Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant.

BACKGROUND: Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms. PURPOSE: To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing. METHODS: Case report. RESULTS: A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated "read-through" of the premature stop codon, the resulting missense changes were predicted to be "possibly damaging" in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window. CONCLUSIONS: This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants.

A novel homozygous c.67C>T variant in retinol binding protein 4 (RBP4) associated with retinitis pigmentosa and childhood acne vulgaris.

BACKGROUND: The retinol binding protein 4 (RBP4) is essential in delivering retinol to the retinal pigment epithelium and normal functioning of the visual cycle. Homozygous mutations in the RBP4 gene lead to severe retinitis pigmentosa that is phenotypically indistinguishable from retinitis pigmentosa caused by other recessive mutations. METHODS: Case Report. PURPOSE: To report a novel homozygous RBP4 c.67 C > T variant in a case of retinitis pigmentosa associated with severe childhood acne vulgaris. RESULTS: A 49-year old Caucasian man with a family history of retinitis pigmentosa, presented with low vision and night blindness from early childhood. Fundus examination showed findings typical of recessive retinitis pigmentosa. Next generation sequencing analysis revealed a novel homozygous RBP4 c.67 C > T variant. Examination of patient's back showed widespread scaring and hyperpigmentation secondary to severe childhood-onset acne vulgaris. Patient's affected brother, positive for the same homozygous variant, also had a history of severe acne vulgaris whereas the unaffected brother did not, confirming that mutations in RBP4 segregated with the acne vulgaris phenotype in this family. CONCLUSIONS: We describe a case of retinitis pigmentosa associated with acne vulgaris and highlight the role of this systemic manifestation of retinol deficiency in confirming pathogenicity of the novel variant. Given the small size of the genomic RBP4 DNA (0.6kb), gene therapy using an adeno-associated viral vector with subretinal delivery has great potential to treat this severe childhood-onset blinding retinal disease. In addition, ubiquitous expression of RBP4 supports the development of in vitro functional assays to test the vector potency for clinical use.

First-in-human study of the safety and viability of intraocular robotic surgery.

Microsurgery of the retina would be dramatically improved by instruments that offer supra-human precision. Here, we report the results of a first-in-human study of remotely controlled robot-assisted retinal surgery performed through a telemanipulation device. Specifically, 12 patients requiring dissection of the epiretinal or inner limiting membrane over the macula were randomly assigned to either undergo robot-assisted-surgery or manual surgery, under general anaesthesia. We evaluated surgical success, duration of surgery and amount of retinal microtrauma as a proxy for safety. Surgical outcomes were equally successful in the robotic-surgery and manual-surgery groups. Differences in the amount of retinal microtrauma between the two groups were statistically insignificant, yet dissection took longer with robotic surgery (median time, 4 min 5 s) than with manual surgery (1 min 20 s). We also show the feasibility of using the robot to inject recombinant tissue plasminogen activator under the retina to displace sight-threatening haemorrhage in three patients under local anaesthesia. A safe and viable robotic system for intraocular surgery would enable precise and minimally traumatic delivery of gene therapy or cell therapy to the retina.

Misdiagnosis of X-linked retinitis pigmentosa in a choroideremia patient with heavily pigmented fundi.

Inherited retinal diseases are thought to be the leading cause of sight loss in the working age population. Mutations found in the RPGR and CHM genes cause retinitis pigmentosa (RP) and choroideremia, respectively. In the first instance, an X-linked family history of visual field loss commonly raises the suspicion of one of these two genes. In choroideremia, the classic description of a white fundal reflex secondary to the widespread chorioretinal degeneration was made over a hundred years ago in Caucasians. But, it is not so obvious in heavily pigmented fundi. Hence, the clinical diagnosis of CHM in non-Caucasian patients may be challenging in the first stages of the disease. Here we report a case of a Southeast Asian gentleman who has a family history of X-linked retinal degeneration and was found to have a confirmed in-frame deletion of 12 DNA nucleotides in exon 15 of the RPGR gene. Later in life, however, his fundal appearance showed unusual areas of circular pigment hypertrophy and clumping. He was therefore tested for carrying a disease-causing mutation in the CHM gene and a null mutation was found. Since gene therapy trials are ongoing for both of these conditions, it has now become critically important to establish the correct genetic diagnosis in order to recruit suitable candidates. Moreover, this case demonstrates the necessity to remain vigilant in the interpretation of genetic results which are inconsistent with clinical features.

