1 in the retina and its newly discovered systemic manifestations, and discuss the therapeutic strategies for tackling this disease, including the outcomes from recent clinical trials.

REP1 deficiency causes systemic dysfunction of lipid metabolism and oxidative stress in choroideremia.

Choroideremia (CHM) is an X-linked recessive chorioretinal dystrophy caused by mutations in CHM, encoding for Rab escort protein 1 (REP1). Loss of functional REP1 leads to the accumulation of unprenylated Rab proteins and defective intracellular protein trafficking, the putative cause for photoreceptor, retinal pigment epithelium (RPE), and choroidal degeneration. CHM is ubiquitously expressed, but adequate prenylation is considered to be achieved, outside the retina, through the isoform REP2. Recently, the possibility of systemic features in CHM has been debated; therefore, in this study, whole metabolomic analysis of plasma samples from 25 CHM patients versus age- and sex-matched controls was performed. Results showed plasma alterations in oxidative stress-related metabolites, coupled with alterations in tryptophan metabolism, leading to significantly raised serotonin levels. Lipid metabolism was disrupted with decreased branched fatty acids and acylcarnitines, suggestive of dysfunctional lipid oxidation, as well as imbalances of several sphingolipids and glycerophospholipids. Targeted lipidomics of the chmru848 zebrafish provided further evidence for dysfunction, with the use of fenofibrate over simvastatin circumventing the prenylation pathway to improve the lipid profile and increase survival. This study provides strong evidence for systemic manifestations of CHM and proposes potentially novel pathomechanisms and targets for therapeutic consideration.

Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia.

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.

Peculiar Clinical Findings in Young Choroideremia Patients: A Retrospective Case Review.

PURPOSE: To report peculiar clinical findings in young choroideremia (CHM) patients. METHODS: We retrospectively reviewed young (age <20 years at the first evaluation) CHM patients examined at the Regional Reference Center for Hereditary Retinal Degenerations at the Eye Clinic in Florence between 2012 and 2018. We took into consideration patients with ophthalmological examinations, fundus color photographs, fundus autofluorescence (FAF) images, optical coherence tomography (OCT) scans, full-field electroretinograms, and Goldmann visual fields. RESULTS: In our series, we studied 8 young CHM patients (average age 13.8 years, median age 12.5, range 10-20) for a total of 16 eyes. Visual acuity (VA) was 20/20 in 7 patients and 20/25 in both eyes of 1 patient. We identified a peculiar central FAF pattern (detectable in 3 patients), characterized by reduced central hypo-autofluorescence. Long OCT scans showed different forms of parapapillary retinal involvement from the mildest to the most severe form when the macula is still preserved. In 3 patients, at the time of atrophic changes at the posterior pole, it was possible to detect a progressive reduction of foveal pigmentation during follow-up. We found mutations of the CHM gene in all 6 patients who had been screened. CONCLUSIONS: CHM is a progressive retinal disorder which involves both the peripheral and the central retina. Using a multimodal imaging approach, we described peculiar central abnormalities underlying the early involvement of the central retina in young CHM patients with a good VA.

Classifying the molecular functions of Rab GTPases in membrane trafficking using deep convolutional neural networks.

Deep learning has been increasingly used to solve a number of problems with state-of-the-art performance in a wide variety of fields. In biology, deep learning can be applied to reduce feature extraction time and achieve high levels of performance. In our present work, we apply deep learning via two-dimensional convolutional neural networks and position-specific scoring matrices to classify Rab protein molecules, which are main regulators in membrane trafficking for transferring proteins and other macromolecules throughout the cell. The functional loss of specific Rab molecular functions has been implicated in a variety of human diseases, e.g., choroideremia, intellectual disabilities, cancer. Therefore, creating a precise model for classifying Rabs is crucial in helping biologists understand the molecular functions of Rabs and design drug targets according to such specific human disease information. We constructed a robust deep neural network for classifying Rabs that achieved an accuracy of 99%, 99.5%, 96.3%, and 97.6% for each of four specific molecular functions. Our approach demonstrates superior performance to traditional artificial neural networks. Therefore, from our proposed study, we provide both an effective tool for classifying Rab proteins and a basis for further research that can improve the performance of biological modeling using deep neural networks.

