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The neuronal ceroid lipofuscinoses (NCLs), often referred to as Batten disease, are inherited lysosomal storage disorders that represent the most common neurodegeneration during childhood. Symptoms include seizures, vision loss, motor and cognitive decline, and premature death. The development of brain-directed treatments for NCLs has made noteworthy progress in recent years. Clinical trials are currently ongoing or planned for different forms of the disease. Despite these promising advances, it is unlikely that therapeutic interventions targeting the brain will prevent loss of vision in patients as retinal cells remain untreated and will continue to degenerate. Here, we demonstrate that Cln3 Δ ex7/8 mice, a mouse model of CLN3 Batten disease with juvenile onset, suffer from a decline in inner retinal function resulting from the death of rod bipolar cells, interneurons vital for signal transmission from photoreceptors to ganglion cells in the retina. We also show that this ocular phenotype can be treated by adeno-associated virus (AAV)-mediated expression of CLN3 in cells of the inner retina, leading to significant survival of bipolar cells and preserved retinal function. In contrast, the treatment of photoreceptors, which are lost in patients at late disease stages, was not therapeutic in Cln3 Δ ex7/8 mice, underlining the notion that CLN3 disease is primarily a disease of the inner retina with secondary changes in the outer retina. These data indicate that bipolar cells play a central role in this disease and identify this cell type as an important target for ocular AAV-based gene therapies for CLN3 disease.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety., Competing Interests: Disclosure: D.A. Thompson, MeiraGTx (F), filed patent on gene therapy (P); A. Iannaccone, Editas Medicine (C), Astellas (C), Roivant Pharma (C), IQVIA (C), Gyroscope/Orbit Biomedical (C), Rhythm Pharmaceuticals (C), Applied Genetic Technologies Corp (F), Allergan (F), Acucela (F), ProQR Therapeutics (F), Foundation Fighting Blindness Clinical Research Institute (F), Alia Therapeutics (S); R.R. Ali, MeiraGTx (S), filed patents on gene and cell therapies (P); V.Y. Arshavsky, None; I. Audo, None; J.W.B. Bainbridge, MeiraGTx (S), filed patents on gene and cell therapies (P); C.G. Besirli, MeiraGTx (F), Spark Therapeutics (F); D.G. Birch, Nightstar Therapeutics/Biogen (C), Applied Genetic Technologies Corp (S), Nacuity Pharmaceuticals (C), Editas Medicine (C), Acucela (S), Foundation Fighting Blindness (C), ProQR Therapeutics (C); K.E. Branham, ProQR Therapeutics (C); A.V. Cideciyan, Patents on gene therapies for RPGR, RHO, and NPHP5-associated IRDs (P); ProQR Therapeutics (F), IVERIC bio (F); S.P. Daiger, Applied Genetic Technologies Corp (S); D. Dalkara, Patent on AAV virions with variant capsid and methods of use (P); J.L. Duncan, 4D Therapeutics (C), Applied Genetic Technologies Corp (C), Editas Medicine (C), Gyroscope (C), Horama (C), Nightstar Therapeutics/Biogen (C), ProQR Therapeutics (C), SparingVision (S), Spark Therapeutics (C), Vedere Bio (C), Acucela (F), Allergan (F), Neurotech (F), Second Sight Medical Products (F); A.T. Fahim, None; J.G. Flannery, None; R. Gattegna, None; J.R. Heckenlively, None; E. Heon, None; K.T. Jayasundera, Editas Medicine (C); N.W. Khan, None; H. Klassen, jCyte (S, I); B.P. Leroy, Bayer (C), Nightstar Therapeutics/Biogen (C), IVERIC bio (C), Novartis Pharma (C), RegenX Bio (C), Vedere Bio (C); Novartis (R), Spark Therapeutics (R); GenSight Biologics (F), ProQR Therapeutics (F); R.S. Molday, Applied Genetic Technologies Corp (S); D.C. Musch, Belite Bio (S), Mactel Group NTMT-03 trial (S), NEI intramural branch clinical trials (S); M.E. Pennesi, Adverum (C), Allergan (C), Astellas (C), Pharmaceuticals (C), Biogen (C), IVERIC bio (C), Novartis (C), Regenix Bio (C), Spark Therapeutics (C), Wave Biosciences (C), Eyevensys (S), Horama (S), Nayan (S), Nacuity Pharmaceuticals (S, I), Ocugen (S, I), Verede (S), ProQR Therapeutics (S), Gensight (S), Applied Genetic Technologies Corp (F), Sanofi (F), Foundation Fighting Blindness (F), ProQR Therapeutics (F), Allergan (F); S.M. Petersen-Jones, None; E.A. Pierce, Astellas (C), Merck (C), Sanofi-Genzyme (C), Wave Therapeutics (C); GenSight Biologics (S), Odylia Therapeutics (S), Opsis Therapeutics (S), Sana Biotechnology (S); CRISPR Therapeutics (F), Spark Therapeutics (F); Editas Medicine (F), MeiraGTx (F), ProQR Therapeutics (F), filed patents on gene and genetic therapies (P); R.C. Rao, None; T.A. Reh, Nayan Pharmaceuticals (C, I); J.A. Sahel, Pixium Vision (S, I), GenSight Biologics (S, I), SparingVision (S, I); Prophesee (I), Chronolife (I); D. Sharon, None; P.A. Sieving, None; E. Strettoi, None; P. Yang, Astellas (C), Applied Genetic Technologies Corp (C); D.N. Zacks, ONL Therapeutics (C, I, P), (Copyright 2020 The Authors.)

