Insight into Leber congenital amaurosis: potential for gene therapy

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1 For reprint orders, please contact Insight into Leber congenital amaurosis: potential for gene therapy Expert Rev. Ophthalmol. 6(2), (2011) Dania Qatarneh 1, Hemal Mehta 1 and Vickie Lee 1 1 Ophthalmology Department, Central Middlesex Hospital, Acton Lane, Park Royal, London, NW10 7NS, UK Author for correspondence: Tel.: Fax: vickielee@mac.com Leber congenital amaurosis describes a group of congenital retinal degenerations that cause significant visual loss. Advances in molecular genetics have helped identify mutations in 15 different genes, which together account for 70% of Leber congenital amaurosis cases. These genes are inherited in a predominantly autosomal recessive manner and encode a variety of retinal functions. These genes are exciting new targets for treatment aimed at improving visual function, notably with animal models such as the Briard RPE knockout dog and, more recently, human gene therapy trials. Keywords: gene therapy Leber congenital amaurosis Leber congenital amaurosis (LCA) represents a group of hereditary retinal dystrophies, typically causing severe visual loss, first described by Theodore Leber in It has a worldwide prevalence of three in 100,000 newborn babies [1]. The burden of disease is significant; LCA accounts for approximately 20% of children attending blind schools in the world [2]. Advances in the field of human genetics have placed this disease at the center stage, with gene mutations identified in approximately 70% of LCA patients [3]. The prospect of gene-replacement therapy in LCA has subsequently become a real possibility. Clinical features While no universally agreed upon diagnostic criteria are available, the following features are highly suggestive: Severe visual impairment presenting in infancy, frequently before the age of 6 months. Patients with LCA usually do not achieve visual acuity better than 20/400 [4]; Extinguished or severely reduced scotopic and photopic electroretinogram (ERG) [5]. Normal ERG responses rule out a diagnosis of LCA. Interestingly, studies of heterozygotes for some of the known LCA mutations found cone ERG dysfunction in GUCY2D heterozygotes [6] and rod ERG dysfunction in AIPL1 mutation hetero zygotes [7]. Visual evoked responses are variable in this condition. Associated features: Sensory nystagmus and sluggish pupillary reactions; The oculo digital sign, characterized by poking, rubbing and/or pressing of the eyes [8]; Family history typically consistent with autosomal recessive inheritance. Fundus abnormalities: Optic disc abnormalities may also be apparent manifesting as disc swelling, drusen or even peripapillary neovascularization [3]; Other abnormalities are keratoconus, cataract, ptosis, macular coloboma and high hyperopia, which have all been linked to the disease [9]. Syndromic forms of LCA Ocular symptoms that typically occur in LCA may be the first presentation of systemic syndrome (Table 1). This calls for a multidisciplinary approach for diagnosis and management of this disease with the involvement of pediatricians and clinical geneticists. There is some evidence of unifying genetic mutations that may account for these diverse clinical phenotypes. For example, CEP290 mutations have been implicated in both LCA and Joubert syndrome [10]. LCA & juvenile retinitis pigmentosa Leber congenital amaurosis represents the severe end of a spectrum of inherited retinal dystrophies /EOP Expert s Ltd ISSN

2 Qatarneh, Mehta & Lee Table 1. Extraocular features of systemic syndromes associated with Leber congenital amaurosis. Syndrome Alstrom syndrome Conorenal syndromes Joubert syndrome Infantile neuronal ceroid-lipofuscinosis Zellweger syndrome (i.e., represents the most severe end of peroxismal biogenesis disorders) Extraocular features Hearing loss, diabetes mellitus, dilated cardiomyopathy, seizures and developmental delay Cerebellar hypoplasia, renal impairment and cone-shaped digital epiphyses Juvenile cystic kidney disease and cerebellar hypoplasia with episodic apnoea Developmental regression and epilepsy and early death Sensorineural deafness, developmental delay, hepatic dysfunction, hypotonia and early death that are often difficult to discriminate, and shares several important clinical features with Juvenile retinitis pigmentosa (RP). Juvenile RP initially displays a milder phenotype and is