Ординатура / Офтальмология / Английские материалы / Retinal and Vitreoretinal Diseases and Surgery_Boyd, Cortez, Sabates_2010

rAAV-Mediated Gene Therapy for Corneal Diseases

The cornea is a transparent tissue chiefly involved in protecting the structure and function of the eye. As the outermost ocular structure, the cornea protects the eye from any physical and pathogenic injury due to the external environment. The cornea also plays an immunoprotective role through its expression of inhibitors that prevent activation of pro-inflammatory factors, as well as through its secretion of cytokines. The protective function of the cornea is regulated by the five cellular layers that comprise the cornea, including the epithelium, Bowman’s membrane, stroma, Descemet’s membrane and endothelium. Direct accessibility of the corneal epithelium to the external environment facilitates more convenient vector delivery methods for gene therapy, such as non-invasive, topical administration; however, invasive vector delivery methods, including injection into the anterior chamber, have yielded greater transduction efficiencies.14 While gene therapy holds great potential in the treatment of inherited corneal endothelial diseases, as well as in the prevention of corneal allograft rejection,15 the most extensive research concerning rAAV-mediated gene therapy in the cornea has been conducted in experimental models of acquired corneal endothelial disorders.

Acquired Corneal Diseases

Ocular neovascularization is a threatening condition in all of the tissues of the

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eye that it can affect, and particularly so in the avascular cornea. The transparent and immunoprotective nature of the cornea is compromised in corneal neovascularization, which can be induced by a wide variety of factors including inflammation, infection, degeneration, and by both direct trauma to the cornea and indirect trauma to the limbus structure that borders it. Treatment approaches focus on the regulation of corneal angiogenesis to counter the development of new blood vessels from pre-existing pathological vasculature. The homeostatic mechanism behind the regulation of corneal angiogenesis involves both proand anti-angiogenic factors, many of which have been used in conjunction with rAAV-mediated gene therapy approaches. Specifically, the rAAV-mediated gene transfer of angiogenesis inhibitors such as angiostatin and endostatin has been shown to reduce and even inhibit corneal neovascularization.12,16

Recently, Cheng et al. observed a regression in corneal neovascularization due to experimental alkali burn-induced corneal angiogenesis in rats given a subconjunctival injection of rAAV-angiostatin. After establishing efficient rAAV-mediated transduction by GFP transgene expression, the experimental rats were divided into two groups and treated with either a blank rAAV control vector or a rAAV-angiostatin vector. In both cases, the viral vector was administered by subconjunctival injection three weeks prior to the induction of the alkali burn-induced corneal angiogenesis. Corneal neovascularization was observed one week after injury induction and subsequently quantified by calculating the area of marked neovascular engorgement. The prolonged exposure of

the limbal and conjunctival vasculature (the site at which corneal neovessels originate) to transgene expression was determined to account for the observed regression in corneal neovascularization of the rAAV-angiostatin treated group.12

The corneal endothelium has presented challenges for gene therapy in terms of both efficient and prolonged gene transduction, which rAAV-mediated gene transfer has been able to overcome. Until recently, however, in vivo regulation of transgene expression proved difficult even for rAAV-mediated gene therapy. In 2002, Tsai et al. demonstrated prominent transgene expression in the corneal endothelium in vivo when induced by inflammation. Following pre-treatment with the rAAV vector containing the LacZ reporter gene, inflammation was induced by intravitreal injection of lipopolysaccharide (LPS) in the ocular anterior chambers of New Zealand rabbits. A direct correlation was observed between transient ocularanterior segmentinflammation induced by the lipopolysaccharide injection and increases in LacZ gene expression in the rabbit corneal endothelial cells. Results revealed a peak in inflammation one day after LPS injection; the inflammation concurrently activated transgene expression of LacZ in approximately 90% of corneal endothelial cells. Furthermore, a second LPS-injection, given 60 days after the first, elicited a dramatic reactivation of transgene expression to levels once again nearing 90% of endothelial cells, even after transgene expression diminished as inflammation subsided following the first LPS injection. This finding of increased transgene expression following transient LPS-induced inflammation has expanded the implications of rAAV-mediated gene therapy

for the treatment of other acquired corneal diseases including keratitis, anterior uveitis, and corneal graft rejection.15

