Correction: Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65.

The authors originally published this article under the incorrect license type; this has now been corrected and is published under the CC-BY license.

In situ regeneration of retinal pigment epithelium by gene transfer of E2F2: a potential strategy for treatment of macular degenerations.

The retinal pigment epithelium (RPE) interacts closely with photoreceptors to maintain visual function. In degenerative diseases such as Stargardt disease and age-related macular degeneration, the leading cause of blindness in the developed world, RPE cell loss is followed by photoreceptor cell death. RPE cells can proliferate under certain conditions, suggesting an intrinsic regenerative potential, but so far this has not been utilised therapeutically. Here, we used E2F2 to induce RPE cell replication and thereby regeneration. In both young and old (2 and 18 month) wildtype mice, subretinal injection of non-integrating lentiviral vector expressing E2F2 resulted in 47% of examined RPE cells becoming BrdU positive. E2F2 induced an increase in RPE cell density of 17% compared with control vector-treated and 14% compared with untreated eyes. We also tested this approach in an inducible transgenic mouse model of RPE loss, generated through activation of diphtheria toxin-A gene. E2F2 expression resulted in a 10-fold increase in BrdU uptake and a 34% increase in central RPE cell density. Although in mice this localised rescue is insufficiently large to be demonstrable by electroretinography, a measure of massed retinal function, these results provide proof-of-concept for a strategy to induce in situ regeneration of RPE for the treatment of RPE degeneration.

Ranibizumab pretreatment in diabetic vitrectomy: a pilot randomised controlled trial (the RaDiVit study).

PurposeOur aim was to evaluate the impact of intravitreal ranibizumab pretreatment on the outcome of vitrectomy surgery for advanced proliferative diabetic retinopathy. The objective was to determine the feasibility of a subsequent definitive trial and estimate the effect size and variability of the outcome measure.Patients and methodsWe performed a pilot randomised double-masked single-centre clinical trial in 30 participants with tractional retinal detachment associated with proliferative diabetic retinopathy. Seven days prior to vitrectomy surgery, participants were randomly allocated to receive either intravitreal ranibizumab (Lucentis, Novartis Pharmaceuticals UK Ltd, Frimley, UK) or subconjunctival saline (control). The primary outcome was best-corrected visual acuity 12 weeks following surgery.ResultsAt 12 weeks, the mean (SD) visual acuity was 46.7 (25) ETDRS letters in the control group and 52.6 (21) letters in the ranibizumab group. Mean visual acuity improved by 14 (31) letters in the control group and by 24 (27) letters in the ranibizumab group. We found no difference in the progression of tractional retinal detachment prior to surgery, the duration of surgery, or its technical difficulty. Vitreous cavity haemorrhage persisted at 12 weeks in two of the control group but none of the ranibizumab group.ConclusionRanibizumab pretreatment may improve the outcome of vitrectomy surgery for advanced proliferative diabetic retinopathy by reducing the extent of post-operative vitreous cavity haemorrhage. However, the effect size appears to be modest; we calculate that a definitive study to establish a minimally important difference of 5.9 letters at a significance level of P<0.05 would require 348 subjects in each arm.

Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65.

Leber congenital amaurosis is a group of inherited retinal dystrophies that cause severe sight impairment in childhood; RPE65-deficiency causes impaired rod photoreceptor function from birth and progressive impairment of cone photoreceptor function associated with retinal degeneration. In animal models of RPE65 deficiency, subretinal injection of recombinant adeno-associated virus (AAV) 2/2 vectors carrying RPE65 cDNA improves rod photoreceptor function, and intervention at an early stage of disease provides sustained benefit by protecting cone photoreceptors against retinal degeneration. In affected humans, administration of these vectors has resulted to date in relatively modest improvements in photoreceptor function, even when retinal degeneration is comparatively mild, and the duration of benefit is limited by progressive retinal degeneration. We conclude that the demand for RPE65 in humans is not fully met by current vectors, and predict that a more powerful vector will provide more durable benefit. With this aim we have modified the original AAV2/2 vector to generate AAV2/5-OPTIRPE65. The new configuration consists of an AAV vector serotype 5 carrying an optimized hRPE65 promoter and a codon-optimized hRPE65 gene. In mice, AAV2/5-OPTIRPE65 is at least 300-fold more potent than our original AAV2/2 vector.

RPE65 gene therapy slows cone loss in Rpe65-deficient dogs.

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Restoration of vision after transplantation of photoreceptors.

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.

Pilot randomised controlled trial of face-down positioning following macular hole surgery.