characterized by nyctalopia, visual-field narrowing and eventual visual acuity loss, with or without nystagmus. Seven of the known LCA disease genes, including CRX, CRB1, RPE65, RDH12, LRAT, MERTK and TULP1, have already been linked to the clinical appearance of juvenile RP in other subjects [11 16]. Prognosis A number of longitudinal natural history studies of visual function have attempted to address the question of visual prognosis in patients with LCA. Heher et al. studied 22 LCA patients with 1 14 years follow-up. In total, 77% of patients were found to have stable visual acuity, vision worsened in 18% and improved in 5% [9]. They found that the subgroup of patients with macular colobomas developed progressive visual loss. Cataracts and keratoconus were recognized as additional factors contributing to visual impairment in older patients. Fulton et al. investigated 14 LCA patients with follow-up of 2 15 years, using grating acuities and dark-adapted visual thresholds as the main outcome measure [17]. Visual capabilities varied widely with measurable grating acuities ranging from 0.16 to six cycles per degree (median: 1.27 cycles per degree or ~20/500) and dark-adapted thresholds elevated to log units (median: 2.33 log units). Vision was stable in the majority by longitudinal measures, but increased in a few and deteriorated in others in this small study neither fundus appearance nor degree of hyperopia predicted visual function. Brecelj et al. recorded ERG and visual evoked potentials to whiteflash stimulation in nine children at least twice [18]. Patients were followed up over 1 5 years. A total of 55% of patients had stable visual evoked potential, 11% had worsened and 33% recovered. Genetic basis of disease Over 400 mutations in 15 genes with a predominantly autosomal recessive mode of inheritance have been implicated in the development of LCA [11 16,19 34] (Table 2). Besides LCA5 and CEP290, LCA genes are preferentially expressed in the retina and are involved in nonsyndromic forms of the disease [3]. These causative genes were identified using a variety of techniques; notably classic linkage studies, identity-by-descent mapping and the candidate gene approach. Together, they are responsible for approximately 70% of LCA cases. Functional studies of these genes indicate their involvement in diverse pathways underlying the disease, such as development (CRB1 and CRX ), phototransduction (GUCY2D and AIPL1), vitamin A metabolism (RPE65, LRAT and RDH12), and cilium formation and function (CEP290, TULP1, RPGRIP1 and LCA5). The function of SPATA7 remains unknown, but recent work by Wang et al. suggested that it may have a role in vesicular transport [19]. There have also been sporadic reports of autosomal dominant inheritance, which has mainly been associated with a CRX mutation leading to defective development [35]. Genotype phenotype correlations Many studies have described a correlation between certain retinal appearances and changes in longitudinal visual function with specific genetic mutations. Hanein et al. published the genotype phenotype association on a pool of 179 unrelated subjects with different LCA mutations [36]. Patients with mutations of AIPL1, RGRIP1 and GUCY2D were found to have a cone rod dystrophy and suffer from photophobia, whereas patients with mutations in CRB1, CRX, RPE65 and TULP1 had a rod cone dystrophy and corresponding night blindness. They concluded that it is possible to divide patients into two main groups. The first group (LCA-I) included patients whose symptoms fit the traditional definition of LCA, that is, a severe congenital stationary cone rod dystrophy characterized by photophobia, severe hyperopia and visual acuity reduced to light perception or, at best, perception of hand movements; while the second group (LCA-II) included patients with congenital progressive rod cone dystrophy characterized by night blindness, variable refractive errors ranging from moderate hyperopia to high myopia and measurable dark-adapted visual thresholds. Other groups (e.g., Galvin et al. [37]) found considerable overlap in phenotypic expression of six genetic subtypes (AIPL1, CRB1, CRX, GUCY2D, RPE65 and RPGRIP1) in their LCA cohort. However, phenotypic trends were noted in the patients visual acuities and posterior segment findings within genotypes. Leber congenital amaurosis patients with the RPE65 genotype appear to have measurable visual function with transient improvement followed by a slow deterioration over the measured 20 years [38 40]. Photophobia does not seem to be a feature in such patients who had relatively preserved peripheral vision. Koenekoop et al. 204 Expert Rev. Ophthalmol. 6(2), (2011)