In a more recent study by Tsai et al., the effect of cell-specific and inducible-expression systems on the level and timing of transgene expression in the treatment of experimental uveitis was investigated. Uveitis is a recurrent, intraocular inflammatory condition that can severely compromise vision. In previous gene therapy studies investigating the therapeutic potential of interleukin-1 receptor antagonist (IL-1Ra) as a treatment for uveitis, the efficiency of intraocular gene transfer by the chosen vector delivery systems was limited. Therefore, Tsai et al. employed the use of the rAAV vector encoding IL-1Ra cDNA to elicit transgene expression in the eyes of New Zealand white rabbits. The rAAV-IL-1Ra vector, as well as a control vector encoding the LacZ reporter gene were administered intravitreally. The therapeutic potential of rAAV-IL-1Ra was assessed after induction of experimental uveitis by intravitreal injection of rAAV-IL-1α at both 10and 100-day timepoints following rAAV-IL-1Ra delivery. Using methods of immunohistochemistry, ELISA, and RT-PCR, Tsai et al. witnessed recovery from experimental uveitis by transgene expression following a single administration of rAAV-IL-1Ra at both the 10-day and 100-day timepoints.17

The demonstration of efficient rAAVmediated transduction of corneal cells in both in vivo and ex vivo conditions has broadened the possible applications of rAAV-mediated gene therapy in corneal diseases to include inherited, iatrogenic, and metabolic diseases of the cornea.8 The work done by Liu et

al., 2008 encompassed an investigation of the tropism of a variety of rAAV serotype vectors in organ-cultured human corneas. The efficiency of transduction was marked by expression of the GFP reporter gene, which encodes the green fluorescent protein (GFP), and was delivered via the rAAV vectors. The observed transduction was extensive, reaching of a variety corneal cells including those of the epithelium, endothelium, and keratocytes. Their findings hold great promise for treating a wide spectrum of corneal disorders; from a group of corneal dystrophies where the causative gene has been deduced to a single mutation in the transforming growth factor-ß-induced gene, TGFBI/BIGH3, to others diseases where the genetic components remain either unknown or are multi-faceted. As demonstrated by the work of Liu et al. 2008, rAAV-mediated gene therapy can be used to target specific corneal cells, in which it could alter the expression levels of known mutant genes.

rAAV-Mediated Gene Therapy for Optic Neuropathies

The optic nerve is a unique ocular tissue because it originates in the eye yet functions in the nervous system by carrying the electrical impulses it receives from the retina to the brain. The axons of retinal ganglion cells converge into fiber bundles along the base of the inner retina, forming the optic nerve. Optic neuropathies encompass all conditions in which the optic nerve incurs any damage. Ocular rAAV-mediated gene therapy for the optic nerve involves both gene replacement and gene addition approaches, to which optic

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neuropathies such as Leber’s hereditary optic neuropathy and glaucoma are amenable.2,1

Inherited Optic Neuropathies

Leber’s hereditary optic neuropathy (LHON). Leber’s hereditary optic neuropathy (LHON) is a common mitochondrial disease characterized by staggered, bilateral vision loss. As a maternally inherited disease, LHON primarily affects men during early adulthood. The disease is predominantly caused by three separate point mutations in genes that encode for the subunits of nicotinamide adenine dinucleotide: ubiquinone oxidoreductase, or complex I, an enzyme involved in the oxidative phosphorylation pathway.2 Guy et al. recently implemented rAAV-mediated gene therapy to replace the G11778A mutation, which encodes for the ND4 subunit of complex I, and accounts for approximately 50% of all LHON cases. Guy et al. employed the use of an allotopic expression system for rAAV-mediated gene transfer of this G11778A mutation to the mitochondrial genome, since as a viral vector, the rAAV vector cannot directly transfer exogenous genes to the mitochondrial genome. The allotopic expression system expresses a nuclear-encoded version of a mitochondrial gene that encodes for a cytoplasmically expressed protein tagged with amitochondrial-targetingpeptide.18,2 Although Guy et al. were successful in restoring the cellular respiration deficit caused by LHON in vitro, an animal model for LHON does not exist, which limits investigation of the rAAV-mediated allotopic ND4 gene therapy approach in vivo.