Novel non-contiguous exon duplication in choroideremia.

The importance of establishing a genetic diagnosis in patients with a choroideremia phenotype has been underscored by the advent of gene replacement therapy for this condition. Here, we describe a complex imbalance at the CHM locus in a male patient with classical disease. At the DNA level, this imbalance consists of 2 non-contiguous duplications (exons 1-2 and 9-12). Further characterization suggests the generation of 2 independent CHM transcriptional units, one of which may produce a deleted form of the Rab escort protein 1 protein. Expression of such a type of aberrant protein in photoreceptors may have important implications when considering gene therapy for this disorder.

Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina.

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery-intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.

Technique of retinal gene therapy: delivery of viral vector into the subretinal space.

PurposeSafe and reproducible delivery of gene therapy vector into the subretinal space is essential for successful targeting of the retinal pigment epithelium (RPE) and photoreceptors. The success of surgery is critical for the clinical efficacy of retinal gene therapy. Iatrogenic detachment of the degenerate (often adherent) retina in patients with hereditary retinal degenerations and small volume (eg, 0.1 ml) subretinal injections pose new surgical challenges.MethodsOur subretinal gene therapy technique involved pre-operative planning with optical coherence tomography (OCT) and autofluorescence (AF) imaging, 23 G pars plana vitrectomy, internal limiting membrane staining with Membrane Blue Dual (DORC BV, Zuidland, Netherlands), a two-step subretinal injection using a 41 G Teflon tipped cannula (DORC) first with normal saline to create a parafoveal bleb followed by slow infusion of viral vector via the same self-sealing retinotomy. Surgical precision was further enhanced by intraoperative OCT (Zeiss Rescan 7000, Carl Zeiss Meditec AG, Jena, Germany). Foveal functional and structural recovery was evaluated using best-corrected Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, microperimetry and OCT.ResultsTwo patients with choroideremia aged 29 (P1) and 27 (P2) years, who had normal and symmetrical levels of best-corrected visual acuity (BCVA) in both eyes, underwent unilateral gene therapy with the fellow eye acting as internal control. The surgeries were uncomplicated in both cases with successful detachment of the macula by subretinal vector injection. Both treated eyes showed recovery of BCVA (P1: 76-77 letters; P2: 84-88 letters) and mean threshold sensitivity of the central macula (P1: 10.7-10.7 dB; P2: 14.2-14.1 dB) to baseline within a month. This was accompanied by normalisation of central retinal thickness on OCT.ConclusionsHerein we describe a reliable technique for subretinal gene therapy, which is currently used in clinical trials to treat choroideremia using an adeno-associated viral (AAV) vector encoding the CHM gene. Strategies to minimise potential complications, such as avoidance of excessive retinal stretch, air bubbles within the injection system, reflux of viral vector and post-operative vitritis are discussed.

Electronic retinal implant surgery.

Blindness due to outer retinal degeneration still remains largely untreatable. Photoreceptor loss removes light sensitivity, but the remaining inner retinal layers, the optic nerve, and indeed the physical structure of the eye itself may be unaffected by the degenerative processes. This provides the opportunity to restore some degree of vision with an electronic device in the subretinal space. In this lecture I will provide an overview of our experiences with the first-generation retinal implant Alpha IMS, developed by Retina Implant AG and based on the technology developed by Eberhart Zrenner as part of a multicentre clinical trial (NCT01024803). We are currently in the process of running a second NIHR-funded clinical trial to assess the next-generation device. The positive results from both studies to date indicate that the retinal implant should be included as a potential treatment for patients who are completely blind from retinitis pigmentosa. Evolution of the technology in future may provide further opportunities for earlier intervention or for other diseases.

Outcomes following cataract surgery in choroideremia.