SLC4A3 has been shown to cause retinal degeneration in a genetically engineered knockout mouse, and in a naturally occurring form of canine progressive retinal atrophy considered to be the equivalent of retinitis pigmentosa in humans (RP). This study was undertaken to investigate if SLC4A3 coding variants were implicated in human retinal degeneration. SLC4A3 exons were amplified and sequenced in 200 patients with autosomal recessive retinal degeneration who had no known molecular diagnosis for their condition, which included 197 unrelated individuals with suspected RP and three individuals with other forms of retinal disease. Three rare variants were identified that were predicted to be potentially pathogenic, however each variant was heterozygous in a single patient and therefore not considered disease-causing in isolation. Of these three variants, SNP-3 was the rarest, with an allele frequency of 7.06 x 10(-5) (>46,000 exomes from the ExAC database). In conclusion, no compound heterozygous or homozygous potentially pathogenic variants were identified that would account for recessive RP or retinal degeneration in this cohort, however the possibility remains that the rare variants identified could be acting with as yet undiscovered mutations in introns or regulatory regions. SLC4A3 remains an excellent candidate gene for human retinal degeneration, and with the advent of whole exome and whole genome sequencing of cohorts of molecularly unsolved patients with syndromic and non-syndromic forms of retinal degeneration, SLC4A3 may yet be implicated in human disease.

Understanding phenotype-genotype correlations in retinal degeneration is a major challenge. Mutations in CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the Crb1(rd8/rd8) mutation, we compared the retinal pathology of Crb1(rd8/rd8)/J inbred mice with that of two Crb1(rd8/rd8) lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated with photoreceptor death, Müller and microglia activation and telangiectasia-like vascular remodelling-features that were stable in the inbred, variable in the second, but virtually absent in the third Crb1(rd8/rd8) line, even at 12 months of age. This suggests that the Crb1(rd8/rd8) mutation is necessary, but not sufficient for the development of these degenerative features. By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified. This may carry one or more genetic modifiers for the manifestation of the retinal pathology associated with mutations in Crb1. This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers., (© The Author 2014. Published by Oxford University Press.)

Inherited retinal disease, which includes conditions such as retinitis pigmentosa (RP), affects about 1/3000 of the population in the Western world. It is characterized by gradual loss of vision and results from mutations in any one of 60 or so different genes. There are currently no effective treatments, but many of the genes have now been identified and their functions elucidated, providing a major impetus to develop gene-based treatments. Many of the disease genes are photoreceptor- or retinal pigment epithelium (RPE) cell specific. Since adeno-associated viral (AAV) vectors can be used for efficient gene transfer to these two cell types, we are developing AAV-mediated gene therapy approaches for inherited retinal degeneration using animal models that have defects in these cells. The retinal degeneration slow (rds or Prph2Rd2/Rd) mouse, a model of recessive RP, lacks a functional gene encoding peripherin 2, which is a photoreceptor-specific protein required for the formation of outer segment discs. We have previously demonstrated restoration of photoreceptor ultrastructure and function by AAV-mediated gene transfer of peripherin 2. We have now extended our assessment to central visual neuronal responses in order to show an improvement of central visual function. The Royal College of Surgeons (RCS) rat, provides another model of recessive RP. Here the defect is due to a defect in Mertk, a gene that is expressed in the RPE and encodes a receptor tyrosine kinase that is thought to be involved in the recognition and binding of outer segment debris. The gene defect results in the inability of the RPE to phagocytose the shed outer segments from photoreceptor cells. The resulting accumulation of debris between the RPE and the neuroretina leads to progressive loss of photoreceptor cells. AAV-mediated delivery of Mertk to the RPE results in reduction of debris indicating that the phagocytosing function of the RPE is restored and delays the degeneration of the photoreceptor cells 3-4 months. Our results, along with those of other groups support the use of AAV vectors for the treatment of inherited retinal degeneration.