Optic neuritis. Optic neuritis is a condition that causes inflammation of the optic nerve. People suffering from multiple sclerosis, an autoimmune disease of the central nervous system, are often susceptible to progressive visual loss due to recurrent episodes of optic neuritis. Specifically, in optic neuritis, autoimmune-mediated oxidative injury targets oligodendrocytes, cells that function as neuroglia in the central nervous system and produce the myelin sheath that surrounds retinal ganglion cell axons. The axonal demyelination that results in optic neuritis contributes to axonal degeneration and subsequent neuronal degeneration. Qi et al., in their extensive work, have utilized the experimental autoimmune encephalomyelitis (EAE) rat model of multiple sclerosis in conjunction with rAAV-mediated gene therapy to probe the mechanisms that underlie the autoimmune-mediated oxidative injury observed in optic neuritis. Based on prior findings elucidating the damage-inducing role of reactive oxygen species (ROS), including superoxide and hydrogen peroxide in the EAE disease model,19 Qi et al. employed the use of the ROS scavengers superoxide dismutase (SOD) and catalase to develop an antioxidant gene therapy approach.20

ROS are inducers of optic nerve demyelination and have also been found to interfere with the permeability of the bloodbrain barrier in EAE. The ROS scavengers counteract the oxidative damage done by ROS, with SOD functioning in the dismutation of superoxide to hydrogen peroxide, and catalase detoxifying hydrogen peroxide to water and oxygen.21,20 The cDNA for extracellular superoxide dismutase (ECSOD) and catalase

was cloned into respective rAAV vectors, which were used to infect retinal ganglion cells of the rat EAE model by intravitreal injection. Relevant findings following transgene expression using both the rAAV-ECSOD and rAAV-catalase vectors included decreases in retinal ganglion cell loss by 29%, optic nerve demyelination by 36%, and axonal loss by 44%.21 Earlier results obtained by Qi et al. also demonstrated up to a 78% reduction in optic nerve demyelination in the EAE model.20 The promising results elicited by the rAAV-mediated gene transfer of extracellular superoxide dismutase and catalase serve to demonstrate the great therapeutic potential as well as highlight the advantageous features of rAAV-mediated gene transfer for optic neuritis. rAAV-mediated gene transfer allows for a direct treatment approach, as antioxidant genes were delivered directly to the oligodendrocytes, whereas previous treatment approaches, such as catalase protein delivery, have been limited by incomplete penetration through the blood-brain barrier. Additionally, the long-term transgene expression enabled by the rAAV-mediated delivery of the ROS scavenger genes promoted long-term suppression of optic nerve demyelination and subsequent axonal degeneration.

Acquired Optic Neuropathies

Optic nerve trauma. Optic nerve trauma can result from any condition in which the retinal ganglion cells undergo axotomy, including transection of the optic nerve, which can ultimately lead to both neuronal and retinal ganglion cell death. Retinal ganglion cells experiencing optic nerve trauma are deprived

of brain-derived neurotrophic factor (BDNF), upon which they depend for survival. To better understand how optic nerve trauma can be treated, experimental optic nerve transection models have been developed.2 rAAV-mediated gene transfer has been used as part of the treatment approach for optic nerve transection in a murine model. The ability of the rAAV vector to sustain long-term gene transduction proved advantageous for the work of Cheng et al., who investigated the effect of upregulation of TrkB expression on retinal ganglion cell survival. Cheng et al. used rAAV-mediated gene therapy to transfer the gene encoding for TrkB, a BDNF receptor expressed by retinal ganglion cells, to the retinal ganglion cells in an optic nerve transection rat model. The researchers also supplemented TrkB transgene expression with direct administration of BDNF to the TrkB receptors. An increase in neuronal survival following optic nerve transection was observed.22