Molecular genetics characterization and homology modeling of the CHM gene mutation: A study on its association with choroideremia.

Choroideremia (CHM) is a rare form of X-linked chorioretinal dystrophy that is caused by mutations in the CHM gene. Mutations in the Rab escort protein-1 (REP-1), an ubiquitously encoded protein of the CHM gene, lead to prenylation and vesicle trafficking deficiency in the protein, resulting in the progressive degeneration of choriocapillaris, retinal pigment epithelium (RPE), and photoreceptors. Despite previous studies concerning this disease, no effective diagnostic tests or established therapeutic interventions currently exist for CHM. In this paper, we reviewed the pathogenic effects of synonymous hotspot mutation in the CHM gene and the genotypic-phenotypic associations in families with CHM. In addition, we employed a combination of molecular dynamics simulations and principal component analysis to gain insight into the underlying molecular basis of these deleterious and disease-causing hotspot mutation analogs. These computer predictions provide strong evidence that the C > T nonsynonymous hotspot mutations of CHM spectrum contribute to overall RPE retinopathy. These findings increase our understanding of the CHM pathogenesis, which may potentially define a new approach in developing novel symbiotic strategies for genetic diagnosis and specific treatment of inherited retinal diseases.

Use of induced pluripotent stem cell models to probe the pathogenesis of Choroideremia and to develop a potential treatment.

Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degeneration resulting from mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. The primary retinal cell type leading to CHM is unknown. In this study, we explored the utility of induced pluripotent stem cell-derived models of retinal pigmented epithelium (iPSC-RPE) to study disease pathogenesis and a potential gene-based intervention in four different genetically distinct forms of CHM. A number of abnormal cell biologic, biochemical, and physiologic functions were identified in the CHM mutant cells. We then identified a recombinant adeno-associated virus (AAV) serotype, AAV7m8, that is optimal for both delivering transgenes to iPSC-RPEs as well as to appropriate target cells (RPE cells and rod photoreceptors) in the primate retina. To establish the proof of concept of AAV7m8 mediated CHM gene therapy, we developed AAV7m8.hCHM, which delivers the human CHM cDNA under control of CMV-enhanced chicken ß-actin promoter (CßA). Delivery of AAV7m8.hCHM to CHM iPSC-RPEs restored protein prenylation, trafficking and phagocytosis. The results confirm that AAV-mediated delivery of the REP1-encoding gene can rescue defects in CHM iPSC-RPE regardless of the type of disease-causing mutation. The results also extend our understanding of mechanisms involved in the pathophysiology of choroideremia.

Novel mutation in the choroideremia gene and multi-Mendelian phenotypes in Spanish families.

AIMS: We aimed to accurately diagnose several retinitis pigmentosa (RP) patients with complex ocular phenotypes by combining massive sequencing genetic diagnosis and powerful clinical imaging techniques. METHODS: Whole-exome sequencing (WES) of selected patients from two RP families was undertaken. The variants identified were validated by Sanger sequencing and cosegregation analysis. Accurate clinical re-evaluation was performed using electrophysiological and visual field records as well as non-invasive imaging techniques, such as swept-source optical coherence tomography and fundus autofluorescence. RESULTS: The WES results highlighted one novel and one reported causative mutations in the X-linked choroideremia gene (CHM), which challenged the initial RP diagnosis. Subsequent clinical re-evaluation confirmed the choroideremia diagnosis. Carrier females showed different degrees of affectation, even between twin sisters, probably due to lyonization. A severe multi-Mendelian phenotype was associated with coincidental dominant pathogenic mutations in two additional genes: PAX6 and PDE6B. CONCLUSIONS: Genetic diagnosis via massive sequencing is instrumental in identifying causative mutations in retinal dystrophies and additional genetic variants with an impact on the phenotype. Multi-Mendelian phenotypes previously ascribed to rare syndromes can thus be dissected and molecularly diagnosed. Overall, the combination of powerful genetic diagnosis and clinical non-invasive imaging techniques enables efficient management of patients and their prioritisation for gene-specific therapies.

