Juvenile retinoschisis is caused by mutations in the RS1 gene, which encodes for retinoschisin, an extracellular protein integral to the retina for cellular adhesion and tissue stability.33,3 Many studies investigating rAAVmediated gene therapy in a RS1 knockout mouse model of human X-linked retinoschisis have demonstrated improvements in retinal function. Few studies, however, have shown restoration of the structural integrity of the retina. Min et al. found significant improvements in both retinal structure and function of 15-day old Rs1h-deficient mice following subretinal delivery of human RS1 cDNA via the rAAV5 vector. The structure and function of the retina in the Rs1h-deficient mice was restored to levels comparable to those in wild-type mice and was long-term, persisting for up to a year.33,34

Achromatopsia. Achromatopsia is a rare autosomal recessive congenital disorder that can render patients completely colorblind. Additionally, affected individualsoftenpresent with poor visual acuity, photophobia, and/ or nystagmus.31 In general, achromatopsia is characterized by slow progression of cone photoreceptor dysfunction that can lead to one of two known forms, complete or incomplete achromatopsia. The two forms bear phenotypic resemblance, with the only difference being that incomplete achromatopsia patients have slightly better visual acuity and cone function. The two forms, however, are often genetically heterogeneous: three different genes have been found to be implicated for achromatopsia-associated mutations. Of these three cone-specific genes (CNGB3, CNGA3, and GNAT2), only CNGA3 has been associated with both forms of achromatopsia.31,1

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Although both murine and canine models of achromatopsia exist, results of rAAV2/5-mediated gene therapy interventions in the naturally occurring dog model of achromatopsia are still at the preliminary stage. However, the rAAV2-mediated gene replacement of GNAT2 in 2-3 week old Gnat2-deficient mice has resulted in marked improvements in achromatopsic pathology. Cone electrophysiology and visual acuity, which are both directly dependent on cone photoreceptor function, were restored to wildtype levels for as long as 7 months following subretinal administration of rAAV2-GNAT2 in the Gnat2-deficient mouse model.1 Improving cone function is the goal of rAAV-mediated gene therapy for achromatopsia; it has been noted that success of gene therapy in as a potential therapeutic may depend on the age at which patient receives treatment.3

Stargardt disease. Stargardt disease is the most common form of juvenile macular dystrophy and has a recessive inheritance pattern. Degeneration due to Stargardt disease usually presents itself within the second decade of a patient’s life and is characterized by progressive loss of central vision due to subretinal deposition of lipofuscin-like material in the macula.1,3 ABCA4, the large gene responsible for the development of Stargardt disease, encodes for the ABCA4 transporter protein. ABCA4 functions in the transport of a visual cycle retinoid byproduct, known as N-retinylidinephosphatidylethanolamine (N-RPE), across the outer segment membrane of photoreceptor discs to the retinal pigment epithelium. When any one of the nearly 400 mutations found to be associated with Stargardt disease are present in the ABCA4

gene, the dysfunctional transporter protein product triggers a cascade that first causes N-RPE to accumulate in the outer segment membrane of photoreceptor discs. The build up of N-RPE then leads to generation of the lipofuscin component N-retinylidene- N-retinylethanolamine (A2E), which causes lipofuscin deposits to form in the subretinal space between the retinal pigment epithelium and photoreceptors, and ultimately leads to photoreceptor degeneration.31,35

FurtherinvestigationofStargardtdisease at the experimental level has been enabled through the development of an Abca4-/- knockout mouse model. An increased accumulation of both A2E and lipofuscin was observed in the retinal pigment epithelium of Abca4-/- mice, as was subsequent photoreceptor degeneration and abnormal electrical activity of photoreceptors.31,1 Until recently, gene therapy approaches for Stargardt disease were limited due to the large size of the ABCA4 cDNA and limited packaging capacity of rAAV vectors. However, rAAV-mediated gene therapy was successfully implemented in the Abca4-/- mouse model using rAAV2/5- based vectors, which have been shown to efficiently package recombinant genomes up to 9kb in size. Following subretinal injection of the rAAV2/5 vector encoding the Abca4 murine gene in the Abca4-/- mouse model, a significant improvement was observed in retinal morphology and function owing to a corresponding reduction in lipofuscin accumulation.1,3

