Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

ROP

ROP

ROP

ROP

ROP

ROP

CNV laser

Prph2Rd2/Rd2*

Rs1−/−

Rs1−/−

GC1−/−

Rpe65 −/−

rd12

Rpgrip−/−

was used to deliver PEDF under the control of the CMV promoter (Auricchio, Behling, et al., 2002). These were the first studies to show that intravitreal AAV conferred stable and effective expression of an antiangiogenic transgene.

VEGF has been demonstrated to be a key mediator of retinal neovascularization. VEGF isoforms bind two tyrosine kinase receptors expressed on the surface of endothelial cells, VEGF receptor 1 (FLT1) and VEGF receptor 2 (KDR) (De Vries et al., 1992; Gitay-Goren et al., 1992; Jakeman et al., 1992).

When activated by VEGF, these receptors stimulate endothelial cell proliferation and migration, vasopermeability, and vasculature organization and modeling. VEGF has been shown to be upregulated by hypoxia in various types of neovascular retinopathies (Adamis et al., 1994; Aiello et al., 1994), as well as in animal models of retinal ischemia (Miller et al., 1994; Pierce et al., 1995; Shima et al., 1996). Various anti-VEGF strategies have been developed that target either the expression of the protein or its interaction with its receptors. Antisense oligonucleotides, soluble VEGF receptors, receptor mimics, and small interfering RNAs targeting VEGF have all been shown to inhibit neovascularization in mouse models (Aiello et al., 1995; Robinson et al., 1996; Reich et al., 2003). However, the transient nature of all of these reagents may well hinder their therapeutic application.

In vitro studies have shown that VEGF peptides derived from exons 6 and 7 inhibit VEGF-mediated angiogenesis

(Soker et al., 1997; Jia et al., 2001). These peptides were found to block the interaction of VEGF with the KDR receptor and coreceptor, neuropilin-1 (NPN1), thereby inhibiting VEGF-induced mitogenesis and migration of endothelial cells ( Jia et al., 2001). Taking advantage of the antiangiogenic properties of these peptides by delivering both to the ROP mouse retina via an AAV serotype 2 vector under the control of a CBA promoter effectively inhibited retinal neovascularization (Deng et al., 2005). In this study, vector was administered to newborn pups prior to exposing them to the hyperoxic environment.

A related study sought to prevent ocular neovascularization by expressing a soluble version of the VEGF receptor 1, SFLT1, in the ROP mouse retina. AAV2 was used to deliver SFlt1 under the control of the CMV promoter to the intravitreal space of ROP mouse eyes (Bainbridge et al., 2002). In this case, vector was delivered after the mouse was exposed to the hyperoxic environment. Utilizing a secretable gene product expressed by retinal cells not normally responsible for production of SFLT1 allowed vector injection into the vitreous rather than the subretinal space, a much less challenging technique. Treatment resulted in a 50% reduction of retinal neovascularization, with efficacy quantified as previously described.

Another vascular retinopathy characterized by early-onset ocular neovascularization is Sorsby’s fundus dystrophy. Mutations in the gene for tissue inhibitor of matrix metallo- proteinase-3 (TIMP3) have been identified in Sorsby’s

patients (Weber et al., 1994). The antiangiogenic properties of TIMP3 in vitro (Anand-Apte et al., 1997) prompted AAV1-mediated delivery of this transgene, under the control of the CMV promoter, to the subretinal space of newborn pups prior to placing them in the hyperoxic environment. The number of neovascular retinal endothelial cell nuclei in eyes treated with AAV1-Timp3 was significantly less than in those injected with control vector (AAV1-GFP or AAV1- lacZ), proving that TIMP3 exerts angiostatic effects in vivo (Auricchio, Behling, et al., 2002).

The same study also addressed whether endostatin exhibited anti-angiogenic properties in the ROP mouse. Endostatin, encoded by a natural fragment of collagen XVIII (Col18a1), has been shown to inhibit ocular angiogenesis when secreted intravascularly from hepatocytes transduced by viral vectors (O’Reilly et al., 1997; Mori et al., 2001). Using the same protocol as in their TIMP3 experiment, it was shown that AAV1-mediated endostatin expression, driven by the CMV promoter, was also able to reduce angiogenesis in the ROP mouse (Auricchio, Behling, et al., 2002).