3 Insight into Leber congenital amaurosis: potential for gene therapy also describe a relatively preserved retinal appearance in patients with GUCY2D mutations [41], but these patients had worse visual outcome than those with RPE65 mutations [42]. AIPL1 mutations were found to cause a severe and progressive form of LCA [7]. Most of the patients studied developed maculopathy and had marked bone spicule pigmentary retinopathy. Keratoconus and cataract were also noted in a large subset. Patients who harbor the RPGRIP1 mutation have progressive loss of vision associated with photophobia in addition to high hyperopia [36]. Those with a CRB1 mutation have significant night blindness. keratoconus and a fundus appearance of preserved para-arteriolar retinal pigment epithelium are also highly suggestive of a CRB1 mutation [14]. CRX mutations are associated with stable visual function [38]. The inheritance is reported to follow an autosomal dominant pattern. RDH12 mutations lead to a clinical picture similar to that of RPE65 mutations. Patients may display transient improvement in visual function with eventual development of macular coloboma and visual deterioration. Loss of vision in RDH12 patients occurs at an earlier age than those with an RPE65 mutation. Such variation in the course of the disease highlights the importance of genetic testing in order to provide the patient and their families with a more accurate prediction of their prognosis. Animal models of LCA Numerous animal models have been developed in order to study the phenotypic effects of certain LCA mutations and potential therapeutics. This has allowed intervention in different cell lineages and at varying stages of the disease process. Table 2. Genes implicated in Leber congenital amaurosis. Study (year) Gene Function Identification method (RetNet) Mutation frequency (RetNet) Perrault et al. (1996) AIPL1 Phototransduction Linkage analysis Accounts for 5 10% of recessive LCA den Hollander et al. (2006) CEP290 Transport across Lotery et al. (2001), den Hollander et al. (2001) Homozygosity and linkage mapping Ref. [21] 20% of LCA [22] CRB1 Photoreceptor development Linkage mapping 9 13% of LCA [13] Freund et al. (1998) CRX (AD) Photoreceptor development Mutation analysis and causes Sohocki et al. (2000) GUCY2D Phototransduction Linkage analysis 10 20% of recessive LCA Bowne et al. (2002) and (2006) IMPDH1 (AD LCA) Unknown den Hollander et al. (2007) LCA5 Transport across Mutation analysis and linkage mapping Identity-by-descent mapping and linkage mapping [14] 1 3% of LCA [15] [23] NA [16,24] NA [25] Thompson et al. (2001) LRAT Retinoid cycle Mutation analysis NA [26] Gal et al. (2000) MERTK Failure to phagocytose outer segment Mutation analysis NA [27] Friedman et al. (2006) RD3 Unknown Mutation analysis NA [28] Janecke et al. (2004) RDH12 Retinoid cycle Linkage analysis 4% of recesssive LCA Aguirre et al. (1998), Gu et al. (1997), Marlhens et al. (1997), Morimura et al. (1998) Dryja et al. (2001), Gerber et al. (2001) Zhang et al. (2003), Wang et al. (2009) RPE65 Retinoid cycle Mutation analysis 6 16% of LCA [30] RPGRIP1 Transport across [29] [11] [31] [32] Mutation analysis 4 6% of LCA [33] SPATA7 Unknown Homozygosity mapping NA [34] Hagstrom et al. (1998) TULP1 Transport across LCA: Leber congenital amaurosis; NA: Not available. Mutation analysis and linkage mapping [12] [19] NA [35] 205

4 Qatarneh, Mehta & Lee Mouse models Both naturally occurring and man-made models exist. The four naturally occurring models are CEP290 (rd16), CRB1 (rd8), RD3 (rd3) and RPE65 (rd12). The remaining man-made models have knock-out or induced mutations in all other genes that are known to cause LCA in humans. Degeneration of rods, and in some cases cones, was noted in all but three of these models (Crb1, Gucy2D and RDH12). However, in the case of GUcy2d, ERG activity was nevertheless significantly reduced [43 45]. Chicken model The chicks have naturally occurring deletions in GUCY2D, which are responsible for replenishing cgmp. At hatching, their retinae show no degeneration, but 7 10 days later, degeneration is noted, starting from the central retina and progressing towards the periphery. At approximately 7 months, the entire layer