Purpose: To assess the progression in functional and structural measures over a five-year period in patients with retinal dystrophy caused by RLBP1 gene mutation. Methods: This prospective, noninterventional study included patients with biallelic RLBP1 mutations from two clinical sites in Sweden and Canada. Key assessments included ocular examinations, visual functional measures (best-corrected visual acuity [BCVA], contrast sensitivity [CS], dark-adaptation [DA] kinetics up to six hours for two wavelengths [450 and 632 nm], Humphrey visual fields [HVF], full-field flicker electroretinograms), and structural ocular assessments. Results: Of the 45 patients enrolled, 38 completed the full five years of follow-up. At baseline, patients had BCVA ranging from −0.2 to 1.3 logMAR, poor CS, HVF defects, and prominent thinning in central foveal thickness. All patients had extremely prolonged DA rod recovery of approximately six hours at both wavelengths. The test-retest repeatability was high across all anatomic and functional endpoints. Cross-sectionally, poorer VA was associated with older age (right eye, correlation coefficient [CC]: 0.606; left eye, CC: −0.578; P < 0.001) and HVF MD values decreased with age (right eye, CC: −0.672, left eye, CC: −0.654; P < 0.001). However, no major changes in functional or structural measures were noted longitudinally over the five-year period. Conclusions: This natural history study, which is the first study to monitor patients with RLBP1 RD for five years, showed that severely delayed DA sensitivity recovery, a characteristic feature of this disease, was observed in all patients across all age groups (17–69 years), making it a potentially suitable efficacy assessment for gene therapy treatment in this patient population.

Intravitreal (IVT) injection has become the standard route for drug administration in retinal diseases. However, the ability to measure biodistribution of ocular therapeutics in large species remains limited, due to the invasive nature of some techniques or their lack of spatial information. The aim of this study was to develop in cynomolgus monkeys a non-invasive fluorescence imaging technology that enables tracking of IVT-dosed drugs and could be easily translated into humans. Here, we show a proof-of-concept for labeled ranibizumab with observed half-lives of 3.34 and 4.52 days at the retina and in the vitreous, respectively. We further investigate a long acting anti-VEGF antibody, which remains as an agglomerate with some material leaking out until the end of the study at Day 35. Overall, we were able to visualize and measure differences in the in vivo behavior between short and long-acting antibodies, demonstrating the power of the technology for ocular pharmacokinetics.

PURPOSE:Mononuclear phagocytes (MNPs) are present in neovascular age-related macular degeneration (nv AMD) which is also called choroidal neovascularization (CNV). The number and phenotype of the MNPs depend upon the local environment in the CNV and effect of nv AMD therapy. We investigated ocular cell infiltration and conditions that modulate angiogenesis in a laser-induced mouse CNV model.METHODS:We developed assays to quantify MNPs in our established mouse CNV model. One MNP assay quantified the number of subretinal cells peripheral to the CNV lesions. A second assay semiquantitatively assesses the number of MNPs localized to the CNV lesion. We used these assays to measure the effect of toll-like receptor-2 (TLR-2) activation, anti-vascular endothelial growth factor (VEGF) therapy, and chemokine (C-C motif) ligand 2 (Ccl2) genetic deletion on MNP infiltration after laser injury.RESULTS:Laser injury induced blood vessel growth and infiltration of MNPs. Systemic administration of a TLR-2 activating peptide increased laser-induced CNV area, MNP cell numbers, and MNP density over the CNV lesions. Systemic administration of a VEGF antibody reduced CNV area, while Ccl2 genetic deletion increased CNV area. Despite the change in amount of angiogenesis, MNP infiltration was, surprisingly, unchanged in these 2 conditions.CONCLUSIONS:MNP quantification provides biological insights for candidate AMD therapies. The number of infiltrating MNP cells does not correlate with the amount of laser-induced CNV area.

Protein drugs that neutralize vascular endothelial growth factor (VEGF), such as aflibercept or ranibizumab, rescue vision in patients with retinal vascular diseases. Nonetheless, optimal visual outcomes require intraocular injections as frequently as every month. Here we report a method to extend the intravitreal half-life of protein drugs as an alternative to either encapsulation or chemical modifications with polymers. We combine a 97-amino-acid peptide of human origin that binds hyaluronan, a major macromolecular component of the eye's vitreous, with therapeutic antibodies and proteins. When administered to rabbit and monkey eyes, the half-life of the modified proteins is increased ∼3-4-fold relative to unmodified proteins. We further show that prototype long-acting anti-VEGF drugs (LAVAs) that include this peptide attenuate VEGF-induced retinal changes in animal models of neovascular retinal disease ∼3-4-fold longer than unmodified drugs. This approach has the potential to reduce the dosing frequency associated with retinal disease treatments.