Glaucoma. Glaucoma is a progressive optic neuropathy that likely results from the interaction of multiple genetic and environmental factors. While the predisposing genetic factors of glaucoma remain unknown, mutations in the gene encoding the myocilin protein have been found to cause autosomal dominant juvenile primary open-angle glaucoma, as well as nearly 3% of adult-onset open-angle glaucoma cases.23 Many factors, including elevated intraocular pressure (IOP), can predispose an individual to developing the optic nerve head damage and eventual death of retinal ganglion cells characteristic of glaucoma. The implications

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for rAAV-mediated gene therapy in the treatment of glaucoma are pervasive, given that gene therapy targets for glaucoma can range from structures to cell types, including the trabecular meshwork, ciliary body, retinal ganglion cells, and Müller cells.23

Elevated intraocular pressure, which is widely known as the hallmark of glaucoma, is often accompanied by the accumulation of BDNF and its TrkB receptor at the optic nerve head. BDNF transport from the brain to the retinal ganglion cells of the inner retina is therefore interrupted, leading to BDNF deprivation of the retinal ganglion cells, and subsequent neuronal and retinal ganglion cell death. Previous work conducted by Ko et al.24 demonstrated the limited survival of retinal ganglion cells in an experimental rat model of glaucoma following intravitreal injection of BDNF in conjunction with an intraperitoneal injection of a nonspecific free radical scavenger. Recognizing the limitations of multiple intravitreal injections of BDNF, however, Martin and Quigley used rAAVmediated gene transfer to transfect retinal ganglion cells, also in a rat model of glaucoma. Using intravitreal delivery of rAAVBDNF, a rAAV vector in which the cDNA for BDNF was enclosed, a 38% rescue of retinal ganglion cells from BDNF deprivation was witnessed.2 The ability of rAAV-BDNF to slow the rate of retinal ganglion cell death, and thereby the overall progression of glaucoma, in a rat model highlights the relevance of rAAV-mediated gene therapy as a potential treatment for both polygenic diseases and those with unknown etiologies.

Another rAAV-mediated gene therapy approach implemented for the treatment of experimentalglaucomainvolvescaspaseinhibitors. Activated caspase enzymes are intrinsic to the initiation and regulation of apoptosis in retinal ganglion cells, specifically caspase-8 and caspase-3. McKinnon et al. investigated the role of modulating the activation of these caspase enzymes in increased retinal ganglion cell survival and subsequent optic nerve survival. The gene encoding baculoviral IAP repeat-containing protein-4 (BIRC4), a potent caspase inhibitor, was packaged into a rAAV vector and delivered to rat eyes by unilateral, intravitreal injection.25 Ocular hypertension was then induced in the treated rat eyes to simulate the elevated intraocular pressure characteristic of glaucoma. Following a 12week exposure to increased intraocular pressure, the rat optic nerve axons pretreated with rAAV-BIRC4 were counted and compared to balanced salt solution-treated control groups. On average 50% of the optic nerve axons in rat eyes expressing the BIRC4 transgene had been protected, compared with the control glaucoma eyes. The greater promotion of retinal ganglion cell survival by the rAAVmediated transgene expression of the BIRC4 caspase inhibitor demonstrates the therapeutic potential that interrupting apoptosis of retinal ganglion cells has in the treatment of glaucoma. As a chronic optic neuropathy, however, glaucoma likely requires transgene expression for longer than even the yearlong expression that rAAV-mediated gene therapy can currently permit.25,2