SLC4A3 has been shown to cause retinal degeneration in a genetically engineered knockout mouse, and in a naturally occurring form of canine progressive retinal atrophy considered to be the equivalent of retinitis pigmentosa in humans (RP). This study was undertaken to investigate if SLC4A3 coding variants were implicated in human retinal degeneration. SLC4A3 exons were amplified and sequenced in 200 patients with autosomal recessive retinal degeneration who had no known molecular diagnosis for their condition, which included 197 unrelated individuals with suspected RP and three individuals with other forms of retinal disease. Three rare variants were identified that were predicted to be potentially pathogenic, however each variant was heterozygous in a single patient and therefore not considered disease-causing in isolation. Of these three variants, SNP-3 was the rarest, with an allele frequency of 7.06 x 10(-5) (>46,000 exomes from the ExAC database). In conclusion, no compound heterozygous or homozygous potentially pathogenic variants were identified that would account for recessive RP or retinal degeneration in this cohort, however the possibility remains that the rare variants identified could be acting with as yet undiscovered mutations in introns or regulatory regions. SLC4A3 remains an excellent candidate gene for human retinal degeneration, and with the advent of whole exome and whole genome sequencing of cohorts of molecularly unsolved patients with syndromic and non-syndromic forms of retinal degeneration, SLC4A3 may yet be implicated in human disease.

Introduction: Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit. Sources of data: Pubmed and clinicaltrials.gov. Areas of agreement: Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration. Areas of controversy: The magnitude of vision improvement from RPE65- LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration. Growing points: The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials. Areas timely for developing research: Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes. [ABSTRACT FROM AUTHOR]

Stem cell therapies are being explored as potential treatments for retinal disease. How to replace neurons in a degenerated retina presents a continued challenge for the regenerative medicine field that, if achieved, could restore sight. The major issues are: (i) the source and availability of donor cells for transplantation; (ii) the differentiation of stem cells into the required retinal cells; and (iii) the delivery, integration, functionality, and survival of new cells in the host neural network. This review considers the use of induced pluripotent stem cells (iPSC), currently under intense investigation, as a platform for cell transplantation therapy. Moreover, patient-specific iPSC are being developed for autologous cell transplantation and as a tool for modeling specific retinal diseases, testing gene therapies, and drug screening. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx+ photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling. S TEM C ELLS 2013;31:408-414 [ABSTRACT FROM AUTHOR]

Retinal dystrophies are inherited disorders of photoreceptor and retinal pigment epithelial function that may result in severe visual impairment. Advances in molecular genetics have helped identify many of the gene defects responsible, and progress in gene transfer technology has enabled therapeutic strategies to be developed and applied. The first human clinical trials of gene therapy for RPE65 associated retinal dystrophy have shown promising initial results and have helped prepare the way for further trials of gene therapy for inherited retinal disorders. The results of these trials will provide further insight into the safety and efficacy of gene therapy for a range of currently untreatable and debilitating eye disorders. [ABSTRACT FROM AUTHOR]

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling agerelated macular degeneration. We generated and characterised a mouse ''knock-in'' model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct. [ABSTRACT FROM AUTHOR]

The authors reflect on the potential of stem cells to treat retinal diseases. They believe the best approach for photoreceptor cell replacement is to generate photoreceptor precursors from human embryonic stem (hES) cell lines and transplant immature cells into the subretinal space where they can integrate into the host retinal circuity to produce new photoreceptors. They believe the most challenging goal is to replace retinal neurons which have died with other functional neurons.

