Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium.
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ABSTRACT:
SUBMITTER: Park SW
PROVIDER: S-EPMC4544934 | biostudies-other | 2015 Aug
REPOSITORIES: biostudies-other
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Project description:PurposeTo test whether increased light transmission (hypertransmission) through subretinal drusenoid deposits (SDD) into the choroid in age-related macular degeneration (AMD) represented retinal pigment epithelium (RPE) degeneration.DesignCross-sectional study.MethodsNineteen eyes of 12 patients with early- to intermediate-stage AMD and 18 eyes of 12 normal subjects were evaluated with color fundus photography, optical coherence tomography (OCT), and high-resolution adaptive optics scanning laser ophthalmoscopy (AOSLO) at baseline and 24 months later. SDD were classified using an OCT-based 3-stage grading system. Hypertransmission beneath SDD into the choroid was examined in OCT. SDD microstructure was assessed with AOSLO. To characterize the hypertransmission-associated chorioretinal degeneration, choroidal thickness and photoreceptor length were measured in OCT at 1 mm and 2 mm superior, inferior, temporal, and nasal to the foveal center.ResultsOCT disclosed hypertransmission beneath stage 3 SDD in 8 eyes. These lesions showed a distinctive regressing structure in AOSLO, compared with stage 3 lesions without hypertransmission. The phenomenon persisted at follow-up, and new hypertransmission developed as SDD advanced. In eyes with hypertransmission, choroids were thinner than those of normal eyes at all sites (by 44%-56%, P ≤ .0028) and those of eyes with SDD but without hypertransmission at superior and temporal sites (by 31%-46%, P ≤ .039). Photoreceptors were significantly shorter than those in normal eyes (by 6%-26%, P ≤ .0379).ConclusionsHypertransmission into the choroid, accompanied with SDD regression and thinning of choroid and photoreceptor layers, indicates RPE degeneration associated with advanced stages in the SDD life cycle.
| S-EPMC5337135 | biostudies-literature
Project description:Ocular gene therapy has been extensively explored in recent years as a therapeutic avenue to target diseases of the cornea, retina and retinal pigment epithelium (RPE). Adeno-associated virus (AAV)-mediated gene therapy has shown promise in several RPE clinical trials but AAVs have limited payload capacity and potential immunogenicity. Traditionally however, non-viral alternatives have been plagued by low transfection efficiency, short-term expression and low expression levels. Recently, these drawbacks have begun to be overcome by the use of specialty carriers such as polylysine, liposomes, or polyethyleneimines, and by inclusion of suitable DNA elements to enhance gene expression and longevity. Recent advancements in the field have yielded non-viral vectors that have favorable safety profiles, lack immunogenicity, exhibit long-term elevated gene expression, and show efficient transfection in the retina and RPE, making them poised to transition to clinical applications. Here we discuss the advancements in nanotechnology and vector engineering that have improved the prospects for clinical application of non-viral gene therapy in the RPE.
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Project description:Advanced age-related macular degeneration (AMD) may lead to geographic atrophy or fibrovascular scar at macular, dysfunctional retinal microenvironment, and cause profound visual loss. Recent clinical trials have implied the potential application of pluripotent cell-differentiated retinal pigment epithelial cells (dRPEs) and membranous scaffolds implantation in repairing the degenerated retina in AMD. However, the efficacy of implanted membrane in immobilization and supporting the viability and functions of dRPEs, as well as maintaining the retinal microenvironment is still unclear. Herein we generated a biomimetic scaffold mimicking subretinal Bruch's basement from plasma modified polydimethylsiloxane (PDMS) sheet with laminin coating (PDMS-PmL), and investigated its potential functions to provide a subretinal environment for dRPE-monolayer grown on it. Firstly, compared to non-modified PDMS, PDMS-PmL enhanced the attachment, proliferation, polarization, and maturation of dRPEs. Second, PDMS-PmL increased the polarized tight junction, PEDF secretion, melanosome pigment deposit, and phagocytotic-ability of dRPEs. Third, PDMS-PmL was able to carry a dRPEs/photoreceptor-precursors multilayer retina tissue. Finally, the in vivo subretinal implantation of PDMS-PmL in porcine eyes showed well-biocompatibility up to 2-year follow-up. Notably, multifocal ERGs at 2-year follow-up revealed well preservation of macular function in PDMS-PmL, but not PDMS, transplanted porcine eyes. Trophic PEDF secretion of macular retina in PDMS-PmL group was also maintained to preserve retinal microenvironment in PDMS-PmL eyes at 2 year. Taken together, these data indicated that PDMS-PmL is able to sustain the physiological morphology and functions of polarized RPE monolayer, suggesting its potential of rescuing macular degeneration in vivo.