rAAV-Mediated Gene Therapy for Retinal Degenerative Disorders

The retina is the ocular structure predominantly involved in generating vision, converting light into electrical impulses and transmitting these signals to the brain. Therefore, the most severe forms of visual impairment are generally attributed to disorders in which the retina is implicated. The hallmark of all retinal degenerative disorders is the progressive apoptotic loss of the rod and/or cone photoreceptor cells of the retina. Nearly all retinal degeneration is either inherited or gene-based, making gene replacement therapy for such ocular diseases a potentially viable treatment option. Ocular gene therapy approaches in the retina include gene replacement, gene silencing, and gene addition, all of which target defective genes encoding the expression of proteins vital to photoreceptor function.1,3 The timing of vector and/or gene delivery is very significant in rAAV-mediated gene therapy for retinal degenerative disorders, since retinal degeneration can vary from early and severe to late and progressive. The retinal dystrophies that progress the fastest and have an earlier onset are the most difficult to treat, while slowly progressing degeneration has a wider therapeutic window.4,3 Certain retinal degenerative disorders have been found to be more amenable to treatment than others, such as Leber’s congenital amaurosis, retinitis pigmentosa, and age-related macular degeneration.

Retinal degenerative disorders affect various regions of the retina, which spans nearly the entirety of the interior ocular circumference; therefore the retina can be divided into the peripheral retina and the central retina.

Inherited Degenerative Disorders

of the Peripheral Retina

Leber’s congenital amaurosis (LCA). Leber’s congenital amaurosis (LCA) is one of the most severe forms of an early-onset, inherited retinal degeneration and one of the most extensively studied disease models for retinal gene therapy. There are multiple forms of LCA that all share a common disease progression, featuring the onset of severe visual impairment from birth and a complete loss of vision by early adulthood (Smith et al., 2009). Mutations identified in at least 12 different loci, including those in the RDH12, RPGRIP, LRAT, and RPE65 genes, are currently thought to account for 50% of LCA cases. Mutations in RPE65, in particular, which cause a deficiency in production of the RPE65 enzyme, have provided the most successful example of gene therapy intervention in the treatment of an ocular disease. The RPE65 protein is localized in the retinal pigment epithelium and functions in visual cycle regulation by converting all trans-retinoids to 11-cis retinoids. RPE65 deficiency interrupts this process and causes rod photoreceptors to become dysfunctional, leading to photoreceptor degeneration. Animal models of LCA have greatly facilitated experimental intervention with rAAV-mediated gene replacement, such as the RPE65-deficient murine model.

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More significant, however, were the results of rAAV-mediated gene therapy in the larger, spontaneous RPE65-null model of LCA in the Swedish Briard dog, which demonstrated a persistent improvement in vision over an eight-year period following only a single administration of rAAV-vector. Rod photoreceptor function was restored following subretinal injection of either the rAAV2or rAAV4-vectored canine RPE65 gene.1,3

The promising results of pre-clinical research helped to launch LCA as the first ocular disease treated in gene therapy clinical trials. Since 2007, the safety and efficacy of rAAV-mediated gene therapy for treatment of the RPE65-deficient form of LCA has continued to be investigated in three separate Phase I clinical trials. In each of the three clinical trials, a subretinal injection of rAAV2/2-vectored RPE65 was administered to three RPE65-deficient LCA patients, who were between the ages of 17 and 26 years old and suffered from varying degrees of visual impairment due to LCA. Additionally, procedural differences between each of the three trials featured divergence in promoter types, either a RPE-specific RPE65 or chicken β actin (CBA) promoter, in surgical protocols and also different volumes of rAAV vector used for injection. Changes in vision due to rAAV-mediated treatment in LCA patients compared to control patients were also evaluated by a variety of measures including pupillometry, microperimetry, and Early Treatment Diabetic Retinopathy Study acuity testing.26,27,28 Despite these differences, all three Phase I trials revealed improvements in retinal sensitivity.