PurposeTo present a case series of cataract surgery outcomes in choroideremia eyes with an emphasis on the safety of this common operation in advanced stages of the disease.MethodsA single centre retrospective interventional case series comprising six patients with varying degrees of visual loss secondary to choroideremia underwent cataract surgery at a single tertiary eye hospital. Pre- and post-operative best-corrected Snellen visual acuity, spectral domain optical coherence tomography (SD-OCT), and slit lamp examination were performed together with fundus autofluorescence (FAF) and colour fundus photographs.The prevalence of intra- or post-operative complications, post-operative visual outcome, and change in central macular thickness were recorded.ResultsThe pre-operative best-corrected Snellen visual acuity in the operated eyes ranged from 6/12 (20/40) to PL. All but one patient had either an objective or a subjective improvement in visual acuity. There was no evidence of retinal phototoxicity or post-operative cystoid macular oedema (CMO). Three patients developed early capsular fibrosis.ConclusionsAlthough the residual functioning retina in choroideremia patients may be potentially vulnerable, this report finds no evidence of iatrogenic vision loss after uncomplicated cataract surgery. This suggests that cataract surgery may be performed safely in choroideremia patients, although a guarded prognosis for visual improvement should be emphasized in the informed consent.

[Gene therapy for retinal dystrophies]. / Gentherapie bei Netzhautdystrophien.

Genetic mutations are the cause of inherited retinal dystrophies. The underlying genetic basis of these diseases suggests that a gene therapy approach is logical either to replace or reduce the expression of defective genes. The first proof-of-concept clinical studies in patients with Leber's congenital amaurosis have suggested that retinal gene therapy is safe and potentially effective, at least for specific disease entities. In contrast to pharmacological treatment gene therapy has the advantage of being able to express a protein within specific cell populations and is a potentially definitive therapy. Besides replacing deficient genes in inherited diseases, additional strategies that might broaden the application of retinal gene therapy are also being developed. These include the permanent expression of neuroprotective substances or photosensitive molecules (so-called optogenetics). This overview discusses the current clinical strategies and potential problems of retinal gene therapy.

Manipulation of the recipient retinal environment by ectopic expression of neurotrophic growth factors can improve transplanted photoreceptor integration and survival.

Degeneration of the neural retina is the leading cause of untreatable blindness in the developed world. Stem cell replacement therapy offers a novel strategy for retinal repair. Postmitotic photoreceptor precursors derived from the early postnatal (P) retina are able to migrate and integrate into the adult mouse retina following transplantation into the subretinal space, but it is likely that a large number of these cells would be required to restore vision. The adult recipient retina presents a very different environment to that from which photoreceptor precursor donor cells isolated from the developing postnatal retina are derived. Here we considered the possibility that modulation of the recipient environment by ectopic expression of developmentally regulated growth factors, normally present during photoreceptor development, might enhance the migration and integration of transplanted cells into the adult neural retina. Adeno-associated viral (AAV) vectors were used to introduce three growth factors previously reported to play a role in photoreceptor development, IGF1, FGF2, and CNTF, into the adult retina, prior to transplantation of P4 cells derived from the Nrl.GFP(+ve) neural retina. At 3 weeks posttransplantation the number of integrated, differentiated photoreceptor cells present in AAV-mediated neurotrophic factor-treated eyes was assessed and compared to control treated contralateral eyes. We show, firstly, that it is possible to manipulate the recipient retinal microenvironment via rAAV-mediated gene transfer with respect to these developmentally relevant growth factors. Moreover, when combined with cell transplantation, AAV-mediated expression of IGF1 led to significantly increased levels of cell integration, while overexpression of FGF2 had no significant effect on integrated cell number. Conversely, expression of CNTF led to a significant decrease in cell integration and an exacerbated glial response that led to glial scarring. Together, these findings demonstrate the importance of the extrinsic environment of the recipient retina for photoreceptor cell transplantation and show for the first time that it is possible to manipulate this environment using viral vectors to influence photoreceptor transplantation efficiency.

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.

Sources of retinal pigment epithelium (RPE) for replacement therapy.

Stem cells, with their capacity to regenerate and replace diseased tissues, have recently been proposed as having great potential in the treatment of age-related macular degeneration (AMD). A stem cell therapeutic approach could operate to replace either the retinal pigment epithelium (RPE), the neurosensory retina or a combination of both. From the scientific perspective, RPE replacement alone is likely to be far more straightforward than rebuilding the complex circuitry of the neurosensory retina. Furthermore, recent advances with induced pluripotent stem cells have raised the real possibility of transplanting healthy 'young' autologous RPE into patients with early signs of AMD. At this stage, however, it is useful to reconsider some of the earlier clinical studies that used suspensions of autologous RPE cells harvested from the peripheral retina. These showed that isolated RPE cell suspensions had little capacity to recreate a monolayer on the diseased Bruch's membrane of AMD. To counter this problem, researchers from Southampton in the UK report the use of a synthetic polymer alternative to Bruch's membrane, which could provide a scaffold for future RPE derived from stem cells or possibly reopen opportunities for autologous RPE cells harvested from the peripheral retina.