has degenerated. Interestingly, ERG responses are not detectable in these chicks certainly not before morphological changes are noted. It is hypothesized that this is due to absent cgmp causing closure of cgmp-gated cation channels resulting in hyperpolarization, thus mimicking constant light stimulation [46,47]. Briard dog model Multiple mutations in the RPE65 gene are associated with severe, early-onset recessive LCA in humans (LCA-2 or RPE65- LCA) [3,48]. The close similarities in clinical characteristics of disease resulting from RPE65 gene defects in humans and dogs make the RPE65 -/- Briard dog model valuable for the evaluation of gene-replacement therapy. The defective RPE65 gene was replaced with a normal copy by a subretinal injection of adenoviral-associated viral (AAV) vector. This resulted in substantial restoration of rod function confirmed by dark- and light-adapted ERG responses, and improved psychophysical outcomes. By contrast, intravitreal injections did not consistently show any improvement in visual function. Both al and postreceptoral function in both rod and cone systems improved with subretinal therapy. ERG responses remained stable in the follow-up periods of 3 and 7.5 years [49,50]. Subretinal AAV RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Le Meur et al. compared the use of two different vectors (recombinant [r]aav2/4- and raav2/2-) and found that raav2/4 displayed faster onset in restoration of retinal function. Although both vectors were of the same titer (1011 vector genomes/ml [vg/ml]), they could not exclude that the ratio between total particles and transducing units was different, given that the vectors were produced by different facilities [51]. Le Meur et al. had another interesting finding in a dog treated at a later age of 30 months [51]. The older dog did not recover retinal function nor vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65 -/- dogs by gene-replacement therapy [52]. Gene therapy & the eye The eye offers an ideal environment for gene therapy owing to a number of factors. Its relatively small size allows the use of small volumes of vector, which has positive implications on both cost and adverse effects. Its compartmentalization enables the delivery to specific cell types, which in themselves are relatively stable allowing for a longer lasting effect from their transfection. The blood retinal and aqueous barriers make the eye an immune-privileged site with relative protection against immune-mediated destruction of the viral vectors. Moreover, the transparency of ocular media allows the visualization of vector delivery and noninvasive monitoring of the transfected tissue. Most importantly, the effect of gene therapy can be monitored both clinically and functionally using readily available tests. LCA & gene therapy Leber congenital amaurosis was specifically chosen in recent clinical trials as a target disease for fulfilling numerous criteria. The disease is caused by a loss-of-function mutation in a number of identified genes. The hallmark retinal degeneration in those with the defective RPE65 gene typically occurs after the third decade of life, so younger patients have relatively stable retinal structure, despite the onset of symptoms, providing an opportunity for protection with early intervention. The most significant advances have been reported in the case of the RPE65 gene highly expressed in the retinal pigment epithelium. RPE65 is required for the conversion of all-trans-retinoids to 11-cis-retinal. In its absence, the visual pathway malfunctions with the absence of active visual pigment and eventual visual loss. The first significant study of gene therapy in LCA involved the previously described Briard dog model. Although the retinal dystrophy caused by defects in RPE65 is severe, features of the disorder suggest that it may respond to gene-replacement therapy. There is useful visual function in childhood, and retinal imaging suggests that cell death occurs late in the disease process [53]. Gene therapy therefore has the potential to improve visual function as well as preserve existing vision. Human trials Recently, results from three clinical trials with human subjects have been published [54 56]. All three are Phase I trials aimed at establishing the safety and dose ranging of these treatments. In each study, three LCA sufferers received a subretinal injection of an AAV2 vector expressing RPE65. Different promoter sequences