Rab Escort Protein Research Articles

Retinal Patterns and the Role of Autofluorescence in Choroideremia.

Choroideremia is a monogenic inherited retinal dystrophy that manifests in males with night blindness, progressive loss of peripheral vision, and ultimately profound sight loss, commonly by middle age. It is caused by genetic defects of the CHM gene, which result in a deficiency in Rab-escort protein-1, a key element for intracellular trafficking of vesicles, including those carrying melanin. As choroideremia primarily affects the retinal pigment epithelium, fundus autofluorescence, which focuses on the fluorescent properties of pigments within the retina, is an established imaging modality used for the assessment and monitoring of affected patients. In this manuscript, we demonstrate the use of both short-wavelength blue and near-infrared autofluorescence and how these imaging modalities reveal distinct disease patterns in choroideremia. In addition, we show how these structural measurements relate to retinal functional measures, namely microperimetry, and discuss the potential role of these retinal imaging modalities in clinical practice and research studies. Moreover, we discuss the mechanisms underlying retinal autofluorescence patterns by imaging with a particular focus on melanin pigment. This could be of particular significance given the current progress in therapeutic options, including gene replacement therapy.

An RXLR effector disrupts vesicle trafficking at ER-Golgi interface for Phytophthora capsici pathogenicity

Phytophthora species, an oomycetes plant pathogen, secrete effectors into plant cell throughout their life cycle for manipulating host immunity to achieve successful colonization. However, the molecular mechanisms underlying effector-triggered necrotic cell death remain elusive. In this study, we identified an RXLR effector (Pc12) from Phytophthora capsici which contributes to virulence and induces necrosis by triggering a distinct endoplasmic reticulum (ER) stress response through its interaction with Rab13-2. The necrotic cell death induced by Pc12 did not exhibit conventional effector-triggered immunity (ETI)-mediated hypersensitive cell death, including the involvement of NLR downstream signaling components and transcriptional reprogramming of defense-related genes. Instead, it alters the localization of ER-resident proteins and confines secretory proteins within the ER. Pc12 directly interacts with Rab13-2, which is primarily localized to the ER and Golgi apparatus, resulting in a diminished Rab13-2 signal on the Golgi apparatus. Furthermore, Rab13-2 exhibits increased affinity for its interactor, Rab escort protein 1 (REP1), in the presence of Pc12. Structural predictions revealed that a specific residue of Rab13-2 is crucial for binding to the C-terminus of Pc12. Substitution of this residue reduced its interaction with Pc12 and impaired P. capsici infection, while maintaining its interaction with REP1 and prenylated Rab acceptor (PRA1). These findings provide insight into how a pathogen effector induces a distinct form of necrotic cell death to facilitate colonization of the host plant by disrupting the recycling of Rab13-2, a protein involved in vesicle trafficking at the ER-Golgi interface.

An Open-Label Phase II Study Assessing the Safety of Bilateral, Sequential Administration of Retinal Gene Therapy in Participants with Choroideremia: The GEMINI Study.

Choroideremia, an incurable, progressive retinal degeneration primarily affecting young men, leads to sight loss. GEMINI was a multicenter, open-label, prospective, two-period, interventional Phase II study assessing the safety of bilateral sequential administration of timrepigene emparvovec, a gene therapy, in adult males with genetically confirmed choroideremia (NCT03507686, ClinicalTrials.gov). Timrepigene emparvovec is an adeno-associated virus serotype 2 vector encoding the cDNA of Rab escort protein 1, augmented by a downstream woodchuck hepatitis virus post-transcriptional regulatory element. Up to 0.1 mL of timrepigene emparvovec, containing 1 × 1011 vector genomes, was administered by subretinal injection following vitrectomy and retinal detachment. The second eye was treated after an intrasurgery window of <6, 6-12, or >12 months. Each eye was followed at up to nine visits over 12 months. Overall, 66 participants received timrepigene emparvovec, and 53 completed the study. Visual acuity (VA) was generally maintained in both eyes, independent of intrasurgery window duration, even after bilateral retinal detachment and subretinal injection. Bilateral treatment was well tolerated, with predominantly mild or moderate treatment-emergent adverse events (TEAEs) and a low rate of serious surgical complications (7.6%). Retinal inflammation TEAEs were reported in 45.5% of participants, with similar rates in both eyes; post hoc analyses found that these were not associated with clinically significant vision loss at month 12 versus baseline. Two participants (3.0%) reported serious noninfective retinitis. Prior timrepigene emparvovec exposure did not increase the risk of serious TEAEs or serious ocular TEAEs upon injection of the second eye; furthermore, no systemic immune reaction or inoculation effect was observed. Presence of antivector neutralizing antibodies at baseline was potentially associated with a higher percentage of TEAEs related to ocular inflammation or reduced VA after injection of the first eye. The GEMINI study results may inform decisions regarding bilateral sequential administration of other gene therapies for retinal diseases.

