A Retrospective Longitudinal Study of 460 Patients with ABCA4-Associated Retinal Disease.

PURPOSE: To investigate the distribution of genotypes and natural history of ABCA4-associated retinal disease in a large cohort of patients seen at a single institution. DESIGN: Retrospective, single-institution cohort review. PARTICIPANTS: Patients seen at the University of Iowa between November 1986 and August 2022 clinically suspected to have disease caused by sequence variations in ABCA4. METHODS: DNA samples from participants were subjected to a tiered testing strategy progressing from allele-specific screening to whole genome sequencing. Charts were reviewed, and clinical data were tabulated. The pathogenic severity of the most common alleles was estimated by studying groups of patients who shared 1 allele. Groups of patients with shared genotypes were reviewed for evidence of modifying factor effects. MAIN OUTCOME MEASURES: Age at first uncorrectable vision loss, best-corrected visual acuity, and the area of the I2e isopter of the Goldmann visual field. RESULTS: A total of 460 patients from 390 families demonstrated convincing clinical features of ABCA4-associated retinal disease. Complete genotypes were identified in 399 patients, and partial genotypes were identified in 61. The median age at first vision loss was 16 years (range, 4-76 years). Two hundred sixty-five families (68%) harbored a unique genotype, and no more than 10 patients shared any single genotype. Review of the patients with shared genotypes revealed evidence of modifying factors that in several cases resulted in a > 15-year difference in age at first vision loss. Two hundred forty-one different alleles were identified among the members of this cohort, and 161 of these (67%) were found in only a single individual. CONCLUSIONS: ABCA4-associated retinal disease ranges from a very severe photoreceptor disease with an onset before 5 years of age to a late-onset retinal pigment epithelium-based condition resembling pattern dystrophy. Modifying factors frequently impact the ABCA4 disease phenotype to a degree that is similar in magnitude to the detectable ABCA4 alleles themselves. It is likely that most patients in any cohort will harbor a unique genotype. The latter observations taken together suggest that patients' clinical findings in most cases will be more useful for predicting their clinical course than their genotype. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.

Rhodopsin (RHO) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOpCRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

Long-term functional and structural outcomes in X-linked retinoschisis: implications for clinical trials.

Introduction: X-linked retinoschisis (XLRS) is an inherited retinal disease (IRD) caused by pathogenic mutations in the retinoschisin gene, RS1. Affected individuals develop retinal layer separation, leading to loss of visual acuity (VA). Several XLRS gene therapy trials have been attempted but none have met their primary endpoints. An improved understanding of XLRS natural history and clinical outcomes may better inform future trials. Here, we report the long-term functional and structural outcomes of XLRS and the relevance of RS1 genotypes to the visual prognosis of affected individuals. Methods: A retrospective chart review of patients with molecularly confirmed X-linked retinoschisis was performed. Functional and structural outcomes, and RS1 genotype data, were included for analysis. Results: Fifty-two patients with XLRS from 33 families were included in the study. Median age at symptom onset was 5 years (range 0-49) and median follow-up was 5.7 years (range 0.1-56.8). Macular retinoschisis occurred in 103 of 104 eyes (99.0%), while peripheral retinoschisis occurred in 48 of 104 eyes (46.2%), most often in the inferotemporal quadrant (40.4%). Initial and final VA were similar (logMAR 0.498 vs. 0.521; p = 0.203). Fifty of 54 eyes (92.6%) developed detectable outer retinal loss by age 20, and 29 of 66 eyes (43.9%) had focal or diffuse outer retinal atrophy (ORA) by age 40. ORA but not central subfield thickness (CST) was associated with reduced VA. Inter-eye correlation was modest for VA (r-squared = 0.03; p = 0.08) and CST (r-squared = 0.15; p = 0.001). Carbonic anhydrase inhibitors (CAIs) improved CST (p = 0.026), but not VA (p = 0.380). Eight of 104 eyes (7.7%) had XLRS-related retinal detachment (RD), which was associated with poorer outcomes compared to eyes without RD (median final VA 0.875 vs. 0.487; p <0.0001). RS1 null genotypes had greater odds of at least moderate visual impairment at final follow-up (OR 7.81; 95% CI 2.17, 28.10; p = 0.002) which was independent of age at onset, initial CST, initial ORA, or previous RD. Discussion: Overall, long-term follow-up of XLRS patients demonstrated relatively stable VA, with presenting CST, development of ORA, and null RS1 mutations associated with poorer long-term visual outcomes, indicating a clinically relevant genotype-phenotype correlation in XLRS.