The use of AAV-RPE65 vectors for gene supplementation has achieved spectacular success as a treatment for individuals with autosomal recessive retinal disease caused by biallelic mutations in the visual cycle gene RPE65. However, the efficacy of this approach in treating autosomal dominant retinitis pigmentosa (adRP) associated with a monoallelic mutation encoding a rare D477G RPE65 variant has not been studied. Although lacking a severe phenotype, we now find that knock-in mice heterozygous for D477G RPE65 (D477G KI mice) can be used to evaluate outcomes of AAV-RPE65 gene supplementation. Total RPE65 protein levels, which are decreased in heterozygous D477G KI mice, were doubled following subretinal delivery of rAAV2/5.hRPE65p.hRPE65. In addition, rates of recovery of the chromophore 11-cis retinal after bleaching were significantly increased in eyes that received AAV-RPE65, consistent with increased RPE65 isomerase activity. While dark-adapted chromophore levels and a-wave amplitudes were not affected, b-wave recovery rates were modestly improved. The present findings establish that gene supplementation enhances 11-cis retinal synthesis in heterozygous D477G KI mice and complement previous studies showing that chromophore therapy results in improved vision in individuals with adRP associated with D477G RPE65. [ABSTRACT FROM AUTHOR]

The first report of in vivo gene delivery to the retina dates back to 1987 when a retroviral vector was injected intraocularly in newborn mice. Later came the observation that retinal cells could be successfully transduced using adenoviral and then adeno-associated and lentiviral vectors. By 2000, it had become clear that the eye, compared to other organs and tissues, provides a number of advantages for in vivo gene therapy with regard to safety, efficacy, and route to clinical application. This has prompted the development of many successful proof-of-concept studies in animal models. The demonstration that sight could be restored in a large-animal model with a congenital form of blindness was a major landmark that opened the door to the first-in-human trials for recessively inherited blinding conditions. With these first human studies demonstrating safety as well as some efficacy, retinal gene therapy has now come of age. Rapid clinical development has highlighted various new challenges, including the treatment of patients with advanced photoreceptor degeneration or dominantly inherited retinal dystrophies and those with defects in large genes. Yet, given the progress over the last 25 years, a bright future is expected for retinal gene therapy. [ABSTRACT FROM AUTHOR]

Purpose: To assess excision of choroidal new vessels (CNV) combined with autologous transplantation of the equatorial retinal pigment epithelium (RPE) as a means of restoring vision for patients with acute neovascular age-related macular degeneration (AMD). Design: Prospective interventional cohort study. Participants: Twelve patients were recruited into an ethics committee approved trial with informed consent between 2004 and 2005. All had <6 months of acute visual loss owing to subfoveal neovascular AMD and were ineligible for photodynamic therapy. Methods: Patients underwent submacular removal of CNV through a single retinotomy. A full-thickness patch graft of RPE, Bruch’s membrane, and choroid was harvested from the superior equatorial retina and transplanted into the subfoveal space. The graft was flattened under heavy liquid, before silicone oil exchange. Removal of silicone oil and cataract surgery were performed 3 months later. All patients underwent cataract grading, full refraction, optical coherence tomography, fundus autofluorescence, and fluorescein and indocyanine angiography preoperatively and again 6 months postoperatively. Retinal pigment epithelium samples from 3 patients were tested for ex vivo gene transfer using a recombinant lentiviral vector. Main Outcome Measures: Six months after surgery, successful transplantation was determined by the presence of a pigmented subfoveal graft showing RPE autofluorescence and choroidal reperfusion. Visual outcome was assessed by subjective refraction and microperimetry of the retina overlying the graft. Results: Successful viable grafts were seen in 11 patients. Three patients had good visual function on the grafts, with mean logarithm of the minimum angle of resolution (logMAR) improving from 0.88 to 0.79 and maintained beyond 1 year. Operative complications occurred in 8 patients, including retinal detachment in 5 patients and hemorrhage affecting the graft in 4 patients. The mean visual acuity over the whole cohort fell from logMAR 0.82 to 1.16. The excised RPE choroid could also be genetically modified outside the eye with a viral vector applied within the time frame of the operation. Conclusions: Autologous RPE transplantation can in principle restore vision in neovascular AMD, but surgical complications remain high. The possibility for ex vivo gene transfer to the free graft of RPE may widen the scope of this procedure to include gene therapy or adjunctive molecular treatments for AMD. [Copyright &y& Elsevier]