Althoughpreclinicalexperimentationsuggested the greater amenability of younger subjects to rAAV-mediated gene therapy, the success of the three independent Phase I trials has provided even stronger evidence of the need to investigate the specific therapeutic window for rAAV-mediated gene replacement in LCA patients.29,1,3

In a one-year follow up for the first LCA clinical trial to become a Phase 1/2 safety and efficacy trial, none of the patients involved experienced adverse effects, and one patient demonstrated an unexpected gain in visual function. To better understand this patient’s improved vision, investigators quantified the patient’s foveal fixation in response to a series of dim targets that were contrasted with a range of luminances. One eye was used as a control, while the other eye was treated by rAAV-mediated gene therapy. Results revealed that foveal fixation was similar in both eyes, however a shift in foveal fixation to the region of the retina occurred in the treated eye. The researchers surmise that additional clustering of cone photoreceptors at the superotemporal region of the retina formed a pseudo-fovea, which accounted for the increased cone function and improved vision. This latest finding has introduced yet another application of rAAV-mediated gene therapy in the treatment of congenital blindness.30

Retinitis pigmentosa (RP). Retinitis pigemtosa (RP) is one of the most prevalent inherited retinal diseases, affecting nearly 1 in 3000, and has a variety of inheritance

patterns including dominant, recessive, and X-linked.31 Initially, affected individuals present with night-blindness due to degeneration of their rod photoreceptor cells, which can lead to tunnel vision. Complete vision loss in an affected individual can arise once cone photoreceptor cells degenerate. Many of the genesencodingthenumerousproteinsinvolved in the phototransduction pathway are susceptible to mutation and lead to the defects in phototransduction in RP. For example, the X-linked form of RP, which accounts for 1520% of all RP cases, is caused by a defect in the retinitis pigmentosa GTPase regulator (RPGR) gene. The RPGR protein product is thought to regulate the protein distribution in theconnectingciliumbydirecting orrestricting protein transport to the photoreceptor outer segment. Interestingly, this pathway is also implicated in the form of LCA caused by a defect in the RPGRIP gene by mislocalizing the RPGR protein, which has been found to anchor to the connecting cilium.31,3

The rapid degeneration of rod photoreceptors in the autosomal recessive form of RP is caused by null mutations in several genes encoding proteins involved in both phototransduction and photoreceptor outer segment regulation. In the murine model of autosomally recessive retinal degeneration, PDE6Brd1 is an allele of the gene that encodes for the β subunit of the rod cGMP phosphodiesterase (βPDE), an ezyme vital to the phototransduction cascade. Meanwhile, another frequently used model for autosomal recessive RP is in mice containing a spontaneous mutation in the gene encoding the

MER protein tyrosine kinase (MERTK), which is localized in the retinal pigment epithelium and is required for the phagocytosis of photoreceptor debris. Many other spontaneous retinal degenerative animal models exist for recessively inherited RP, which have all contributed to the understanding of autosomally inherited RP progression.31,3

While there are many challenges to treating early onset, rapidly degenerating retinal dystrophies such as the X-linked and recessive forms of RP with gene replacement therapy, the mutational heterogeneity of autosomal dominantly inherited diseases provides the greatest challenge to implementing rAAV-mediated gene therapy, which has extensive potential as a treatment for monogenic diseases. Unlike the aforementioned primary gene defects for the X-linked and autosomal recessive forms of RP, no single causative gene has been identified for autosomal dominant RP. Rather, more than 200 mutations in the RHO gene, which encodes the photoreactive pigment absorbed by rod photoreceptor cells (rhodopsin, or RHO), have been found to account for the dominantly inherited form of RP. The complexity of treating dominantly inherited retinal disorders lies in the fact that several mutations in one gene can cause the same RP disease phenotype. Therefore, implementing a treatment that is independent of the mutation and that can still correct the resulting defect has been the focus of investigative efforts. Specifically, the recent successes in the implementation of gene replacement with gene suppression have renewed the possibilities for rAAV-mediated gene therapy in the treatment of RP.32 While

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gene knockout strategies have long been used forgenesuppression,thedevelopmentofRNAi technology has introduced the possibility for gene knockdown using shRNA and siRNA.