The Ocular Trauma Score Underestimates Visual Recovery for the Most Severe Open-Globe Injuries.

PURPOSE: To compare visual outcomes after open-globe injury (OGI) with those predicted by the Ocular Trauma Score (OTS), and to investigate the effect of treatment with pars plana vitrectomy (PPV). DESIGN: Retrospective cohort study. SUBJECTS: Patients presenting with OGI to an academic United States ophthalmology department from 2017 to 2020. METHODS: Best-corrected visual acuity (VA) measurements at the most recent follow-up were compared with final VA predicted by the OTS, based on preoperative injury characteristics. The most recently measured VA of patients treated with PPV during initial OGI repair (primary PPV group) was compared with patients treated with PPV after initial OGI repair (secondary PPV group) and patients never treated with PPV (No PPV group). MAIN OUTCOME MEASURES: Best-corrected VA in the injured eye at last follow-up; secondary outcome measures included the occurrence of vitreous hemorrhage at any time, occurrence of retinal detachment at any time, rates of additional surgery, and rates of enucleation. RESULTS: One-hundred and thirty-three subjects with OGI were identified and analyzed. The overall rate of PPV was 32%. Predictors of worse VA at last follow-up included older age (P = 0.047) and worse presenting VA (P < 0.001). Visual acuity outcomes for eyes in OTS categories 2 to 5 did not significantly differ from OTS predictions. However, eyes in OTS category 1 had a higher likelihood of last follow-up VA of light perception (LP) to hand motion (46% in the study cohort vs. 15% predicted by the OTS, P = 0.004) and a lower likelihood of no LP (33% vs. 74%, P < 0.001). The secondary PPV group had the worst VA at presentation among the 3 groups (P = 0.016), but VA at last follow-up did not significantly differ between the study groups (P = 0.338). CONCLUSIONS: The most severe OGIs (i.e., OTS category 1) had better visual outcomes than predicted by the published OTS expectations, and secondary PPV was associated with significant visual improvement despite poor prognostic predictions. Evaluation by a vitreoretinal surgeon should be considered for all patients with severe OGI, especially those in OTS category 1. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

RNA-based therapies in inherited retinal diseases.

Inherited retinal diseases (IRDs) are a genetically and phenotypically heterogeneous group of genetic eye disorders. There are more than 300 disease entities, and together this group of disorders affects millions of people globally and is a frequent cause of blindness or low-vision certification. However, each type is rare or ultra-rare. Characteristically, the impaired vision in IRDs is due to retinal photoreceptor dysfunction and loss resulting from mutation in a gene that codes for a retinal protein. Historically, IRDs have been considered incurable and individuals living with these blinding conditions could be offered only supportive care. However, the treatment landscape for IRDs is beginning to evolve. Progress is being made, driven by improvements in understanding of genotype-phenotype relationships, through advances in molecular genetic testing and retinal imaging. Alongside this expanding knowledge of IRDs, the current era of precision medicine is fueling a growth in targeted therapies. This has resulted in the first treatment for an IRD being approved. Several other therapies are currently in development in the IRD space, including RNA-based therapies, gene-based therapies (such as augmentation therapy and gene editing), cell therapy, visual prosthetics, and optogenetics. RNA-based therapies are a novel approach within precision medicine that have demonstrated success, particularly in rare diseases. Three antisense oligonucleotides (AONs) are currently in development for the treatment of specific IRD subtypes. These RNA-based therapies bring several key advantages in the setting of IRDs, and the potential to bring meaningful vision benefit to individuals living with inherited blinding disorders. This review will examine the increasing breadth and relevance of RNA-based therapies in clinical medicine, explore the key features that make AONs suitable for treating genetic eye diseases, and provide an overview of the three-leading investigational AONs in clinical trials.

Restoration of Cone Sensitivity to Individuals with Congenital Photoreceptor Blindness within the Phase 1/2 Sepofarsen Trial.