Choroideremia: melanopsin-mediated postillumination pupil relaxation is abnormally slow.

PURPOSE: To investigate the rod-cone and melanopsin pupillary light response (PLR) pathways in choroideremia. METHODS: Eight patients with choroideremia and 18 healthy age-matched controls underwent chromatic pupillometry by applying blue (463 nm) and red light (643 nm) at 100 lux intensity to the right eye while recording pupil diameters. Absolute baseline pupil size (mm), normalized maximal pupil constriction and the early and late postillumination pupillary dilation, from 0 to 10 seconds and 10 to 30 seconds after the end of illumination, respectively, were determined. Postillumination responses to blue light were considered to be primarily driven by melanopsin activation of the intrinsic photosensitive retinal ganglion cells. RESULTS: Baseline pupil diameters were comparable in patients with choroideremia and control subjects (p = 0.48). The maximum pupil constriction in patients with choroideremia was severely weakened in red light but only mildly weakened in blue light (p < 0.05). Postillumination dilation of the pupil was normal after red illumination but extremely protracted after blue illumination. Also, in contrast to healthy subjects, no abrupt change in the dilation curve was seen in the patients after the end of blue illumination, the early-phase dilation being completely abolished (p < 0.01). CONCLUSION: Rod-cone-driven pupil responses were decreased as expected in an outer retinal degeneration, and near-normal pupil constriction in blue light supports that the melanopsin system is normal. In contrast, the lack of brisk early-phase dilation after blue illumination in choroideremia is remarkable and may be interpreted to mean that the absence of photoreceptor inhibition promotes a tonic contraction of the pupil.

The Spectrum of CHM Gene Mutations in Choroideremia and Their Relationship to Clinical Phenotype.

PURPOSE: We report the underlying genotype and explore possible genotypic-phenotypic correlations in a large cohort of choroideremia patients. METHODS: We studied prospectively a cohort of 79 patients diagnosed within a tertiary referral service for patients with retinal dystrophies. Phenotypic evaluation consisted of clinical examination, including visual acuity and residual retinal area by fundus autofluorescence (FAF). Genotype was established by sequencing. We also investigated whether particular genotypes were associated with more severe phenotypes by performing analysis of covariance (ANCOVA), with visual acuity and FAF as the dependent variables and age as the covariant. RESULTS: A total of 74 (94%) of patients in our cohort had causative mutations by sequencing, the majority of which were anticipated to be null. Of these, 35 (47%) had insertions and deletions, 13 (18%) had mutations predicted to affect splicing, and 26 (35%) had single point mutations. In the latter case, 13 of 21 (62%) pedigrees with single point mutations were C to T transitions at C-phosphate-G (CpG) dinucleotides. These mutations were spread across 5 of only 24 CpG dinucleotides in the entire CHM cDNA. Furthermore, these 5 locations are the only sites at which C to T transitions result in a stop codon. No clear evidence was found for genotype-phenotype correlation except in the instance of a patient with a large deletion involving neighbouring sequences. CONCLUSIONS: In patients with a diagnosis of choroideremia made by a specialty service, there is a high likelihood of establishing a genetic diagnosis. The majority of causative mutations appear to be null and, therefore, may benefit from gene replacement therapy. A disproportionate number of single point mutations observed were C to T transitions, consistent with the evolutionary decay of CpG dinucleotides through methylation and subsequent deamination. Hence, the development of choroideremia in such patients may represent the unwanted consequence of human evolution; de novo mutations are predicted to arise at these sites in future generations. (ClinicalTrials.gov number, NCT01461213.).

Genetic analysis and clinical phenotype of two Indian families with X-linked choroideremia.