Age-related macular degeneration (AMD). Age-related macular degeneration (AMD)istheleadingcauseofvisuallossamong the elderly population in many countries, including the United States. While there is no known single causative factor of AMD, both genetics and the environment are thought to play predominant roles in development of the disease. Central vision loss is characteristic of AMD since the macula deteriorates as the diseaseprogresses. Consequently,experimental investigation of AMD has been complicated by the fact that most of the mammalian models available lack the macular region of the retina. However, numerous murine models of AMD have greatly elucidated the physiology and genetics behind AMD.36

AMD is phenotypically heterogeneous, manifesting in one of two forms: the “dry,” non-exudative form or the “wet,” exudative form of AMD. “Dry” AMD is the more prevalent of the two forms and causes a milder phenotype, in which a yellowishwhite substance called drusen is deposited just beneath the retina in the space between the retinal pigment epithelium and Bruch’s membrane. The deposition of drusen causes death of the retinal pigment epithelial cells and subsequent degeneration of the photoreceptors. Eventually, geographic atrophy occurs as the retina thins and vision worsens. Wet AMD is easily differentiated from dry AMD; wet AMD progresses rapidly and results in much more severe vision loss due to neovascularization of the choroid. The associated visual impairment progresses rapidly

once the blood leaks from the neovasculature into the subretinal region and thus damages the retina.31 Although both the phenotypic and environmental heterogeneity inherent between the two forms of AMD contribute to notions suggesting the two forms should be treated as two distinct diseases, recent genetic studies have linked polymorphisms in the complement fact H (CFH) gene to both the wet and dry forms of AMD.22

Gene therapy approaches for treatment of dry AMD are limited; however, the recent finding of a genetic variant in the gene encoding the toll-like 3 (TLR3) receptor has expanded the possibilities for therapeutic intervention. Specifically, Yang et al. tested for an association between the functional TLR3 variant, which involves the substitution of phenylalanine for leucine at amino acid 412, with AMD. Their results demonstrated an association between the TLR3 variant and protection against the geographic atrophy indicative of dry AMD, which is thought to be mediated by suppression of retinal pigment epithelial cell death. However, no association with the choroidal neovascularization (CNV) phenotype of wet AMD was observed.37 On the other hand, several rAAV-mediated gene therapy approaches have been implemented in the treatment of an experimental model of choroidal neovascularization. rAAV-mediated gene therapy has been used in both the induction and inhibition of choroidal neovascularization in the rat disease model. First, induction of choroidal neovascularization was adapted in the development of the rat model of wet AMD through subretinal delivery of

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rAAV-vector encoding vascular endothelial growth factor (VEGF), a pro-angiogenic factor. Subsequently, the injected rats presented with the common symptoms of exudative AMD, including subretinal neovascularization, photoreceptor degeneration, and blood leakage of the neovasculature.38

rAAV-mediated gene therapy has also been used to inhibit experimental models of choroidal neovascularization in a variety of animal models including mice, rats, and monkeys.39,40 Mori et al. have observed a reduction in both the development of CNV, as well as the regression of already developed CNV in a murine model following subretinal and intravitreal delivery of rAAV-vector encoding the pigment epithelial derived factor (PEDF), an anti-angiogenic, neurotrophic protein.39 The many pre-clinical studies using rAAV-mediated gene therapy, especially in the investigation of wet AMD, hold great promise for clinical trials using rAAV-mediated gene therapy. Clinical trials employing the use of alternative gene therapy methods to treat wet AMD are currently underway.41 A few trials focus on gene suppression of VEGF and its receptor using siRNA targeting. Another trial employingadenoviralvector-mediateddelivery of PEDF has successfully been completed. As mechanisms of AMD pathology continue to be unveiled, such as the role of inflammatory pathways in the progression of AMD and the susceptibility of retinal pigment epithelial cells to damage, the potential of intervention with rAAV-mediated gene therapy for use in clinical trials is significant.