Another potent inhibitor of angiogenesis is angiostatin, a proteolytic fragment of plasminogen. Kringle domains 1 through 3 of angiostatin (K1K3), has been shown to have angiostatic effects in tumor bearing mice (O’Reilly et al., 1994, 1996; Wu et al., 1997). AAV2 was used to deliver K1K3 under the control of a CBA promoter to either the subretinal or intravitreal space of newborn mouse pups with contralateral eyes receiving only PBS (Raisler et al., 2002). In ROP mice, the number of inner retinal vascular endothelial cells treated intravitreally or subretinally with AAV2-CBA-K1K3 was again significantly less than in control eyes.

Therapy for choroidal neovascularization

The laser-induced choroidal neovascularization protocol was developed first in nonhuman primates (Ryan, 1982). Subsequently used in other mammalian species, including mouse (Miller et al., 1990), this model employs the use of a laser to rupture Bruch’s membrane, which leads to recruitment of inflammatory and neovascular mediators promoting the proliferation of choroidal vessels into the retina proper. This pathology mimics that seen in the late stages of wet AMD. The extent of CNV is measured in the mouse 2–4 weeks post laser treatment in choroidal flat mounts prepared from animals that have been perfused with a fluorescent dye (Raisler et al., 2002). In a study using the laser CNV mouse model to test the effects of the antiangiogenic protein PEDF, mice were given intravitreal or subretinal injections of AAV2 containing a CBA promoter driving expression of either PEDF or GFP as a control (Mori et al., 2002). At 4–6 weeks post injection, the Bruch’s membranes of these mice were

ruptured by laser photocoagulation at three sites per eye. After 14 days, the area of CNV at the laser hole was measured by imaging choroidal flat mounts of mice that had been perfused with fluorescein-labeled dextran. Mice treated with AAV-CBA-PEDF before laser treatment, either subretinally or intravitreally, showed significantly smaller mean areas of CNV than mice injected with control vector. Thus, the principle of AAV-vectored antiangiogenic gene therapy utilizing soluble factors has been well established in mouse models for both retinal and choroidal neovascularization.

Gene replacement therapy

Gene replacement strategies are primarily designed for autosomal recessive diseases in which a defective gene is complemented, in trans, with a wild-type counterpart that will produce a normal functional protein, thereby preventing the pathology. It is usually important to express the desired transgene in the cell type in which it is normally expressed, and autosomal recessive retinal diseases are no exception. A number of successful retinal gene replacement studies have been performed in mice using genes that all play significant roles in the structural integrity or function of the retina. Several of these proof-of-principle mouse studies hold great promise for human application.

Therapy for Structural Defects in Retinal Disease

The oldest and most studied mouse model for structural disorders of the retina is the retinal degeneration slow (rds) or peripherin-2 (Prph2Rd2/Rd2) mouse. The Prph2Rd2/Rd2 mouse is homozygous for a null mutation (large DNA insertion) in the Prph2 gene, encoding a photoreceptor-specific membrane glycoprotein (Travis et al., 1989) necessary for the stabilization of disc rims in photoreceptor outer segments. As a consequence of its absence, the Prph2Rd2/Rd2 mouse is incapable of developing normal photoreceptor discs and outer segments (Sanyal and Jansen, 1981; Molday 1994). In addition to loss of photoreceptor function, rhodopsin is downregulated, and photoreceptors eventually suffer apoptotic cell death (Reuter and Sanyal, 1984; Nir et al., 1990; Chang et al., 1993). In humans, mutations in PRPH2 cause autosomal dominant retinitis pigmentosa and macular dystrophy. As in the Prph2Rd2/Rd2 mouse, these human diseases are characterized by progressive photoreceptor cell death and loss of visual function (Gregory-Evans and Bhattacharya, 1998). AAV2 carrying a normal mouse Prph2 under the control of a bovine rhodopsin promoter was subretinally injected at P10 into Prph2Rd2/Rd2 mice and resulted in preservation of vestigial rhodopsin-containing outer segments and improved photoreceptor function (Ali et al., 2000). However, subsequent experiments noted that ultrastructural improvements in photoreceptors depended on the age at which animals were

treated (Sarra et al., 2001). Also, in all cases, there was no significant slowing of the rate of photoreceptor cell loss. Attempts at promoting photoreceptor cell survival in the Prph2Rd2/Rd2 mouse via AAV delivered neurotrophic factors are discussed later.