Purpose: To understand consequences of reconstituting cone photoreceptor function in congenital binocular blindness resulting from mutations in the centrosomal protein 290 (CEP290) gene. Design: Phase 1b/2 open-label, multicenter, multiple-dose, dose-escalation trial. Participants: A homogeneous subgroup of 5 participants with light perception (LP) vision at the time of enrollment (age range, 15-41 years) selected for detailed analyses. Medical histories of 4 participants were consistent with congenital binocular blindness, whereas 1 participant showed evidence of spatial vision in early life that was later lost. Intervention: Participants received a single intravitreal injection of sepofarsen (160 or 320 µg) into the study eye. Main Outcome Measures: Full-field stimulus testing (FST), visual acuity (VA), and transient pupillary light reflex (TPLR) were measured at baseline and for 3 months after the injection. Results: All 5 participants with LP vision demonstrated severely abnormal FST and TPLR findings. At baseline, FST threshold estimates were 0.81 and 1.0 log cd/m2 for control and study eyes, respectively. At 3 months, study eyes showed a large mean improvement of -1.75 log versus baseline (P < 0.001), whereas untreated control eyes were comparable with baseline. Blue minus red FST values were not different than 0 (P = 0.59), compatible with cone mediation of remnant vision. At baseline, TPLR response amplitude and latency estimates were 0.39 mm and 0.72 seconds, respectively, for control eyes, and 0.28 mm and 0.78 seconds, respectively, for study eyes. At 3 months, study eyes showed a mean improvement of 0.44 mm in amplitude and a mean acceleration of 0.29 seconds in latency versus baseline (P < 0.001), whereas control eyes showed no significant change versus baseline. Specialized tests performed in 1 participant confirmed and extended the standardized results from all 5 participants. Conclusions: By subjective and objective evidence, intravitreal sepofarsen provides improvement of light sensitivity for individuals with LP vision. However, translation of increased light sensitivity to improved spatial vision may occur preferentially in those with a history of visual experience during early neurodevelopment. Interventions for congenital lack of spatial vision in CEP290-associated Leber congenital amaurosis may lead to better results if performed before visual cortex maturity.

Intravitreal antisense oligonucleotide sepofarsen in Leber congenital amaurosis type 10: a phase 1b/2 trial.

CEP290-associated Leber congenital amaurosis type 10 (LCA10) is a retinal disease resulting in childhood blindness. Sepofarsen is an RNA antisense oligonucleotide targeting the c.2991+1655A>G variant in the CEP290 gene to treat LCA10. In this open-label, phase 1b/2 ( NCT03140969 ), 12-month, multicenter, multiple-dose, dose-escalation trial, six adult patients and five pediatric patients received ≤4 doses of intravitreal sepofarsen into the worse-seeing eye. The primary objective was to evaluate sepofarsen safety and tolerability via the frequency and severity of ocular adverse events (AEs); secondary objectives were to evaluate pharmacokinetics and efficacy via changes in functional outcomes. Six patients received sepofarsen 160 µg/80 µg, and five patients received sepofarsen 320 µg/160 µg. Ten of 11 (90.9%) patients developed ocular AEs in the treated eye (5/6 with 160 µg/80 µg; 5/5 with 320 µg/160 µg) versus one of 11 (9.1%) in the untreated eye; most were mild in severity and dose dependent. Eight patients developed cataracts, of which six (75.0%) were categorized as serious (2/3 with 160 µg/80 µg; 4/5 with 320 µg/160 µg), as lens replacement was required. As the 160-µg/80-µg group showed a better benefit-risk profile, higher doses were discontinued or not initiated. Statistically significant improvements in visual acuity and retinal sensitivity were reported (post hoc analysis). The manageable safety profile and improvements reported in this trial support the continuation of sepofarsen development.

Long-Term Outcomes and Risk Factors for Severe Vision Loss in Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV).

Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is a rare disorder characterized by uveitis, retinal neovascularization, and retinal degeneration. We sought to describe the course of treated and untreated ADNIV and to identify risk factors for severe vision loss. DESIGN: Observational case series. METHODS: Clinical data from ADNIV patients from 4 families seen from 1967 through 2019 at a single academic, tertiary referral center were reviewed. The main outcome measures were visual acuity at baseline and follow-up, as well as risk factors for vision loss. RESULTS: A total of 130 eyes from 65 ADNIV patients (45 female, 20 male; mean age 40.8 years, range 6-77 years) were included. Mean best corrected visual acuity (BCVA) at presentation was LogMAR 0.59 (about Snellen 20/80). Longitudinal analysis included 84 eyes from 42 patients (31 female, 11 male), with mean follow-up of 17.3 years (range 2-43.6 years). Mean BCVA at last follow-up was LogMAR 1.48 (about Snellen 20/600). The disease accelerated in the fifth decade of life, during which the majority of eyes went from normal vision or mild vision loss to at least moderate vision loss (20/70 Snellen equivalent); 25 eyes from 16 patients (29.8%;) showed a steep trajectory of vision loss to no light perception. Tractional retinal detachment was the greatest risk factor for severe vision loss (BCVA <20/200) on multivariable analysis (P < .05). CONCLUSIONS: Patients with ADNIV have a high lifetime risk of severe vision loss. Tractional retinal detachment is an important risk factor for poor vision.

The effect of retinal scaffold modulus on performance during surgical handling.

Emerging treatment strategies for retinal degeneration involve replacing lost photoreceptors using supportive scaffolds to ensure cells survive the implantation process. While many design aspects of these scaffolds, including material chemistry and microstructural cues, have been studied in depth, a full set of design constraints has yet to be established. For example, while known to be important in other tissues and systems, the influence of mechanical properties on surgical handling has not been quantified. In this study, photocrosslinked poly(ethylene glycol) dimethacrylate (PEGDMA) was used as a model polymer to study the effects of scaffold modulus (stiffness) on surgical handling, independent of material chemistry. This was achieved by modulating the molecular weight and concentrations of the PEGDMA in various prepolymer solutions. Scaffold modulus of each formulation was measured using photo-rheology, which enabled the collection of real-time polymerization data. In addition to measuring scaffold mechanical properties, this approach gave insight on polymerization kinetics, which were used to determine the polymerization time required for each sample. Scaffold handling characteristics were qualitatively evaluated using both in vitro and ex vivo trials that mimicked the surgical procedure. In these trials, scaffolds with shear moduli above 35 kPa performed satisfactorily, while those below this limit performed poorly. In other words, scaffolds below this modulus were too fragile for reliable transplantation. To better compare these results with literature values, the compressive modulus was measured for select samples, with the lower shear modulus limit corresponding to roughly 115 kPa compressive modulus. While an upper mechanical property limit was not readily apparent from these results, there was increased variability in surgical handling performance in samples with shear moduli above 800 kPa. Overall, the knowledge presented here provides important groundwork for future studies designed to examine additional retinal scaffold considerations, including the effect of scaffold mechanical properties on retinal progenitor cell fate.

LEBER CONGENITAL AMAUROSIS DUE TO CEP290 MUTATIONS-SEVERE VISION IMPAIRMENT WITH A HIGH UNMET MEDICAL NEED: A Review.

PURPOSE: Leber congenital amaurosis due to CEP290 mutations (LCA10) is an inherited retinal disease that often results in severe visual impairment or blindness in early childhood. Currently, there are no approved treatments, highlighting the considerable unmet medical need associated with LCA10. We aimed to review the clinical characteristics of LCA10, its impact on patients and society, and the investigational treatment strategies currently in development. METHODS: Review of the current literature. RESULTS: LCA10 is an autosomal recessive ciliopathy, for which the CEP290 intronic variant c.2991+1655A>G (p.Cys998X) is the most common mutation. Usually diagnosed in early childhood, most patients with LCA10 have severe visual impairment during their first decade of life, which significantly affects the quality of life and development. LCA10 also has a significant societal burden (direct and indirect costs). RNA editing using antisense oligonucleotides or Staphylococcus aureus CRISPR-associated protein-9 nuclease is currently under investigation for treatment of p.Cys998X LCA10. Specifically, the antisense oligonucleotide therapy QR-110 (sepofarsen) has demonstrated encouraging safety and efficacy data in a first-in-human trial; a phase 3 clinical trial is ongoing. CONCLUSION: Interventions that can preserve or improve vision in patients with LCA10 have considerable potential to improve the patient quality of life and reduce burden of disease.

Genetic Association between MMP9 and Choroidal Neovascularization in Age-Related Macular Degeneration.