OBJECTIVE: This was a pilot randomised controlled trial (RCT) to investigate the effect of post-operative face-down positioning on the outcome of macular hole surgery and to inform the design of a larger definitive study. METHODS: In all, 30 phakic eyes of 30 subjects with idiopathic full-thickness macular holes underwent vitrectomy with dye-assisted peeling of the ILM and 14% perfluoropropane gas. Subjects were randomly allocated to posture face down for 10 days (posturing group) or to avoid a face-up position only (non-posturing group). The primary outcome was anatomical hole closure. RESULTS: Macular holes closed in 14 of 15 eyes (93.3%; 95% confidence interval (CI) 68-100%) in the posturing group and in 9 of 15 (60%; 95% CI 32-84%) in the non-posturing group. In a subgroup analysis of outcome according to macular hole size, all holes smaller than 400 µm closed regardless of posturing (100%). In contrast, holes larger than 400 µm closed in 10 of 11 eyes (91%; 95% CI 58-99%) in the posturing group and in only 4 of 10 eyes (40%; 95% CI 12-74%) in the non-posturing group (Fisher's exact test P=0.02). CONCLUSION: Post-operative face-down positioning may improve the likelihood of macular hole closure, particularly for holes larger than 400 µm. These results support the case for a RCT.

Pilot randomised controlled trial of face-down posturing following phacovitrectomy for macular hole.

BACKGROUND: To gather information on the effect of postoperative face-down posturing following combined phacoemulsification and vitrectomy for macular hole surgery in order to assist in the design of a larger definitive study. METHODS: Thirty phakic patients with stage II-IV full-thickness macular hole had combined phacoemulsification and pars plana vitrectomy with internal limiting membrane peel and 14% perfluoropropane (C(3)F(8)) gas. At the conclusion of surgery, patients were randomised either to face-down posture or to no posture, for 10 days. The primary outcome was macular hole closure. RESULTS: The macular hole was successfully closed in 93.8% of the face-down posture group and in all of the no-posture group. Mean visual improvement was 0.63 (SD=0.21) logMAR units in the face-down group and 0.53 (SD=0.22) in the no posture patients. CONCLUSION: Following combined phacoemulsification and vitrectomy, postoperative face-down posturing appears to make little difference to the final anatomical or visual outcome. If we assume a success rate of 95% in the posturing arm, and that there is no difference between posturing and non-posturing, then 798 patients would be required to be 90% sure that the 95% confidence interval will exclude a difference of more than 5%.

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice.

Insertional mutagenesis following gene therapy with gammaretroviral vectors can cause the development of lymphoproliferation in children with X-linked severe combined immunodeficiency. In experimental studies, recombinant adeno-associated virus (rAAV) vectors have also been reported to increase susceptibility to carcinogenesis. The possibility of vector-induced transformation in quiescent ocular cells is probably significantly lower than in mitotically active cells, but given the increasing number of clinical applications of rAAV and lentiviral vectors for ocular disease, a specific assessment of their oncogenic potential in the eye is important. In this study, we investigated the effect of rAAV2/2 and integrating HIV-1 vectors upon the incidence of ocular neoplasia in p53 tumour-suppressor gene-knockout (p53(-/-)) mice, which are highly susceptible to intraocular malignant transformation. Subretinal injections of high titre rAAV2/2 or integrating HIV-1 vectors induced no tumours in p53(-/-) or p53(+/-) animals, nor significantly affected their natural longevity. We conclude that any insertional events arising from subretinal delivery of these vectors appear insufficient to cause intraocular malignancy, even in highly susceptible animals. These findings support the continued development of these vectors for ocular applications.

Gene supplementation therapy for recessive forms of inherited retinal dystrophies.

Over the last decade, gene supplementation therapy for inherited retinal degeneration has come of age. Early proof-of-concept studies in animal models of disease showed modest, but genuine improvements in retinal function and/or survival. Further development of the vectors used for gene transfer to the retina has led to better treatment efficacy in a wide variety of animal models, leading in 2008 to the initiation of three clinical trials for Leber congenital amaurosis caused by retinal pigment epithelium 65 deficiency. The results from these trials suggest that the treatment of inherited retinal dystrophy by gene therapy can be safe and effective. Here, we examine the progress of gene supplementation therapy in the retina, and discuss the potential for using gene therapy to treat different forms of inherited retinal degeneration.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function.

The purpose of this study was to evaluate whether immune responses interfered with gene therapy rescue using subretinally delivered recombinant adeno-associated viral vector serotype 2 carrying the RPE65 cDNA gene driven by the human RPE65 promoter (rAAV2.hRPE65p.hRPE65) in the second eye of RPE65-/- dogs that had previously been treated in a similar manner in the other eye. Bilateral subretinal injection was performed in nine dogs with the second eye treated 85-180 days after the first. Electroretinography (ERG) and vision testing showed rescue in 16 of 18 treated eyes, with no significant difference between first and second treated eyes. A serum neutralizing antibody (NAb) response to rAAV2 was detected in all treated animals, but this did not prevent or reduce the effectiveness of rescue in the second treated eye. We conclude that successful rescue using subretinal rAAV2.hRPE65p.hRPE65 gene therapy in the second eye is not precluded by prior gene therapy in the contralateral eye of the RPE65-/- dog. This finding has important implications for the treatment of human LCA type II patients.

Targeted disruption of outer limiting membrane junctional proteins (Crb1 and ZO-1) increases integration of transplanted photoreceptor precursors into the adult wild-type and degenerating retina.

Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Müller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 +/- 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1(rd8/rd8) mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 +/- 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 +/- 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho(-/-) mouse; donor cell integration was significantly increased from 313 +/- 58 cells without treatment to 919 +/- 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies.