X-linked juvenile retinoschisis (XLRS) is caused by mutations in the gene for retinioschisin-1 (RS1) (Sauer et al., 1997) that encodes a protein secreted from photoreceptor and bipolar cells as a multimeric complex of identical subunits linked by disulfide bonds (Molday et al., 2001; Reid et al., 2003; Wu and Molday, 2003). Each subunit contains a discoidin domain that has been implicated in cell adhesion and cell-cell interactions (Baumgartner et al., 1998). Patients with XLRS have intraretinal macular voids that form in a spoke-wheel-like pattern, peripheral retinal schisis, and functional changes classically illustrated by a depressed rod ERG b-wave and a fairly normal a-wave. The net effect is a single electronegative ERG waveform. The murine orthologue of the human retinoschisis gene, when knocked out in the mouse (Rs1−/−), mimics some of the pathology seen in human patients, including disorganization of multiple retinal layers, duplication and mislocalization of ganglion cells, shortening of photoreceptor inner and outer segments, and mislocalization of photoreceptor nuclei (Weber et al., 2002). In one study, AAV2 was used to deliver RS1 under the control of a CMV promoter to the intravitreal space of 13-week-old Rs1−/− mice (Zeng et al., 2004). Contralateral eyes received an injection of PBS. Treatment resulted in retinoschisin protein distributed throughout the retina, as well as a reversal of the electronegative waveform through restoration of the normal, positive b-wave. However, treatment failed to improve retinal structure (Zeng et al., 2004). In another study, in order to target RS1 properly to photoreceptors, AAV5 containing human RS1 under the control of the mOP promoter was delivered to the subretinal space of P15 Rs1−/− mice. Treatment led to a normal distribution of retinoschisin and progressive and significant improvement in both retinal function (ERG) and retinal morphology (Min et al., 2005). Photoreceptors were preserved and ERG signals improved for at least 5 months post treatment (Min et al., 2005). Thus, it appears that replacement of either the mouse or human form of RS1 conferred varying levels of synaptic integrity, depending on what AAV serotype, promoter, and delivery route was used. Taken together, these results provide hope that this type of therapy may be useful for XLRS patients.

Therapy for Functional Defects in the Retina

Leber congenital amaurosis. Leber congenital amaurosis (LCA) is an early-onset form of congenital blindness. Thus far, 10 genes associated with LCA have been identified or mapped.

The first to be discovered, and therefore assigned to the LCA1 locus, was the retinal-specific guanylate cyclase gene (GUCY2D) (Perrault et al., 1996). Since its discovery nine others, including RPE65 and RP1GRIP1, have been identified (Marlhens et al., 1998; Perrault et al., 1999; Dryja et al., 2001; Gerber et al., 2001). All LCA patient pedigrees are consistent with an autosomal recessive inheritance pattern, yet each gene has proved to be involved in strikingly different physiological pathways.

A mouse model of LCA1 was created by knocking out guanylate cyclase-1 (GC1) (Yang et al., 1999). Rods in GC1−/− mice do not degenerate, whereas cone loss progresses from central to peripheral retina and peaks between 9 and 16 weeks of age, with very few cones remaining at 6 months of age (Coleman et al., 2004). Cone-mediated ERGs are barely detectable at 1 month of age, while rods exhibit ERG amplitudes 30%–50% of those in congenic control mice but decrease until they plateau at 5 months of age (Yang et al., 1999). Additionally, cone arrestin and cone α-transducin fail to properly translocate between photoreceptor outer and inner segments in response to light. Recent studies have shown that subretinal AAV5-mediated delivery of bovine GC1 under control of either mOP or CBA promoters to P21 GC1−/− mice resulted in restoration of correct lightactivated cone arrestin translocation (Haire et al., 2006). These results suggest that early therapeutic intervention may be required for the optimal treatment of LCA1.