Purpose: To evaluate the first association specific to exudative age-related macular degeneration (AMD) located near the matrix metalloproteinase 9 (MMP9) gene. Design: Genetic association study. Participants: One thousand seven hundred twelve patients with AMD (672 nonexudative, 1040 exudative) of predominantly northern European descent seeking treatment at the University of Iowa Hospitals and Clinics. Methods: We reanalyzed the International AMD Genetics Consortium (IAMDGC) data to validate the association of polymorphisms near MMP9 with exudative AMD and to identify additional associated single nucleotide polymorphisms (SNPs), especially MMP9 coding sequence SNPs. We genotyped a cohort of 1712 AMD patients from Iowa with 3 SNPs identified with our analysis of the IAMDGC cohort using commercially available real-time quantitative polymerase chain reaction (PCR) assays. Firth regression was used to measure the association between MMP9 SNP genotypes and exudative AMD in our cohort of patients from Iowa. In addition, we developed a PCR-based assay to genotype the Iowa cohort at a short tandem repeat polymorphism (STRP) at the MMP9 locus. Main Outcome Measures: Odds ratios and P values for exudative compared with nonexudative AMD patients in the Iowa cohort for MMP9 SNPs (rs4810482, rs17576, and rs17577) and STRP. Results: We identified 3 SNPs in the MMP9 locus (rs4810482, rs17576, and rs17577) that are highly associated with exudative AMD in patient cohorts of the IAMDGC. These MMP9 SNPs also are associated with exudative AMD in the cohort of 1712 AMD patients from Iowa (rs4810482: odds ratio [OR], 0.82; P = 0.010; rs17576: OR, 0.86; P = 0.046; and rs17577: OR, 0.80; P = 0.041). We also genotyped the cohort of AMD patients from Iowa at rs142450006, another MMP9 polymorphism that previously was associated with exudative AMD. We detected a 4bp STRP, (TTTC)n, at the rs142450006 locus that is highly polymorphic and associated significantly with exudative AMD (OR, 0.78; P = 0.016). Conclusions: This study independently confirms and expands an association between the MMP9 locus and exudative AMD, further implicating a role for extracellular matrix abnormalities in choroidal neovascularization.

Comparison Between Graders in Detection of Diabetic Neovascularization With Swept Source Optical Coherence Tomography Angiography and Fluorescein Angiography.

PURPOSE: We compared the ability of ophthalmologists to identify neovascularization (NV) in patients with proliferative diabetic retinopathy using swept-source optical coherence tomography angiography (SS-OCTA) and fluorescein angiography (FA). DESIGN: Retrospective study comparing diagnostic instruments. METHODS: Eyes with proliferative diabetic retinopathy or severe nonproliferative diabetic retinopathy and a high suspicion of NV based on clinical examination were imaged using SS-OCTA and FA at the same visit. Two separate grading sets consisting of scrambled, anonymized SS-OCTA and FA images were created. The ground truth for presence of NV was established by consensus of 2 graders with OCTA experience who did not participate in the subsequent assessment of NV in this study. The 2 anonymized image sets were graded for presence or absence of NV by 12 other graders that included 2 residents, 6 vitreoretinal fellows, and 4 vitreoretinal attending physicians. The percentage of correct grading of NV using SS-OCTA and FA was assessed for each grader and across grader training levels. RESULTS: Forty-seven eyes from 24 patients were included in this study. Overall, the mean percentage of correct NV grading was 87.8% using SS-OCTA with B-scans and 86.2% using FA (P = .92). Assessing each grader individually, there was no statistically significant asymmetry in correct grading using SS-OCTA and FA. CONCLUSIONS: Ophthalmologists across training levels were able to identify diabetic NV with equal accuracy using SS-OCTA and FA. Based on these results, SS-OCTA may be an appropriate standalone modality for diagnosing diabetic NV.

Retinal Tropism and Transduction of Adeno-Associated Virus Varies by Serotype and Route of Delivery (Intravitreal, Subretinal, or Suprachoroidal) in Rats.