and vector titers were used in each of the trials. There were no improvements noted on ERG in any of these trials. However, there was demonstrable improvement in retinal sensitivity in all the studies. The authors suggest that this may have been due to the advanced stage of disease in their subjects. Studies are ongoing and the results will be eagerly awaited to see whether there is a detectable improvement on ERG when treating younger subjects with less advanced disease. Bainbridge et al. gave three young adult patients subretinal injections of ravv2/2 expressing RPE65 cdna under the control of a human RPE65 promoter [54]. They found no clinically significant change in visual acuity or in peripheral visual fields on Goldmann 206 Expert Rev. Ophthalmol. 6(2), (2011)

5 Insight into Leber congenital amaurosis: potential for gene therapy perimetry and no change in retinal responses on ERG. However, one patient showed an improvement in dark-adapted perimetry and visual mobility in low light (assessed at the Pedestrian Accessibility and Movement Environment Laboratory). Maguire et al. investigated the safety of subretinal delivery of raav carrying RPE65 complementary DNA (cdna) in three human subjects [55]. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed. Both subjective and objective measures were used to assess efficacy. Objective measures included pupillary light reflex and nystagmus testing. Subjective measures included standard tests of visual acuity (Early Treatment Diabetic Retinopathy Study [ETDRS]), Goldmann visual-field examination and mobility testing to assess differences in the ability of the patients to navigate a standardized obstacle course. Their results showed gains in visual acuity at 6 weeks, with a slower rate of improvement thereafter. The three patients had reduction in nystagmus and improvement in the pupillary light reflex. They reported no apparent local or systemic adverse events elicited by exposure to the AAV vector. Hauswirth et al. looked at the results of three young adults (aged years) with RPE65-LCA who received a uniocular subretinal injection of vector genomes in 150 µl and were studied with follow-up examinations for 90 days [56]. They did not report any systemic nor local side effects from the intervention. Visual acuity was not significantly different from baseline. However, there was an improvement in dark-adapted full-field sensitivity testing following vector injection. More recently, the age-dependent effects of RPE65 gene therapy for LCA were studied and reported by Maguire et al. [57]. They assessed the retinal and visual function in 12 patients (aged 8 44 years) with RPE65-associated LCA. They were given one subretinal injection of AAV containing the functional RPE65 gene (AAV2 hrpe65v2) in the worst eye at low, medium or high dose for up to 2 years. The patients showed an improvement in both subjective and objective visual measurements assessed by dark adaptometry, pupillometry, ERG, nystagmus and ambulatory behavior. The greatest improvement was noted in the 8-year-old patient who had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. This study shows promise that perhaps early intervention in the pediatric population may significantly minimize the considerable visual loss these patients will face in adulthood. All hallmark human trials used the subretinal method of vector delivery. An intravitreal approach remains to be explored in humans. In the authors opinion, an intravitreal approach would be less invasive, less expensive and probably safer. However, it poses a number of challenges that stand in the way of providing effective treatment delivery. First, the vitreous fluid would significantly dilute the vector concentration compared with a subretinal injection, although a study of macaque eyes showed that AAV is still able to overcome this challenge and lead to retinal transduction [58]. Second, the increase in vitreous viscosity in humans above the age of 40 years could impair the diffusion of the virus in older patients vitreous. In addition, finding a vector that is efficient enough to go through the vitreoretinal barriers and cause successful transduction of s remains to be discovered [59]. However, recent work by Kolstad et al. showed a significant increase in AAV-mediated gene transfer in the diseased compared with normal rat retina [60]. Should this translate into functional improvement in human s, this could raise the hopes for