Modeling Choroideremia Disease with Isogenic Induced Pluripotent Stem Cells.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy causing progressive vision loss due to mutations in the CHM gene, leading to Rab escort protein 1 loss of function. CHM disease is characterized by a progressive degeneration of the choroid, the retinal pigment epithelium (RPE), and the retina. The RPE is a monolayer of polarized cells that supports photoreceptors, providing nutrients, growth factors, and ions, and removes retinal metabolism waste products, having a central role in CHM pathogenesis. Commonly used models such as ARPE-19 cells do not reproduce accurately the nature of RPE cells. Human induced pluripotent stem cells (hiPSCs) can be differentiated into RPE cells (hiPSC-RPE), which mimic key features of native RPE, being more suited to study retinal diseases. Therefore, we took advantage of hiPSCs to generate new human-based CHM models. Two isogenic hiPSC lines were generated through CRISPR/Cas9: a CHM knock-out line from a healthy donor and a corrected CHM patient line using a knock-in approach. The differentiated hiPSC-RPE lines exhibited critical morphological and physiological characteristics of native RPE, including the presence of the tight junction markers Claudin-19 and Zonula Occludens-1, phagocytosis of photoreceptor outer segments, pigmentation, a postmitotic state, and the characteristic polygonal shape. In addition, all the studied cells were able to form retinal organoids. This work resulted in the establishment of isogenic hiPSC lines, representing a new and important CHM cellular model. To our knowledge, this is the first time that isogenic cell lines have been developed to model CHM disease, providing a valuable tool for studying the mechanisms at the onset of RPE degeneration.

Reduced Retinal Pigment Epithelial Autophagy Due to Loss of Rab12 Prenylation in a Human iPSC-RPE Model of Choroideremia.

Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in CHM, encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of CHM, the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited CHM-/- iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. CHM-/- iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in CHM-/- cells by transduction with gene replacement (ShH10-CMV-CHM) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.

Gene therapy for choroideremia using an adeno-associated viral vector encoding Rab escort protein 1: the REGENERATE open-label trial

Background Choroideremia is an X-linked inherited retinal degeneration that begins in childhood with nyctalopia and loss of peripheral vision, and gradually progresses to blindness in adulthood. Choroideremia is caused by null mutations in the CHM gene, which encodes Rab escort protein 1. Objective Assessment of the efficacy and safety of a single subretinal injection of an adeno-associated virus serotype 2 vector encoding Rab escort protein 1 in patients with choroideremia. Design Multicentre open-label clinical trial of a gene therapy for choroideremia using an adeno-associated virus serotype 2-Rab escort protein 1 vector. Setting This study (NCT02407678) was conducted at two NHS eye hospitals in the UK. Participants Males aged 18 years or above, having a clinical diagnosis of choroideremia with genetic confirmation of CHM gene mutation or molecular confirmation of Rab escort protein 1 protein deficiency and having best corrected visual acuity better than or equal to 6/60 (20/200; LogMAR 1.0). Intervention Adeno-associated virus serotype 2-Rab escort protein 1 vector suspension (1 × 1012 vector particles per ml) was supplied by Nightstar Therapeutics (London, UK), now part of Biogen Inc. (Cambridge, MA, USA). Up to 0.1 ml of adeno-associated virus serotype 2-Rab escort protein 1 vector suspension, corresponding to a dose of up to 1 × 1011 vector particles, was administered to the treated eye by subretinal injection. Selection of treated eyes was randomised in participants having relatively symmetrical retinal degeneration. Main outcome measures The primary safety-related outcome was change from baseline in best corrected visual acuity in treated eyes at 24 months post treatment, with prospective efficacy evaluated by comparative change from baseline in best corrected visual acuity in treated and untreated contralateral (control) eyes. Secondary outcomes included comparative change from baseline in mean retinal sensitivity (microperimetry) and retinal anatomy (area of autofluorescence) in treated and control eyes. Visual assessments were conducted by masked assessors. Results The primary efficacy-related outcome (comparative change from baseline in best corrected visual acuity in treated and control eyes at 24 months post treatment) was not statistically different between treated eyes (−2.63 letters, standard error of the mean 2.76) and control eyes (+2.67 letters, standard error of the mean 0.768) in all 30 participants (p = 0.08). Greater loss of visual fields, possibly surgery-induced, was observed in treated eyes. Six serious adverse events were reported in the treated eyes of four participants: one surgery-related and two inflammation-related serious adverse events involving clinically significant decreases in best corrected visual acuity, and three serious adverse events in one participant involving reduction in central retinal sensitivity, but with best corrected visual acuity remaining stable. Limitations No evidence of possible efficacy of the intervention was observed, as a meaningful difference in comparative change from baseline in best corrected visual acuity in treated and control eyes was not discernible at 24 months post treatment. As choroideremia is a very slow degeneration, best corrected visual acuity in control eyes did not decline significantly during the assessment period. Conclusion Although this study has not presented evidence that reduction in visual fields caused by the intervention would be justified by the possible rescue of best corrected visual acuity, a more definitive assessment may be provided by long-term monitoring of trial participants in an observational study (NCT03584165). Trial registration This study is registered as ISRCTN15602229 (www.isrctn.com/) and NCT02407678 (https://clinicaltrials.gov/). Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 12/66/35) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 9. See the NIHR Funding and Awards website for further award information.