Acquired Retinal Degenerative

Disorders

Retinal neovascularization. Retinal neovascularization is a common feature of many ocular diseases, including the neovascular form of age-related macular degeneration, as previously mentioned, and is caused by many of the same aforementioned factors. rAAV-mediated gene therapy for treatment of retinal neovascularization in acquired diseases has far-reaching implications, since acquired diseases such as diabetic retinopathy and AMD account for the majority of cases of irreversible blindness in the world.42 In a long-term study investigating the therapeutic potential of rAAV-mediated expression of a soluble form of the Flt-1 (sFlt-1) receptor in inhibiting the angiogenic action of VEGF, Lai et al. observed a reduction in retinal neovascularization. Additionally,morphologicstudies suggested preservation of retinal structure, unlike the retinal damage that commonly accompanies retinal neovascularization by inducing photoreceptor loss.40

Gene Therapy Simultaneously Targeted to Retinal Rod and Cone Cells

While most of the rAAV-mediated gene therapy interventions for retinal diseases discussed here target either the rod or the cone photoreceptors, it is useful to treat the cell types simultaneously as both are implicated in retinal disease pathology. Furthermore, mutations in specific rod photoreceptor genes

can contribute to cone death as well as rod death, whereas ocular diseases caused by cone-specific mutations result only in cone death. To better understand the mechanism behind non-autonomous cone death, Punzo et al. studied the incidence of cone death in four different mouse models for retinitis pigmentosa. The researchers found a connection between cone survival and insulin release; non-autonomous cone death was triggered when cone photoreceptors were starved from a lack of endogenous insulin.43

Other research efforts seeking to treat both rod and cone photoreceptor cell death have unveiled rAAV-mediated ocular gene therapy approaches that target expression of both cell types. The work of Khani et al. introduced the first well-defined, compact promoter to drive transgene expression in both rods and cones. Khani et al. compared the promoter activity of the human rhodopsin kinase (hRK), a gene which has previously been identified as both rodand cone-specific, with the mouse opsin (mOps) promoter. While both promoter types yielded significant rAAV-mediated transgene expression following subretinal injection, only the hRK promoter demonstrated expression in both rods and cones, whereas the mOps promoter was active only in rod photoreceptors. This primary finding holds great significance for rAAV-mediated treatment of numerous retinal diseases, which has previously been limited by the lack of well-defined, rod/cone-specific promoters.44 The same team of investigators demonstrated proof-of-principle of their

2007 findings in mouse models with defects in the AIPL1 gene. Mutations of this gene show allelic heterogeneity, with a null allele resulting in a presentation similar to that of human LCA in mice, and the hypomorphic allele manifesting similarly to human RP in mice. Sun et al. assessed the role of rAAVmediated gene expression of AIPL1 using hRK to promote both rod and cone photoreceptor survival. Their results exhibited successful transgene expression in both rods and cones from the single hRK promoter.45

Looking Ahead: Promising Prospects for Ocular Gene Therapies

Most recently, the therapeutic potential of rAAV-mediated gene therapy was yet again illustrated by Mancuso et al., who studied the effects of rAAV-mediated intervention on colorblindness in adult primates. Colorblindness is one of the most prevalent X-linked recessive disorders in humans, and is a congenital condition in all male squirrel monkeys. Females of this species often have trichromatic color vision, meaning they possess all the necessary photopigments. The dichromatic male squirrel monkeys, however, lack either the L- or M-photopigment preventing them from distinguishing particular wavelengths. Mancuso et al. tested two adult male squirrel monkeys for color vision deficits using the Cambridge Colour Test. As expected, the two monkeys failed to discriminate between

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red-violet and blue-green. These same two dichromatic squirrel monkeys lacking the L- opsin gene, were then given subretinal injections of an L-opsin coding rAAV 2/5 vector. The L-opsin transgene was co-expressed in a subset of endogenous M-cones in the primates, giving way to a shift in the spectral sensitivity of the M-cone photoreceptors. Mancuso et al. identified this very shift as the component underlying the transition from dichromatic color vision to trichromacy in the red-green colorblind adult primates, and that thereby corrected their colorblindness.46 Many advances in experimental research have also fueled the success of rAAV-mediated gene therapy as a potential treatment for numerous ocular diseases. One such advance is the possibility of regulating the activation and deactivation of rAAV-mediated transgene expression by pharmacologically inducible expression systems.6,14 Intrinsic to any gene-regulated gene delivery system is an inducible promoter and a transactivator. An important consideration is the selection of the pharmacological agent used to induce any promoter system, several of which have already been identified as transgene expression regulators in eukaryotes. The development of pharmacologically regulated rAAV vectors has enormous implications for treatment of a wide variety of ocular diseases. The near future holds even greater promise for rAAVmediated gene therapy in the treatment of ocular diseases.47

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Optical coherence tomography (OCT) 318

Retinal pigment epithelial detachment 312