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Project description:Transplantation of stem cell-derived retinal pigment epithelium (RPE) cells is a promising potential therapy for currently incurable retinal degenerative diseases like advanced dry age-related macular degeneration. In this study, we designed a set of clinically applicable devices for subretinal implantation of RPE grafts, towards the overarching goal of establishing enabling technologies for cell-based therapeutic approaches to regenerate RPE cells. This RPE transplant kit includes a custom-designed trephine for the production of RPE transplants, a carrier for storage and transportation, and a surgical device for subretinal delivery of RPE transplants. Cell viability assay confirmed biocompatibility of the transplant carrier and high preservation of RPE transplants upon storage and transportation. The transplant surgical device combines foldable technology that minimizes incision size, controlled delivery speed, no fluid reflux, curved translucent tip, usability of loading and in vivo reloading, and ergonomic handle. Furthermore, the complementary design of the transplant carrier and the delivery device resulted in proper grasping, loading, and orientation of the RPE transplants into the delivery device. Proof-of-concept transplantation studies in a porcine model demonstrated no damage or structural change in RPE transplants during surgical manipulation and subretinal deployment. Post-operative assessment confirmed that RPE transplants were delivered precisely, with no damage to the host retina or choroid, and no significant structural change to the RPE transplants. Our novel surgical kit provides a comprehensive set of tools encompassing RPE graft manufacturing to surgical implantation rendering key enabling technologies for pre-clinical and clinical phases of stem cell-derived RPE regenerative therapies.
| S-EPMC8895272 | biostudies-literature
Project description:PurposeThe loss of retinal pigment epithelial (RPE) cells is a feature common to age-related macular degeneration (AMD) and retinitis pigmentosa (RP) and multiple early phase clinical trials are underway testing the safety of RPE cell replacement for these diseases. We examined whether transplantation of human neural stem cells into the subretinal space could enhance the endogenous proliferative capacity of the host RPE cell to regenerate.MethodsHuman central nervous system stem cells (HuCNS-SC) were isolated from enzymatically treated brain tissue using flow cytometry. Pigmented dystrophic Royal College of Surgeons (RCS) and S334ter-4 rats treated with oral bromodeoxyuridine (BrdU) received a unilateral subretinal injection of 1.0 × 105 HuCNS-SC cells at either postnatal day 21 or 60. Animals were sacrificed at 90, 120, and 150 days of age. Eyes were fixed processed for cryostat sectioning. Sections were immunostained with Stem101, Ku80, RPE65, OTX1/2, BrdU, and CRALBP antibodies and analyzed via confocal microscopy.ResultsRCS rats that received transplantation of HuCNS-SC had significantly more (approximately 3-fold) Ki67-positive or BrdU-labelled host RPE cells adjacent to the HuCNS-SC graft than controls. Significantly increased host RPE cell proliferation as a result of HuCNS-SC transplantation also was confirmed in S334ter-line 4 transgenic rats with higher proliferation observed in animals with longer posttransplantation periods.ConclusionsThese results suggest that controlled proliferation of endogenous RPE by HuCNS-SC may provide another mechanism by which RPE cell diseases could be treated.Translational relevanceEngaging the capacity for endogenous RPE cell regeneration in atrophic diseases may be a novel therapeutic strategy for degenerative diseases of the RPE and retina.
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Project description:In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.
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Project description:Antibody-mediated rejection is characterized by donor-specific antibody produced by B cells. However, to our knowledge, B cell invasion and antibody in the inflamed retina after transplantation of retinal pigment epithelial (RPE) cells has not been reported. To determine if RPE transplantation could be performed using allografts, we established in vivo immune rejection models with induced pluripotent stem cell (iPSC)-RPE allografts and determined whether RPE-specific antibody could be detected in these models. We detected alloantibodies in the serum from recipient monkeys that had immune attacks in the retina in an immunofluorescent assay using the transplanted iPSC-RPE cells as the antigen. In addition to T cell and antigen-presenting cell immunity, peripheral blood cells and lymph nodes in animal models with allogeneic iPSC-RPE cells also had activated B cells, which were probably secreting alloantibodies. Using serum and transplanted cells, alloreactive antibody can be detected for the diagnosis of immune rejection after transplantation.
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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.
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Project description:The purpose of this study was to evaluate focal damage in the retinal pigment epithelium (RPE) layer in serous retinal pigment epithelium detachment (PED) with multi-contrast optical coherence tomography (OCT), which is capable of simultaneous measurement of OCT angiography, polarization-sensitive OCT and standard OCT images. We evaluated 37 eyes with age-related macular degeneration that had serous PED. Focal RPE damage was indicated by hyper-transmission beneath the RPE-Bruch's membrane band in standard OCT images. Distribution of RPE melanin was calculated using the dataset from multi-contrast OCT. Twenty-four points with hyper-transmission were detected in 21 of the 37 eyes. Standard OCT images failed to show disruption of the RPE-Bruch's membrane band at 5 of the 24 hyper-transmission points. Conversely, multi-contrast OCT images clearly showed melanin defects in the RPE-Bruch's membrane band at all points. Areas of melanin defects with disruption of the RPE-Bruch's membrane band were significantly larger than those without disruption. The volume of intraretinal hyper-reflective foci was significantly larger in eyes with hyper-transmission than that in eyes without hyper-transmission. Multi-contrast OCT is more sensitive than standard OCT for displaying changes at the RPE-Bruch's membrane band when there are small areas of RPE damage.
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Project description: Not available
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