Loss of REP1 impacts choroidal melanogenesis and vasculogenesis in choroideremia

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, however, the involvement of the choroid in disease progression is not fully understood. CHM is caused by mutations in the CHM gene, encoding the ubiquitously expressed Rab escort protein 1 (REP1). REP1 plays an important role in intracellular trafficking of vesicles, including melanosomes. In this study, we examined the ultrastructure of the choroid in chmru848 fish and Chmnull/WT mouse models using transmission electron and confocal microscopy. Significant pigmentary disruptions were observed, with lack of melanosomes in the choroid of chmru848 fish from 4 days post fertilisation (4dpf), and a reduction in choroidal blood vessel diameter and interstitial pillars suggesting a defect in vasculogenesis. Total melanin and expression of melanogenesis genes tyr, tryp1a, mitf, dct and pmel were also reduced from 4dpf. In Chmnull/WT mice, choroidal melanosomes were significantly smaller at 1 month, with reduced eumelanin at 1 year. The choroid in CHM patients were also examined using spectral domain optical coherence tomography (SD-OCT) and OCT-angiography (OCT-A) and the area of preserved choriocapillaris (CC) was found to be smaller than that of overlying photoreceptors, suggesting that the choroid is degenerating at a faster rate. Histopathology of an enucleated eye from a 74-year-old CHM male patient revealed isolated areas of RPE but no associated underlying CC. Pigmentary disruptions in CHM animal models reveal an important role for REP1 in melanogenesis, and drugs that improve melanin production represent a potential novel therapeutic avenue.

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.

Oxidative and Endoplasmic Reticulum Stress Represent Novel Therapeutic Targets for Choroideremia.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy, affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, with no approved therapy. CHM is caused by mutations in the CHM gene, which encodes the ubiquitously expressed Rab escort protein 1 (REP1). REP1 is involved in prenylation, a post-translational modification of Rab proteins, and plays an essential role in intracellular trafficking. In this study, we examined oxidative and endoplasmic reticulum (ER) stress pathways in chmru848 zebrafish and CHMY42X patient fibroblasts, and screened a number of neuroprotectants for their ability to reduce stress. The expression of the oxidative stress markers txn, cat and sod3a, and the ER stress markers bip, atf4 and atf6, were dysregulated in chmru848 fish. The expression of SOD2 was also reduced in CHMY42X fibroblasts, along with reduced BIP and increased CHOP expression. The lack of REP1 is associated with defects in vesicular trafficking, photoreceptor outer segment phagocytosis and melanosome transport, leading to increased levels of stress within the retina and RPE. Drugs targeting oxidative and ER stress pathways represent novel therapeutic avenues.

“Homology modeling of Rab11 protein of retina involved in Choroideremia (Homo sapiens)”

Choroideremia (CHM), is caused by a defect on the X gene which prohibits the body from making RAB Escort Protein-1, or REP-1, which further lead vision lost. The biological role of RAB Escort Protein-1 (REP-1) determine by homology modeling. In this research work the structure of Rab11 was predicted using Geno3d server. Ramachandran plot obtained by Geno 3-D server to estimate the effect of amino acid. The predicted structure was visualized through different visualization tools such as Pymol, Rasmol, and Swiss-pdb viewer. The active site was predicted through Q-site finder for further drug discovery and development for said disease.