potential gene therapy through intravitreal delivery. Genetic testing With genotype phenotype correlations starting to become established, the role of genetic testing to confirm diagnosis and provide suggestions for prognosis is becoming increasingly important. Genetic tests using microarrays are commercially available in order to detect mutations that have been identified in other patients with LCA [61]. The advantage of the microarrays is that they are rapid (4 h), cheap, robust and based on mutations found in patients. Approximately 60% of new LCA patients will have at least one causal mutation identified by the latest versions of the microarrays [41]. The disadvantage is that new mutations will be missed. Full sequencing, although more time consuming and costly, overcomes this problem. Next-generation sequencing methods [62] should allow full sequencing to be more time and cost effective in the future. Expert commentary With recent Phase I trials showing promising results for the safety of gene therapy in LCA, genetic testing and counseling of patients has never been more vital. Ethical dilemmas surround decisions regarding whom to treat and when. On the one hand, waiting for gene therapy until visual loss has become established reduces the therapeutic effect. On the other hand, there is a possibility that pediatric eyes that would not develop severe visual loss would be undergoing a surgical procedure that carries some risk. The large amount of safety data of intravitreal procedures in adults may make the transition to younger age groups more feasible. Identifying which genotypic variants or group of genetic variants predispose to particularly severe visual loss could provide early candidates for gene therapy. Five-year view Molecular genetics have advanced our knowledge of LCA, allowing the clinician to have a better evaluation of the disease and its prognosis. Further research is required to identify the mutations responsible for the remaining 30% of LCA cases. Collaboration between the clinicians and geneticists is also of huge value in finding more genetic phenotypic associations that could further aid the diagnosis and facilitate genetic testing. The recent human trials on RPE65 mutations offer some hope of treatment in a condition that is currently only managed conservatively. Subsequent gene-replacement research into other mutations responsible for LCA would need to be conducted should the current Phase I trials prove successful. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript

6 Qatarneh, Mehta & Lee Key issues Leber congenital amaurosis is an inherited retinal degeneration, which is characterized by significant visual loss in early life. It may occur in the presence of a number of syndromes with or without systemic features. Over 400 mutations in 15 genes with a predominantly autosomal recessive mode of inheritance have been implicated in Leber congenital amaurosis. Animal models with knock-out or induced mutations have been developed to analyze both disease manifestation and the effect of intervention. The Briard dog model had the defective RPE65 gene replaced by a normal copy using an adenoviral vector subretinal injection. There was substantial improvement in rod function. Phase I human trials have established safety and also found some improvement in retinal sensitivity following subretinal injection of viral vectors expressing RPE65. There is a suggestion that earlier intervention would have a better outcome in restoring visual function, but studies are still ongoing. References 1 Alstrom CH, Olson OA. Heredoretinopathia congenitalis monohybrida recessiva autosomalis. Hereditas 43, (1957). 2 Koenekoop RK. An overview of Leber congenital amaurosis: a model to understand human retinal development. Surv. Ophthalmol. 49, (2004). 3 den Hollander AI, Roepman R, Koenekoop RK, Cremers FP. Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog. Retin. Eye Res. 27, (2008). 4 Cremers FP, van den Hurk JA, den Hollander AI. Molecular genetics of Leber congenital amaurosis. Hum. Mol. Genet. 11, (2002) 5 Franceschetti A, Dieterle P. Diagnostic and prognostic importance of the electroretinogram in tapetoretinal degeneration with reduction of the visual field and hemeralopia. Confin. Neurol. 14, (1954) 6 Koenekoop RK, Fishman GA, Iannaccone A et al. Electroretinographic abnormalities in parents of patients with Leber congenital amaurosis who have heterozygous GUCY2D mutations. Arch. Ophthalmol. 