AAV-mediated knockdown of peripherin-2 in vivo using miRNA-based hairpins.

Gene therapy for inherited retinal degeneration in which expression of a mutant allele has a gain-of-function effect on photoreceptor cells is likely to depend on efficient silencing of the mutated allele. Peripherin-2 (Prph2, also known as peripherin/RDS) is an abundantly expressed photoreceptor-specific gene. In humans, gain-of-function mutations in PRPH2 result in both autosomal dominant retinitis pigmentosa and dominant maculopathies. Gene-silencing strategies for these conditions include RNA interference by short hairpin RNAs (shRNAs). Recent evidence suggests that microRNA (miRNA)-based hairpins may offer a safer and more effective alternative. In this study, we used for the first time a virally transferred miRNA-based hairpin to silence Prph2 in the murine retina. The results show that an miRNA-based shRNA can efficiently and specifically silence Prph2 in vivo as early as 3 weeks after AAV2/8-mediated subretinal delivery, leading to a nearly 50% reduction of photoreceptor cells after 5 weeks. We conclude that miRNA-based hairpins can achieve rapid and robust gene silencing after efficient vector-mediated delivery to the retina. The rationale of using an miRNA-based template to improve the silencing efficiency of a hairpin may prove valuable for allele-specific silencing in which the choice for an RNAi target is limited and offers an alternative therapeutic strategy for the treatment of dominant retinopathies.

Prospects for gene therapy of inherited retinal disease.

Gene-based therapies offer the means to address gene defects responsible for inherited retinal disorders. A number of studies in experimental and preclinical models have demonstrated proof-of-principle that gene replacement therapy can mediate significant quantifiable improvements in ocular morphology and visual function. The first results of clinical trials of gene therapy for early-onset severe retinal dystrophy caused by defects in RPE65 show proof-of-concept for efficacy and short-term safety in humans. The challenges for gene therapy of conditions caused by gain-of-abnormal function are being addressed by strategies to knock down expression of the disease allele. Vector-mediated expression of neuroprotective proteins may offer a generic approach for preserving vision in single-gene and multi-gene retinal degenerations. Gene therapy is likely to be most successful where stable expression of the therapeutic transgene can be achieved at an appropriate level in diseases in which retinal development is unaffected and a significant number of target cells survive at the point of intervention.

Lentiviral-vector-mediated expression of murine IL-1 receptor antagonist or IL-10 reduces the severity of endotoxin-induced uveitis.

Uveitis is a sight threatening inflammatory disorder that remains a significant cause of visual loss. We investigated lentiviral gene delivery of interleukin 1 receptor antagonist (IL-1ra) or interleukin (IL)-10 to ameliorate murine endotoxin-induced uveitis (EIU). An human immunodeficiency virus-1-based vector containing the mIL-1ra or mIL-10 cDNA demonstrated high expression of biologically active cytokine. Following administration of Lenti.GFP into the anterior chamber, transgene expression was observed in corneal endothelial cells, trabecular meshwork and iris cells. To treat EIU, mice were injected with Lenti.IL-1ra, Lenti.IL-10 or a combination of these. EIU was induced 14 days after vector administration and mice were culled 12 h following disease induction. Lenti.IL-1ra or Lenti.IL-10-treated eyes showed significantly lower mean inflammatory cell counts in the anterior and posterior chambers compared with controls. The aqueous total protein content was also significantly lower in treated eyes, demonstrating better preservation of the blood-ocular barrier. Furthermore, the treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with controls. The combination of both IL-1ra and IL-10 had no additive effect. Thus, lentiviral gene delivery of IL-1ra or IL-10 significantly reduces the severity of experimental uveitis, suggesting that lentiviral-mediated expression of immunomodulatory genes in the anterior chamber offers an opportunity to treat uveitis.

AAV-mediated gene therapy for retinal disorders: from mouse to man.

A wide range of retinal disorders can potentially be treated using viral vector-mediated gene therapy. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV), because they elicit minimal immune responses and mediate long-term transgene expression in a variety of retinal cell types. Proof-of-concept experiments have demonstrated the efficacy of AAV-mediated transgene delivery in a number of animal models of inherited and acquired retinal disorders. Following extensive preclinical evaluation in large animal models, gene therapy for one form of inherited retinal degeneration due to RPE65 deficiency is now being tested in three concurrent clinical trials. Here, we review different approaches for treating inherited retinal degenerations and more common acquired retinal disorders using AAV-based vectors.

Progress and prospects: gene therapy clinical trials (part 1).

Over the last two decades gene therapy has moved from preclinical to clinical studies for many diseases ranging from single gene disorders such as cystic fibrosis and Duchenne muscular dystrophy, to more complex diseases such as cancer and cardiovascular disorders. Gene therapy for severe combined immunodeficiency (SCID) is the most significant success story to date, but progress in many other areas has been significant. We asked 20 leaders in the field succinctly to summarize and comment on clinical gene therapy research in their respective areas of expertise and these are published in two parts in the Progress and Prospect series.