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.

CHML is an NRF2 target gene that regulates mTOR function.

The transcription factor nuclear factor erythroid 2‐related factor 2 (NRF2) is often highly expressed in non‐small cell lung cancer (NSCLC). Through its target genes, NRF2 enhances cancer progression and chemo/radioresistance, leading to a poorer prognosis in patients with high NRF2 expression. In this study, we identified CHM‐like Rab escort protein (CHML; encoding Rep2) as an NRF2 target gene with an antioxidant response element (ARE) in its promoter region (–1622 to –1612). Analysis of patient data curated by The Cancer Genome Atlas (TCGA) and Oncomine databases revealed that CHML mRNA expression was elevated in lung adenocarcinoma (LUAD) patient tumor tissues and correlated with decreased patient survival. Immunohistochemistry (IHC) analysis of normal versus lung cancer patient tissues revealed that Rep2 protein levels were higher in lung tumors compared with normal tissue, which also correlated with increased levels of NRF2. Importantly, siRNA‐mediated knockdown of CHML/Rep2 in A549 NSCLC cells decreased their ability to proliferate. Mechanistically, Rep2 mediates mTOR function, as loss of Rep2 inhibited, whereas overexpression enhanced, mTOR translocation and activation at the lysosome. Our findings identify a novel NRF2–Rep2‐dependent regulation of mTOR function.

Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

SUMMARYLipid anchors are common post‐translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C‐termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP–Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to describe the structural basis of plant REP–Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule‐specific, and in other cases they are common for a subset of Rabs. In particular, the C‐terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C‐terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.

Choroideremia Gene Therapy.

Choroideremia Gene Therapy.

Molecular Characterization of Choroideremia-Associated Deletions Reveals an Unexpected Regulation of CHM Gene Transcription.

Choroideremia (CHM) is a X-linked recessive chorioretinal dystrophy due to deficiency of the CHM gene product, i.e., Rab escort protein isoform 1 (REP1). To date, gene therapy for CHM has shown variable effectiveness, likely because the underlying pathogenic mechanisms as well as genotype-phenotype correlation are not yet fully known. Small nucleotide variants leading to premature termination codons (PTCs) are a major cause of CHM, but about 20% of patients has CHM gene deletions. To improve understanding of the disease mechanisms, we analyzed molecular features of seven deletions involving the CHM gene sequence. We mapped the deletion breakpoints by using polymerase chain reaction, sequencing and array comparative genomic hybridization; to identify rearrangement-promoting DNA sequences, we analyzed genomic architecture surrounding the breakpoint regions. Moreover, in some CHM patients with different mutation types, we measured transcript level of CHM and of CHML, encoding the REP2 isoform. Scattered along the whole CHM gene and in close proximity to the deletion breakpoints we found numerous repeat elements that generate a locus-specific rearrangement hot spot. Unexpectedly, patients with non-PTC variants had increased expression of the aberrant CHM mRNA; CHML expression was higher than normal in a patient lacking CHM and its putative regulatory sequences. This latest evidence suggests that mechanisms regulating CHM and CHML gene expression are worthy of further study, because their full knowledge could be also useful for developing effective therapies for this hitherto untreatable inherited retinal degeneration.

Expression of Rab Prenylation Pathway Genes and Relation to Disease Progression in Choroideremia.

PurposeChoroideremia results from the deficiency of Rab Escort Protein 1 (REP1), encoded by CHM, involved in the prenylation of Rab GTPases. Here, we investigate whether the transcription and expression of other genes involved in the prenylation of Rab proteins correlates with disease progression in a cohort of patients with choroideremia.MethodsRates of retinal pigment epithelial area loss in 41 patients with choroideremia were measured using fundus autofluorescence imaging for up to 4 years. From lysates of cultured skin fibroblasts donated by patients (n = 15) and controls (n = 14), CHM, CHML, RABGGTB and RAB27A mRNA expression, and REP1 and REP2 protein expression were compared.ResultsThe central autofluorescent island area loss in patients with choroideremia occurred with a mean half-life of 5.89 years (95% confidence interval [CI] = 5.09–6.70), with some patients demonstrating relatively fast or slow rates of progression (range = 3.3–14.1 years). Expression of CHM mRNA and REP1 protein were significantly decreased in all patients. No difference in expression of CHML, RABGGTB, RAB27A, or REP2 was seen between patients and controls. No correlation was seen between expression of the genes analyzed and rates of retinal degeneration. Non-sense induced transcriptional compensation of CHML, a CHM-like retrogene, was not observed in patients with CHM variants predicted to undergo non-sense mediated decay.ConclusionsPatients with choroideremia, who are deficient for REP1, show normal levels of expression of other genes involved in Rab prenylation, which do not appear to play any modifying role in the rate of disease progression.Translational RelevanceThere remains little evidence for selection of patients for choroideremia gene therapy based on genotype.