PURPOSE: This study aims to describe the phenotype and genotype of two Indian families affected with X-linked choroideremia (CHM). MATERIALS AND METHODS: In these two families, the affected individuals and unaffected family members underwent a comprehensive ophthalmic examination including an optical coherence tomography (OCT) and electroretinogram. Blood samples were collected from the families for genetic analysis. Next generation sequencing (NGS) was done using a panel of 184 genes, which covered previously associated genes with retinal dystrophies. Sequencing data were analyzed for the CHM, RPGR, and RP2 genes that have been implicated in CHM and X-linked retinitis pigmentosa (XLRP), respectively. The identified variants were confirmed by Sanger sequencing in available individuals and unrelated controls. RESULTS: In two unrelated male patients, NGS analysis revealed a previously reported 3'-splice site change c.820-1G>C in the CHM gene in the first family and hemizygous mutation c.653G>C (p.Ser218X) in the second family. The asymptomatic family members were carriers for these mutations. Spectral domain-OCT showed loss of outer retina, preservation of the inner retina, and choroidal thinning in the affected males and retinal pigment epithelial changes in the asymptomatic carriers. The identified mutations were not present in 100 controls of Indian origin. There were no potential mutations found in XLRP-associated (RPGR and RP2) genes. CONCLUSION: This report describes the genotype and phenotype findings in patients with CHM from India. The identified genetic mutation leads to lack of Rab escort protein-1 (REP-1) or affects the production of a REP-1 protein that is likely to cause retinal abnormalities in patients.

Neovascularizacion coroidea secundaria a coroideremia / Choroidal neovascularization secondary to choroideremia

CASO CLÍNICO: Varón de 30 años, con nictalopía y disminución de agudeza visual (AV) en ambos ojos, mayor en el ojo izquierdo (OI) de 20/100. En el fondo de ojo se observan lesiones compatibles con coroideremia y en OI, una hemorragia macular. Se confirma una NVC mediante OCT. El diagnóstico de certeza se obtuvo por estudio genético. No se realiza tratamiento porque el paciente no acude. A los 6 meses presenta regresión de la NVC con AV 20/25 en OI. CONCLUSIONES: La NVC asociada a coroideremia es infrecuente. El tratamiento sería la terapia antiangiogénica; sin embargo, es posible la resolución espontánea

CASE REPORT: The case is presented of a 30 year-old man, with night blindness and decreased visual acuity (VA) in both eyes, but more significant in the left eye (LE) of 20/100. Lesions consistent with choroideremia and LE macular hemorrhage was observed in the fundus. CNV was confirmed by OCT. A definitive diagnosis was obtained by genetic study. No treatment was given as the patient did not return. At 6 months there was a regression of CNV with VA 20/25 in the LE. CONCLUSIONS: CNV associated with choroideremia is uncommon. Treatment would antiangiogenic therapy, however spontaneous resolution is possible

Choroideremia Is a Systemic Disease With Lymphocyte Crystals and Plasma Lipid and RBC Membrane Abnormalities.

PURPOSE: Photoreceptor neuronal degenerations are common, incurable causes of human blindness affecting 1 in 2000 patients worldwide. Only half of all patients are associated with known mutations in over 250 disease genes, prompting our research program to identify the remaining new genes. Most retinal degenerations are restricted to the retina, but photoreceptor degenerations can also be found in a wide variety of systemic diseases. We identified an X-linked family from Sri Lanka with a severe choroidal degeneration and postulated a new disease entity. Because of phenotypic overlaps with Bietti's crystalline dystrophy, which was recently found to have systemic features, we hypothesized that a systemic disease may be present in this new disease as well. METHODS: For phenotyping, we performed detailed eye exams with in vivo retinal imaging by optical coherence tomography. For genotyping, we performed whole exome sequencing, followed by Sanger sequencing confirmations and cosegregation. Systemic investigations included electron microscopy studies of peripheral blood cells in patients and in normal controls and detailed fatty acid profiles (both plasma and red blood cell [RBC] membranes). Fatty acid levels were compared to normal controls, and only values two standard deviations above or below normal controls were further evaluated. RESULTS: The family segregated a REP1 mutation, suggesting choroideremia (CHM). We then found crystals in peripheral blood lymphocytes and discovered significant plasma fatty acid abnormalities and RBC membrane abnormalities (i.e., elevated plasmalogens). To replicate our discoveries, we expanded the cohort to nine CHM patients, genotyped them for REP1 mutations, and found the same abnormalities (crystals and fatty acid abnormalities) in all patients. CONCLUSIONS: Previously, CHM was thought to be restricted to the retina. We show, to our knowledge for the first time, that CHM is a systemic condition with prominent crystals in lymphocytes and significant fatty acid abnormalities.

