Correlation of features on OCT with visual acuity and Gass lesion type in Best vitelliform macular dystrophy.

OBJECTIVE: To correlate structural features seen on optical coherence tomography (OCT) with best-corrected visual acuity (BCVA) and Gass lesion type in patients with Best vitelliform macular dystrophy (BVMD). METHODS AND ANALYSIS: This is a retrospective case series of consecutive patients with molecularly confirmed BEST1-associated BVMD. OCT scans were reviewed for lesion status and presence of subretinal pillar, focal choroidal excavation (FCE), intraretinal fluid or atrophy. Available OCT angiography images were used to evaluate for the presence of choroidal neovascularisation (CNV). These features were then correlated with BCVA and Gass lesion type. RESULTS: 95 eyes from 48 patients (mean age 38.9 years, range 4-87) were included. The presence of a pillar (24.2%), FCE (20.0%) and atrophy (7.4%) were associated with poor BCVA (p<0.05). Gass lesion type 1 eyes were correlated with good BCVA (LogMAR <0.4) whereas type 5 eyes had poor BCVA (LogMAR >0.4). Among 65 eyes with longitudinal data (mean follow-up 5.1 years), 7 eyes (10.8%) reverted from higher to lower Gass lesion type; of these, 4 eyes (57.1%) had CNV responsive to intravitreal anti-vascular endothelial growth factor treatment. CONCLUSION: OCT-based structural features are readily identifiable in patients with BVMD and have prognostic importance due to their correlation with BCVA.

Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e., potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR, we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e., analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.

Generation of Xeno-Free, cGMP-Compliant Patient-Specific iPSCs from Skin Biopsy.

This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs, and derive autologous retinal cells via 3-D differentiation. The universal methods described herein for the isolation of dermal fibroblasts and generation of iPSCs can be employed regardless of disease, tissue, or cell type of interest. © 2017 by John Wiley & Sons, Inc.

Two-photon polymerization for production of human iPSC-derived retinal cell grafts.

Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific neurons that are ideal for autologous cell replacement for treatment of neurodegenerative diseases. In the case of retinal degeneration and associated photoreceptor cell therapy, polymer scaffolds are critical for cellular survival and integration; however, prior attempts to materialize this concept have been unsuccessful in part due to the materials' inability to guide cell alignment. In this work, we used two-photon polymerization to create 180µm wide non-degradable prototype photoreceptor scaffolds with varying pore sizes, slicing distances, hatching distances and hatching types. Hatching distance and hatching type were significant factors for the error of vertical pore diameter, while slicing distance and hatching type most affected the integrity and geometry of horizontal pores. We optimized printing parameters in terms of structural integrity and printing time in order to create 1mm wide scaffolds for cell loading studies. We fabricated these larger structures directly on a porous membrane with 3µm diameter pores and seeded them with human iPSC-derived retinal progenitor cells. After two days in culture, cells nested in and extended neuronal processes parallel to the vertical pores of the scaffolds, with maximum cell loading occurring in 25µm diameter pores. These results highlight the feasibility of using this technique as part of an autologous stem cell strategy for restoring vision to patients affected with retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Cell replacement therapy is an important goal for investigators aiming to restore neural function to those suffering from neurodegenerative disease. Cell delivery scaffolds are frequently necessary for the success of such treatments, but traditional biomaterials often fail to facilitate the neuronal orientation and close packing needed to recapitulate the in vivo environment. Here, we use two-photon polymerization to create prototype cell scaffolds with densely packed vertical pores for photoreceptor cell loading and small, interconnected horizontal pores for nutrient diffusion. This study offers a thorough characterization of how two-photon polymerization parameters affect final structural outcomes and printing time. Our findings demonstrate the feasibility of using two-photon polymerization to create scaffolds that can align neuronal cells in 3D and are large enough to be used for transplantation. In future work, these scaffolds could comprise biodegradable materials with tunable microstructure, elastic modulus and degradation time; a significant step towards a promising treatment option for those suffering from late-stage neurodegeneration, including retinal degenerative blindness.

The ClC-3 Cl-/H+ antiporter becomes uncoupled at low extracellular pH.

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH

Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent.

ClC-3 is a member of the ClC family of anion channels/transporters. Recently, the closely related proteins ClC-4 and ClC-5 were shown to be Cl(-)/H(+) antiporters (39, 44). The function of ClC-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant ClC-3. The basic biophysical properties of ClC-3 currents were very similar to those of ClC-4 and ClC-5, and distinct from those of the swelling-activated anion channel. ClC-3 expression induced currents with time-dependent activation that rectified sharply in the outward direction. The reversal potential of the current shifted by -48.3 +/- 2.5 mV per 10-fold (decade) change in extracellular Cl(-) concentration, which did not conform to the behavior of an anion-selective channel based upon the Nernst equation, which predicts a -58.4 mV/decade shift at 22 degrees C. Manipulation of extracellular pH (6.35-8.2) altered reversal potential by 10.2 +/- 3.0 mV/decade, suggesting that ClC-3 currents were coupled to proton movement. Mutation of a specific glutamate residue (E224A) changed voltage dependence in a manner similar to that observed in other ClC Cl(-)/H(+) antiporters. Mutant currents exhibited Nernstian changes in reversal potential in response to altered extracellular Cl(-) concentration that averaged -60 +/- 3.4 mV/decade and were pH independent. Thus ClC-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to ClC-4 and ClC-5.