Characterizing the cellular immune response to subretinal AAV gene therapy in the murine retina

Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45+ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 109 genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation.

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.

Update on Gene Therapy Clinical Trials for Choroideremia and Potential Experimental Therapies.

Background and objectives: Choroideremia (CHM) is an X-linked recessive chorioretinal dystrophy caused by mutations involving the CHM gene. Gene therapy has entered late-phase clinical trials, although there have been variable results. This review gives a summary on the outcomes of phase I/II CHM gene therapy trials and describes other potential experimental therapies. Materials and Methods: A Medline (National Library of Medicine, Bethesda, MD, USA) search was performed to identify all articles describing gene therapy treatments available for CHM. Results: Five phase I/II clinical trials that reported subretinal injection of adeno-associated virus Rab escort protein 1 (AAV2.REP1) vector in CHM patients were included. The Oxford study (NCT01461213) included 14 patients; a median gain of 5.5 ± 6.8 SD (−6 min, 18 max) early treatment diabetic retinopathy study (ETDRS) letters was reported. The Tubingen study (NCT02671539) included six patients; only one patient had an improvement of 17 ETDRS letters. The Alberta study (NCT02077361) enrolled six patients, and it reported a minimal vision change, except for one patient who gained 15 ETDRS letters. Six patients were enrolled in the Miami trial (NCT02553135), which reported a median gain of 2 ± 4 SD (−1 min, 10 max) ETDRS letters. The Philadelphia study (NCT02341807) included 10 patients; best corrected visual acuity (BCVA) returned to baseline in all by one-year follow-up, but one patient had −17 ETDRS letters from baseline. Overall, 40 patients were enrolled in trials, and 34 had 2 years of follow-up, with a median gain of 1.5 ± 7.2 SD (−14 min, 18 max) in ETDRS letters. Conclusions: The primary endpoint, BCVA following gene therapy in CHM, showed a marginal improvement with variability between trials. Optimizing surgical technique and pre-, peri-, and post-operative management with immunosuppressants to minimize any adverse ocular inflammatory events could lead to reduced incidence of complications. The ideal therapeutic window needs to be addressed to ensure that the necessary cell types are adequately transduced, minimizing viral toxicity, to prolong long-term transgenic potential. Long-term efficacy will be addressed by ongoing studies.

Alterations in the Genomic Distribution of 5hmC in In Vivo Aged Human Skin Fibroblasts.

5-Hydroxymethylcytosine (5hmC) is a functionally active epigenetic modification. We analyzed whether changes in DNA 5-hydroxymethylation are an element of age-related epigenetic drift. We tested primary fibroblast cultures originating from individuals aged 22–35 years and 74–94 years. Global quantities of methylation-related DNA modifications were estimated by the dot blot and colorimetric methods. Regions of the genome differentially hydroxymethylated with age (DHMRs) were identified by hMeDIP-seq and the MEDIPS and DiffBind algorithms. Global levels of DNA modifications were not associated with age. We identified numerous DHMRs that were enriched within introns and intergenic regions and most commonly associated with the H3K4me1 histone mark, promoter-flanking regions, and CCCTC-binding factor (CTCF) binding sites. However, only seven DHMRs were identified by both algorithms and all of their settings. Among them, hypo-hydroxymethylated DHMR in the intron of Rab Escort Protein 1 (CHM) coexisted with increased expression in old cells, while increased 5-hydroxymethylation in the bodies of Arginine and Serine Rich Protein 1 (RSRP1) and Mitochondrial Poly(A) Polymerase (MTPAP) did not change their expression. These age-related differences were not associated with changes in the expression of any of the ten-eleven translocation (TET) enzymes or their activity. In conclusion, the distribution of 5hmC in DNA of in vivo aged human fibroblasts underwent age-associated modifications. The identified DHMRs are, likely, marker changes.