Rab GTPase prenylation hierarchy and its potential role in choroideremia disease.

Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab escort protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.

High-resolution images of retinal structure in patients with choroideremia.

PURPOSE: To study retinal structure in choroideremia patients and carriers using high-resolution imaging techniques. METHODS: Subjects from four families (six female carriers and five affected males) with choroideremia (CHM) were characterized with best-corrected visual acuity (BCVA), kinetic and static perimetry, full-field electroretinography, and fundus autofluorescence (FAF). High-resolution macular images were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT). Coding regions of the CHM gene were sequenced. RESULTS: Molecular analysis of the CHM gene identified a deletion of exons 9 to 15 in family A, a splice site mutation at position 79+1 of exon 1 in family B, deletion of exons 6 to 8 in family C, and a substitution at position 106 causing a premature stop in family D. BCVA ranged from 20/16 to 20/63 in carriers and from 20/25 to 5/63 in affected males. FAF showed abnormalities in all subjects. SD-OCT showed outer retinal layer loss, outer retinal tubulations at the margin of outer retinal loss, and inner retinal microcysts. Patchy cone loss was present in two symptomatic carriers. In two affected males, cone mosaics were disrupted with increased cone spacing near the fovea but more normal cone spacing near the edge of atrophy. CONCLUSIONS: High-resolution retinal images in CHM carriers and affected males demonstrated RPE and photoreceptor cell degeneration. As both RPE and photoreceptor cells were affected, these cell types may degenerate simultaneously in CHM. These findings provide insight into the effect of CHM mutations on macular retinal structure, with implications for the development of treatments for CHM. (ClinicalTrials.gov number, NCT00254605.).

CHM/REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice.

BACKGROUND: Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers: photoreceptors, retinal pigment epithelium (RPE) and choroid, caused by the loss of Rab Escort Protein-1 (REP1). As a recessive monogenic disorder, CHM is potentially curable by gene addition therapy. The present study aimed to evaluate the potential use of lentiviral vectors carrying CHM/REP1 cDNA transgene for CHM treatment. METHODS: We generated lentiviral vectors carrying either CHM/REP1 cDNA or EGFP transgene under the control of the elongation factor-1α promoter (EF-1α) or its shortened version EFS. We transduced human (HT1080) and dog (D17) cells, CHM patient's fibroblasts and mouse primary RPE cells in vitro, as well as wild-type and CHM mouse retinas in vivo by subretinal injections. Transgene expression was confirmed by immunoblotting, fluorescence-activated cell sorting, immunofluorescence and confocal microscopy. CHM/REP1 transgene functionality was assessed by an in vitro prenylation assay. RESULTS: Lentiviral vectors with CHM/REP1 and EGFP transgenes efficiently transduced HT1080, D17 and CHM fibroblast cells; CHM/REP1 transgene lead to an increase in prenylation activity. Subretinal injections of lentiviral vectors into mouse retinas resulted in efficient transduction of the RPE (30-35% of total RPE cells transduced after a 1-µl injection), long-term expression for at least 6 months and a decrease in amount of unprenylated Rabs in the CHM RPE. Transduction of neuroretinal cells was restricted to the injection site. CONCLUSIONS: Lentiviral CHM/REP1 cDNA transgene rescues the prenylation defect in CHM mouse RPE and thus could be used to restore REP1 activity in the RPE of CHM patients.