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.

Cell transplantation strategies for retinal repair.

Cell transplantation is a novel therapeutic strategy to restore visual responses to the degenerate adult neural retina and represents an exciting area of regenerative neurotherapy. So far, it has been shown that transplanted postmitotic photoreceptor precursors are able to functionally integrate into the adult mouse neural retina. In this review, we discuss the differentiation of photoreceptor cells from both adult and embryonic-derived stem cells and their potential for retinal cell transplantation. We also discuss the strategies used to overcome barriers present in the degenerate neural retina and improve retinal cell integration. Finally, we consider the future translation of retinal cell therapy as a therapeutic strategy to treat retinal degeneration.

ADVIRC is caused by distinct mutations in BEST1 that alter pre-mRNA splicing.

Autosomal dominant vitreoretinochoroidopathy (ADVIRC), a retinal dystrophy often associated with glaucoma and cataract, forms part of a phenotypic spectrum of 'bestrophinopathies'. It has been shown previously that ADVIRC results from BEST1 mutations that cause exon skipping and lead to the production of shortened and internally deleted isoforms. This study describes a novel ADVIRC mutation and show that it disrupts an exonic splice enhancer (ESE) site, altering the binding of a splicing-associated SR protein. As with previous ADVIRC mutations, the novel c.704T-->C mutation in exon 6 altered normal splicing in an ex vivo splicing assay. Both this and another exon 6 ADVIRC-causing mutation (c.707G-->A) either weakened or abolished splicing in an ESE-dependent splice assay compared with a nearby exon 6 mutation associated with Best disease (c.703G-->C). Gel shift assays were undertaken with RNA oligonucleotides encompassing the ADVIRC and Best disease mutations with four of the most commonly investigated SR proteins. Although SC35, SRp40 and SRp55 proteins all bound to the wild-type and mutated sequences with similar intensities, there was increased binding of ASF/SF2 to the two ADVIRC-mutated sequences compared with the wild-type or Best disease-mutated sequences. The exon skipping seen for these two exon 6 ADVIRC mutations and their affinity for ASF/SF2 suggests that the region encompassing these mutations may form part of a CERES (composite exonic regulatory elements of splicing) site.

Isolation and characterisation of neural progenitor cells from the adult Chx10(orJ/orJ) central neural retina.

Retinal stem cells have been isolated from the ciliary epithelium (CE) of the mammalian retina. However, the central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, we showed that in Chx10(orJ/orJ) mice, dividing cells persist in the adult CNR, suggesting the existence of a dormant progenitor population. Here, we show that these cells are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Müller glial cell markers. Our results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors.

Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors.

Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.

Stem cell therapy and the retina.

Retinal degeneration culminating in photoreceptor loss is the leading cause of untreatable blindness in the developed world. In this review, we consider how photoreceptors might be replaced by transplantation and how stem cells might be optimised for use as donor cells in future clinical strategies for retinal repair. We discuss the current advances in human and animal models of retinal cell transplantation, focussing on stem cell and reproductive cloning biology, in relation to the practical issues of retinal transplantation surgery. Stem and progenitor cells can be isolated from a number of sources including embryonic tissue, adult brain and even the retina, prompting many researchers to investigate the potential for using these cells to generate photoreceptors for transplantation. Nevertheless, several obstacles need to be overcome before these techniques can be applied in a clinical setting. Embryonic or stem cells have so far shown little ability to differentiate into retinal phenotypes when transplanted into the adult retina. We have recently noted, however, that donor cells harvested much later, at the photoreceptor precursor developmental stage, can be transplanted successfully and restore visual function. The current challenge is to understand the developmental processes that guide embryonic or adult stem cells towards photoreceptor differentiation, so that large numbers of these cells might be transplanted at the optimal stage. Future advances in reproductive cloning technology could lead to the successful generation of stem cells from adult somatic cells, thereby facilitating auto-transplantation of genetically identical cells in patients requiring photoreceptor replacement.