120, (2002). 7 Dharmaraj S, Leroy BP, Sohocki MM et al. The phenotype of Leber congenital amaurosis in patients with AIPL1 mutations. Arch. Ophthalmol. 122, (2004). 8 Fazzi E, Signorini SG, Scelsa B et al. Leber s congenital amaurosis: an update. Eur. J. Paediatr. Neurol. 7, (2003). 9 Heher KL, Traboulsi EI, Maumenee IH. The natural history of Leber s congenital amaurosis. Age-related findings in 35 patients. Ophthalmology 99, (1992). 10 Perrault I, Delphin N, Hanein S et al. Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype. Hum. Mutat. 28, 416 (2007). 11 Gu SM, Thompson DA, Srikumari CR et al. Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. Nat. Genet. 17, (1997). 12 Gerber S, Perrault I, Hanein S et al. Complete exon intron structure of the RPGR-interacting protein (RPGRIP1) gene allows the identification of mutations underlying Leber congenital amaurosis. Eur. J. Hum. Genet. 9, (2001). 13 Lotery AJ, Jacobson SG, Fishman GA et al. Mutations in the CRB1 gene cause Leber congenital amaurosis. Arch. Ophthalmol. 119, (2001). 14 den Hollander AI, Heckenlively JR, van den Born LI et al. Leber congenital amaurosis and retinitis pigmentosa with Coats-like exudative vasculopathy are associated with mutations in the crumbs homologue 1 (CRB1) gene. Am. J. Hum. Genet. 69, (2001). 15 Freund CL, Wang QL, Chen S et al. De novo mutations in the CRX homeobox gene associated with Leber congenital amaurosis. Nat. Genet. 18, (1998). 16 Bowne SJ, Sullivan LS, Blanton SH et al. Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa. Hum. Mol. Genet. 11, (2002). 17 Fulton AB, Hansen RM, Mayer DL. Vision in Leber congenital amaurosis. Arch. Ophthalmol. 114, (1996) 18 Brecelj J, Stirn-Kranjc B. ERG and VEP follow-up study in children with Leber s congenital amaurosis. Eye (Lond.) 13(Pt 1), (1999). 19 Wang H, den Hollander AI, Moayedi Y et al. Mutations in SPATA7 cause Leber congenital amaurosis and juvenile retinitis pigmentosa. Am. J. Hum. Genet. 84, (2009). 20 Sohocki MM, Sullivan LS, Mintz-Hittner HA et al. A range of clinical phenotypes associated with mutations in CRX, a transcription-factor gene. Am. J. Hum. Genet. 63, (1998). 21 Perrault I, Rozet JM, Calvas P et al. Retinal-specific guanylate cyclase gene mutations in Leber s congenital amaurosis. Nat. Genet. 14, (1996). 22 den Hollander AI, Koenekoop RK, Yzer S et al. Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am. J. Hum. Genet. 79, (2006). 23 Sohocki MM, Bowne SJ, Sullivan LS et al. Mutations in a new -pineal gene on 17p cause Leber congenital amaurosis. Nat. Genet. 24, (2000). 24 Bowne SJ, Sullivan LS, Mortimer SE et al. Spectrum and frequency of mutations in IMPDH1 associated with autosomal dominant retinitis pigmentosa and Leber congenital amaurosis. Invest. Ophthalmol. Vis. Sci. 47, (2006). 25 den Hollander AI, Koenekoop RK, Mohamed MD et al. Mutations in LCA5, encoding the ciliary protein Lebercilin, cause Leber congenital amaurosis. Nat. Genet. 39, (2007). 26 Thompson DA, Li Y, McHenry CL et al. Mutations in the gene encoding lecithin retinol acyltransferase are associated with early-onset severe retinal dystrophy. Nat. Genet. 28, (2001). 27 Gal A, Li Y, Thompson DA et al. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat. Genet. 26, (2000). 208 Expert Rev. Ophthalmol. 6(2), (2011)

7 Insight into Leber congenital amaurosis: potential for gene therapy 28 Friedman JS, Chang B, Kannabiran C et al. Premature truncation of a novel protein, RD3, exhibiting subnuclear localization is associated with retinal degeneration. Am. J. Hum. Genet. 79, (2006). 29 Janecke AR, Thompson DA, Utermann G et al. Mutations in RDH12 encoding a cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy. Nat. Genet. 36, (2004). 30 Aguirre GD, Baldwin V, Pearce-Kelling S et al. Congenital stationary night blindness in the dog: common mutation in the RPE65 gene indicates founder effect. Mol. Vis. 4, 23 (1998). 31 Marlhens F, Bareil C, Griffoin JM et al. Mutations in RPE65 cause Leber s congenital amaurosis. Nat. Genet. 17, (1997). 32 Morimura H, Fishman GA, Grover SA et al. Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis. Proc. Natl Acad. Sci. USA 95, (1998). 33 Dryja TP, Adams SM, Grimsby JL et al. Null RPGRIP1 alleles in patients with Leber congenital amaurosis. Am. J. Hum. Genet. 68, (2001). 34 Zhang X, Liu H, Zhang Y et al. A novel gene, RSD-3/HSD-1, encodes a meioticrelated protein expressed in rat and human testis. J. Mol. Med. 81, (2003). 