Viral-mediated gene augmentation offers tremendous promise for the treatment of inherited retinal diseases. The development of effective gene therapy requires an understanding of the vector's tissue-specific behavior, which may vary depending on serotype, route of delivery, or target species. Using an ex vivo organotypic explant system, we previously demonstrated that retinal tropism and transduction of adeno-associated virus type 2 (AAV2) vary significantly depending on serotype in human eyes. However, the ex vivo system has limited ability to assess route of ocular delivery, and relatively little literature exists on tropic differences between serotypes and routes of delivery in vivo. In this study, we demonstrate that retinal tropism and transduction efficiency of five different AAV2 serotypes (AAV2/1, AAV2/2, AAV2/6, AAV2/8, and AAV2/9) expressing enhanced green fluorescent protein driven by a cytomegalovirus promoter vary greatly depending on serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats. With subretinal delivery, all serotypes successfully transduced the retinal pigmented epithelium and outer nuclear layer (ONL), with AAV2/1 displaying the highest transduction efficiency and AAV2/2 and AAV2/6 showing lower ONL transduction. There was minimal transduction of the inner retina through subretinal delivery for any serotype. Tropism by suprachoroidal delivery mirrored that of subretinal delivery for all AAV serotypes but resulted in a wider distribution and greater ONL transduction. With intravitreal delivery, retinal transduction was seen primarily in the inner retina (retinal nerve fiber, ganglion cell, and inner nuclear layers) for AAV2/1 and AAV2/6, with AAV2/6 showing the highest transduction. When compared with data from human explant models, there are substantial differences in tropism and transduction that are important to consider when using rats as preclinical models for the development of ocular gene therapies for humans.

Attaining functional levels of visual acuity after vitrectomy for retinal detachment secondary to proliferative diabetic retinopathy.

Most patients needing diabetic tractional retinal detachment (TRD) surgery are working-age adults that drive and participate in other vision-dependent activities of daily living. We sought to determine the proportion of patients that achieve functional visual acuity (VA) based on the World Health Organization (WHO) definition of 'low vision' (≤ 20/80) and US driving standards (≥ 20/40) after vitrectomy for diabetic TRD. In this 10-year retrospective review, consecutive patients who underwent primary vitrectomy for TRD from proliferative diabetic retinopathy were studied. 240 eyes in 203 patients met criteria for analysis (38 eyes were lost to follow up at 3 months; 68 at 12 months; 146 at 60 months). While most patients (nearly 80%) having TRD surgery had low vision pre-op, almost half attained VA that was > 20/80 five years post-op. Those most likely to achieve significant visual improvement (p < 0.0001) had concomitant vitreous hemorrhage pre-op. Only 6% of eyes met the US minimum driving standard before surgery based on VA compared to 28% after vitrectomy however this study did not examine visual fields which could warrant additional assessment depending on local requirements. In summary, significant gains in visual acuity are seen after vitrectomy for diabetic TRD that can result in functional improvement in activities of daily living.

A novel, wearable, electronic visual aid to assist those with reduced peripheral vision.

PURPOSE: To determine whether visual-tactile sensory substitution utilizing the Low-vision Enhancement Optoelectronic (LEO) Belt prototype is suitable as a new visual aid for those with reduced peripheral vision by assessing mobility performance and user opinions. METHODS: Sighted subjects (n = 20) and subjects with retinitis pigmentosa (RP) (n = 6) were recruited. The LEO Belt was evaluated on two cohorts: normally sighted subjects wearing goggles to artificially reduce peripheral vision to simulate stages of RP progression, and subjects with advanced visual field limitation from RP. Mobility speed and accuracy was assessed using simple mazes, with and without the LEO Belt, to determine its usefulness across disease severities and lighting conditions. RESULTS: Sighted subjects wearing most narrowed field goggles simulating most advanced RP had increased mobility accuracy (44% mean reduction in errors, p = 0.014) and self-reported confidence (77% mean increase, p = 0.004) when using the LEO Belt. Additionally, use of LEO doubled mobility accuracy for RP subjects with remaining visual fields between 10° and 20°. Further, in dim lighting, confidence scores for this group also doubled. By patient reported outcomes, subjects largely deemed the device comfortable (100%), easy to use (92.3%) and thought it had potential future benefit as a visual aid (96.2%). However, regardless of severity of vision loss or simulated vision loss, all subjects were slower to complete the mazes using the device. CONCLUSIONS: The LEO Belt improves mobility accuracy and therefore confidence in those with severely restricted peripheral vision. The LEO Belt's positive user feedback suggests it has potential to become the next generation of visual aid for visually impaired individuals. Given the novelty of this approach, we expect navigation speeds may improve with experience.

Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

Distribution of Diabetic Neovascularization on Ultra-Widefield Fluorescein Angiography and on Simulated Widefield OCT Angiography.

PURPOSE: Areas of neovascularization (NV) in proliferative diabetic retinopathy (PDR) on ultra-widefield (UWF) fluorescein angiography (FA) were identified and compared with a simulated widefield (WF) swept-source OCT angiography (SS-OCTA) field of view to determine whether the WF SS-OCTA field of view was sufficient for detection of NV in PDR. DESIGN: Retrospective, consecutive case series. METHODS: All patients with PDR and UWF FA imaging at the Bascom Palmer Eye Institute over a period of 5.5 years were identified. UWF FA images were reviewed and sites of NV were identified either as NV of the disc or NV elsewhere. Sites of NV elsewhere were classified by disc-centered retinal quadrants. A simulated WF SS-OCTA montage field of view was overlaid on the UWF FA images to determine whether sites of NV would have been identified by this simulated WF SS-OCTA field of view. RESULTS: A total of 651 eyes with PDR from 433 patients had at least 1 UWF FA with NV. Of the 651 eyes, 50% were treatment-naïve, 9.8% had NV of the disc only, 41.8% had NV elsewhere only, and 48.4% had both NV of the disc and NV elsewhere. NV elsewhere was most prevalent in the superotemporal quadrant and the least prevalent in the nasal quadrants. When the simulated WF SS-OCTA field of view was overlaid on the UWF FA, 98.3% of all eyes, 99.4% of treatment-naive eyes, and 97.2% of previously treated eyes had NV within the WF SS-OCTA field of view. In those eyes with a repeat UWF FA within 6 to 18 months of the first FA, the distribution of NV did not change in either the treatment-naive or previously treated eyes. CONCLUSIONS: NV elsewhere in PDR was most prevalent superotemporally, and 99.4% of treatment-naïve eyes had NV within the simulated WF SS-OCTA field of view. Combined with previous research using WF SS-OCTA to identify NV in PDR, these findings suggest that WF SS-OCTA may be the only imaging modality needed for the diagnosis and longitudinal management of PDR.

Two-photon polymerized poly(caprolactone) retinal cell delivery scaffolds and their systemic and retinal biocompatibility.

Cell replacement therapies are often enhanced by utilizing polymer scaffolds to improve retention or direct cell orientation and migration. Obstacles to refinement of such polymer scaffolds often include challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization of acrylated poly(caprolactone) (PCL) could offer a means of achieving precise microstructural control of a material in a biocompatible platform. In this work, we studied the effect of various formulation and two-photon polymerization parameters on minimum laser power needed to achieve polymerization, resolution, and fidelity to a target 3D model designed to be used for retinal cell replacement. Overall, we found that increasing the concentration of crosslink-able groups decreased polymerization threshold and the size of resolvable features while increasing fidelity of the scaffold to the 3D model. In general, this improvement was achieved by increasing the number of acrylate groups per prepolymer molecule, increasing the acrylated PCL concentration, or decreasing its molecular weight. Resulting two-photon polymerized PCL scaffolds successfully supported human iPSC derived retinal progenitor cells in vitro. Sub-retinal implantation of cell free scaffolds in a porcine model of retinitis pigmentosa did not cause inflammation, infection or local or systemic toxicity after one month. In addition, comprehensive ISO 10993 testing of photopolymerized scaffolds revealed a favorable biocompatibility profile. These results represent an important step towards understanding how two-photon polymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications. STATEMENT OF SIGNIFICANCE: Inherited retinal degenerative blindness results from the death of light sensing photoreceptor cells. To restore high-acuity vision a photoreceptor cell replacement strategy will likely be necessary. Unfortunately, single cell injection typically results in poor cell survival and integration post-transplantation. Polymeric biomaterial cell delivery scaffolds can be used to promote donor cell viability, control cellular polarity and increase packing density. A challenge faced in this endeavor has been developing methods suitable for generating scaffolds that can be used to deliver stem cell derived photoreceptors in an ordered columnar orientation (i.e., similar to that of the native retina). In this study we combined the biomaterial poly(caprolactone) with two-photon lithography to generate a biocompatible, clinically relevant scaffold suitable for retina cell delivery.