Program Number: 3216 Poster Board Number: B0349 Presentation Time: 11:00 AM–12:45 PM EZ Width Reflects Disease Severity in adRP Patients with PRPF31 Gene Mutations Kaylie Webb-Jones1, Martin Klein1, Sara J. Bowne2, Lori S. Sullivan2, Stephen P. Daiger2, 4, David G. Birch1, 3. 1Retina Foundation of the Southwest, Dallas, TX; 2Human Genetics Center, School of Public Health, The Univ. of Texas Health Science Center, Houston, TX; 3Dept. Ophthalmology, Univ. of Texas Southwestern Medical Center, Dallas, TX; 4Ruiz Dept. of Ophthalmology and Visual Science, The Univ. of Texas Health Science Center, Houston, TX. Purpose: Mutations in the pre-mRNA splicing factor gene PRPF31 are one of the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 5.5-8.7% of cases (Sullivan 2006; Daiger 2014). Here we investigate the relationships between visual acuity (VA), kinetic perimetry and the ellipsoid zone (EZ) width in adRP patients with mutations in PRPF31. Methods: Spectral domain optical coherence tomography (SD-OCT) horizontal line scans through the fovea were obtained for both eyes of eighteen RP patients (range: 10-72 yrs; mean: 43 ± 20 yrs) from 10 families with mutations in PRPF31. The EZ band was delineated and its horizontal width was determined (Hood 2011). Visual acuity (VA) was measured with a computerized version of the electronic Early Treatment Diabetic Retinopathy Study (e-ETDRS). Octopus (isopter III) kinetic perimetry was used to measure visual field diameter. Correlations were examined by Pearson’s correlation tests. Results: The mean EZ width was not significantly different between eyes (mean ± 95% CI: 12.0° ± 5.04 OD; 13.1° ± 4.9 OS). Mean VA was also comparable between eyes 0.38 ± 0.0.25 logMAR OD; 0.33 ± 0.18 logMAR OS), therefore OD was selected for correlation analysis. EZ width was significantly correlated with VA (r=-0.724, p<0.001) and visual field diameter (r=0.885, p=0.002). EZ width was also significantly correlated with age (r=-0.586, p<0.001). Conclusions: Previous studies have shown EZ width to be a useful tool to monitor disease progression in adRP, X-linked RP and Usher syndrome IB patients (Birch 2013; Cai, 2014; Sumaroka 2016). Here, we found that EZ width reflects disease severity in that it correlates with age, VA, and visual field diameter. These finding suggest that EZ width or area can be used as a measurement of disease progression in patients with mutations in PRPF31. Commercial Relationships: Kaylie Webb-Jones, None; Martin Klein, None; Sara J. Bowne, None; Lori S. Sullivan, None; Stephen P. Daiger, None; David G. Birch, None Support: Foundation Fighting Blindness, NEI EY09076, NIH EY007142, William Stamps Farish Fund

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

Islet transplantation represents a potentially curative approach for individuals with Type I Diabetes. The requirement for systemic immune suppression to control immune-mediated rejection of transplanted islets and the limited human islet supply represent significant roadblocks to progress for this approach. Islet microencapsulation in alginate offers limited protection in the absence of systemic immunosuppression, but does not support long-term islet survival. The chemokine, CXCL12, can repel effector T cells while recruiting immune-suppressive regulatory T cells (Tregs) to an anatomic site while providing a prosurvival signal for beta-cells. We proposed that coating or encapsulating donor islets with CXCL12 would induce local immune-isolation and protect and support the function of an allo- or xenograft without systemic immune suppression. This study investigated the effect of alginate microcapsules incorporating CXCL12 on islet function. Islet transplantation was performed in murine models of insulin-dependent diabetes. Coating of islets with CXCL12 or microencapsulation of islets with alginate incorporating the chemokine, resulted in long-term allo- and xenoislet survival and function, as well as a selective increase in intragraft Tregs. These data support the use of CXCL12 as a coating or a component of an alginate encapsulant to induce sustained local immune-isolation for allo- or xenoislet transplantation without systemic immunosuppression.