Thelimitedassessmentofretinalfunction afforded by earlier studies focusing on both mutation-specific and mutation-independent suppression of autosomal dominant RP in mouse models using either ribozymes or RNAi has driven the research efforts of Chadderton et al. The researchers first established suppression of RHO in transgenic mice carrying a wild-type human RHO transgene following rAAV2/5 delivery of the RHO-targeting shRNA, shQ1, by subretinal injection. Chadderton et al. then studied the effects of rAAV-shQ1- mediated RHO suppression in conjunction with endogenous RHO gene replacement in transgenic mice carrying a Pro347Ser mutant human RHO transgene. rAAV-shQ1 and the control vector rAAV-shNT were injected into one of each pair of RHO mutant eyes, and retinal structure and function were assessed by histology and eletroretinography (ERG), respectively, at 10 weeks. Significant improvements in both histology and ERG were noted, with the retinal structure of Pro347Ser eyes treated with rAAV-shQ1 showing substantially greater outer nuclear layer thickness than rAAV-shNT-treated eyes at both 5 weeks and 10 weeks post-injection. More importantly, average ERG values for rAAV-shQ1-injected Pro347Ser eyes were twofold greater than for rAAV-shNT-treated eyes at 10 weeks post-injection. The promising results of Chadderton et al. using the twocomponent strategy of gene suppression in

conjunction with gene replacement outlines the great potential of this approach for many other animal models of dominantly inherited disorders such as autosomal dominant RP.32

Mutations in genes that code for proteins in the connecting cilia of photoreceptor cells manifest as RP as well as systemically in syndromes such as Usher and Bardet-Biedl syndromes. These syndromes are also known as ciliopathies, since the resulting dysfunctional protein products of genetic mutations are localized in many, if not all, ciliated cells throughout the body. Consequently, in addition to RP, widespread syndromic defects result from ciliopathies. Individuals suffering from Bardet Biedl Syndrome can present with issues ranging from polydactyly and renal abnormalities to mental retardation and hypogenitalism.31 Usher syndrome is the most common of the RP syndromes; affected individuals present with symptoms that include deafblindness and vestibular dysfunction. The syndrome consists of three subtypes, USH1, USH2, and USH3, which are categorized by their clinical phenotypes and causative genes. Cilial cells are generally implicated in the USH1 form of Usher Syndrome; genes with USH1-associated mutations encode proteins important to the development and regulation of organs of the inner ear as well as to structural and functional integrity of the retina.

Defects in the MYO7A gene have been found to correlate with the USH1B form of Usher syndrome. Mutations in MYO7A are thought to account for the retinal degeneration associated with USH1B in humans, since MYO7A expression is localized in numerous

cell types including retinal pigment epithelial and photoreceptor cells. In the naturally occurring Myo7a-deficient shaker1 mouse model of USH1B, however, affected mice present solely with hearing loss. While retinal degeneration is absent despite the Myo7a-deficieny of shaker mice, defective trafficking of melanosomes in the retinal pigment epithelial cells is thought to be attributable to the MYO7A mutation. Usher syndrome resulting from a defective MYO7A gene seems most amenable to rAAV-mediated gene therapy when the novel rAAV2/5 vector is used, as it facilitates the efficient transfer of larger gene constructs.31,3

Inherited Retinal Degenerative

Disorders of the Central Retina

Within the central retina, which is responsible for central vision, resides the macula. Central to the macula is the fovea, the area of the retina with the greatest density of cone photoreceptor cells. The macula mainly functions in visual acuity and color vision, and therefore degeneration of the portion of retinal pigment epithelium that nourishes macular photoreceptors or the macular photoreceptors themselves significantly reduces central vision.

Retinoschisis. Retinoschisis is a retinal dystrophy in which the structure, and subsequently the function, of the retina are greatly compromised. The X-linked recessive form of retinoschisis, known as juvenile retinoschisis, leads to the degeneration of the central retina. Specifically, the retina separates into several layers, primarily at the fovea, and can potentially lead to a retinal detachment.