35 Hagstrom SA, North MA, Nishina PL et al. Recessive mutations in the gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa. Nat. Genet. 18, (1998). 36 Hanein S, Perrault I, Gerber S et al. Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype phenotype correlations as a strategy for molecular diagnosis. Hum. Mutat. 23, (2004). 37 Galvin JA, Fishman GA, Stone EM, Koenekoop RK. Evaluation of genotypephenotype associations in leber congenital amaurosis. Retina 25, (2005). 38 Dharmaraj SR, Silva ER, Pina AL et al. Mutational analysis and clinical correlation in Leber congenital amaurosis. Ophthalmic Genet. 21, (2000). 39 Lorenz B, Gyurus P, Preising M et al. Early-onset severe rod cone dystrophy in young children with RPE65 mutations. Invest. Ophthalmol. Vis. Sci. 41, (2000). 40 Thompson DA, Gyurus P, Fleischer LL et al. Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration. Invest. Ophthalmol. Vis. Sci. 41, (2000). 41 Koenekoop RK, Lopez I, den Hollander AI et al. Genetic testing for retinal dystrophies and dysfunctions: benefits, dilemmas and solutions. Clin. Experiment. Ophthalmol. 35, (2007). 42 Perrault I, Rozet JM, Ghazi I et al. Different functional outcome of RetGC1 and RPE65 gene mutations in Leber congenital amaurosis. Am. J. Hum. Genet. 64, (1999). 43 Mehalow AK, Kameya S, Smith RS et al. CRB1 is essential for external limiting membrane integrity and morphogenesis in the mammalian retina. Hum. Mol. Genet. 12, (2003). 44 Yang RB, Robinson SW, Xiong WH et al. Disruption of a retinal guanylyl cyclase gene leads to cone-specific dystrophy and paradoxical rod behavior. J. Neurosci. 19, (1999). 45 Maeda A, Maeda T, Imanishi Y et al. Retinol dehydrogenase (RDH12) protects s from light-induced degeneration in mice. J. Biol. Chem. 281, (2006). 46 Ulshafer RJ, Allen CB. Hereditary retinal degeneration in the Rhode Island Red chicken: ultrastructural analysis. Exp. Eye Res. 40, (1985). 47 Semple-Rowland SL, Lee NR, van Hooser JP et al. A null mutation in the guanylate cyclase gene causes the retinal degeneration chicken phenotype. Proc. Natl Acad. Sci. USA 95, (1998). 48 Thompson DA, Gal A. Vitamin A metabolism in the retinal pigment epithelium: genes, mutations, and diseases. Prog. Retin. Eye Res. 22, (2003). 49 Acland GM, Aguirre GD, Ray J et al. Gene therapy restores vision in a canine model of childhood blindness. Nat. Genet. 28, (2001). 50 Acland GM, Aguirre GD, Bennett J et al. Long-term restoration of rod and cone vision by single dose raav-mediated gene transfer to the retina in a canine model of childhood blindness. Mol. Ther. 12, (2005). 51 Le Meur G, Stieger K, Smith AJ et al. Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium. Gene Ther. 14, (2007). 52 Narfstrom K, Katz ML, Bragadottir R et al. Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog. Invest. Ophthalmol. Vis. Sci. 44, (2003). 53 Paunescu K, Wabbels B, Preising MN, Lorenz B. Longitudinal and cross-sectional study of patients with early-onset severe retinal dystrophy associated with RPE65 mutations. Graefes Arch. Clin. Exp. Ophthalmol. 243, (2005). 54 Bainbridge JW, Smith AJ, Barker SS et al. Effect of gene therapy on visual function in Leber s congenital amaurosis. N. Engl. J. Med. 358, (2008). 55 Maguire AM, Simonelli F, Pierce EA et al. Safety and efficacy of gene transfer for Leber s congenital amaurosis. N. Engl. J. Med. 358, (2008). 56 Hauswirth WW, Aleman TS, Kaushal S et al. Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adenoassociated virus gene vector: short-term results of a Phase I trial. Hum. Gene Ther. 19, (2008). 57 Maguire AM, High KA, Auricchio A et al. Age-dependent effects of RPE65 gene therapy for Leber s congenital amaurosis: a Phase 1 dose-escalation trial. Lancet 374, (2009). 58 Morgan JI, Dubra A, Wolfe R et al. In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic. Invest. Ophthalmol. Vis. Sci. 50, (2009). 59 Hellstrom M, Ruitenberg MJ, Pollett MA et al. Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection. Gene Ther. 16, (2009). 60 Kolstad KD, Dalkara D, Guerin K et al. Changes in adeno-associated virusmediated gene delivery in retinal degeneration. Hum. Gene Ther. 21, (2010). 61 Zernant J, Kulm M, Dharmaraj S et al. Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles. Invest. Ophthalmol. Vis. Sci. 46, (2005). 62 Willenbrock H, Salomon J, Sokilde R et al. Quantitative mirna expression analysis: comparing microarrays with nextgeneration sequencing. RNA 15, (2009)