A mouse model of LCA2, the Rpe65 knockout mouse, was generated in 1998 (Redmond et al., 1998). RPE65 is a 61-kd protein that is primarily expressed in the microsomal membrane fraction of the retinal pigmented epithelium (RPE) and is the isomerohydrolase responsible for converting all- trans-retinyl ester to 11-cis-retinol in the visual cycle (Mata et al, 2004; Moiseyev et al., 2005). Rod outer segment discs in the Rpe65−/− mouse are disorganized and rod ERGs abolished, while an apparent cone signal remains. However, this ERG signal is not a true cone response but rather a highly desensitized rod response that is being detected under standard cone assay conditions (Seeliger et al., 2001). Rpe65−/− mice lack rhodopsin but maintain opsin apoprotein, and, owing to their inability to isomerize all-trans-retinal ester to its cis-form, all-trans-retinyl esters accumulate in the RPE (Redmond et al., 1998). AAV1 was used for in utero delivery of human RPE65, driven by a CMV promoter, to Rpe65−/− mice (Dejneka et al., 2004). Gene transfer into E14 fetuses resulted in efficient transduction of the RPE, restoration of ERG signals, and measurable rhodopsin after P6. Some animals responded with nearly normal ERG amplitudes and levels of rhodopsin. A naturally occurring model of LCA2, the rd12 mouse, contains a nonsense mutation in the Rpe65 gene rendering the enzyme dysfunctional (Pang et al., 2005). As was noted in Rpe65−/− mice, rd12 mice have profoundly

diminished rod ERGs, an absence of 11-cis-retinaldehyde and rhodopsin, massive accumulation of retinyl esters in the RPE, and slow photoreceptor degeneration (Pang et al., 2005). Subretinal treatment of P14 mice with a serotype 5 vector containing human RPE65 cDNA, driven by the CBA promoter, resulted in RPE65 expression only in RPE cells, restoration of 11-cis-retinaldehyde, the presence of substantial levels of rhodopsin, and near normal rod and cone ERG amplitudes (Pang et al., 2006). Retinyl ester levels were also reduced to normal, and related to this, funduscopy and retinal morphology remained normal. All parameters of restored retinal health remained stable for at least 7 months. Perhaps most important, visually cued behavioral tests revealed that mice treated in only one eye performed as well as normal, wild-type mice. These results confirm that gene therapy can restore normal vision-dependent behavior in a congenitally blind animal. The promising results obtained in both LCA2 mouse models and the earlier Briard dog results (Acland et al., 2001), coupled with the preclinical safety studies done in rat, dog, and nonhuman primate, have laid the groundwork for upcoming clinical trials for LCA2, likely the first AAV human gene therapy trial in the eye ( Jacobson et al., 2006a, 2006b).

A third mouse model of LCA is the retinitis pigmentosa GTPase regulator interacting protein (Rpgrip) knockout mouse (Hong et al., 2000). RPGRIP is a protein located in the ciliary axoneme of rod and cone photoreceptors (Hong et al., 2000, 2003) that serves to tether retinitis pigmentosa GTPase regulator (RPGR) to this structure (Zhao et al., 2003). In turn, RPGR is proposed to play a role in regulating protein transport through the connecting cilium of photoreceptors to outer segments (Hong et al., 2000). Rpgrip−/− mice display photoreceptor degeneration beginning at P15, as well as early-onset loss of ERG signals. AAV2-mediated delivery of RPGRIP driven by a 236 bp (−218/+17) mOP promoter and delivered to the subretinal space of P18–P20 Rpgrip−/− mice resulted in RPGRIP localized normally to the connecting cilium, photoreceptor preservation, and ERG recovery (Pawlyk et al., 2005).

Lysosomal storage disease. Lysosomes are responsible for the breakdown of intracellular macromolecules and are characterized by the nature of the material stored within. Defects in the enzymes that degrade lysosomal contents can lead to inability to turn over lysosomal material, resulting in a broad spectrum of more than 40 clinical disorders, two of which are mucopolysaccharidosis type VII (MPS VII) and infantile neuronal ceroid lipofuscinosis (INCL).

MPS VII is a member of a group of diseases caused by deficiencies in enzymes responsible for the breakdown of glycosaminoglycans (GAGs). MPS VII is caused by a deficiency in the enzyme β-glucuronidase (Sly et al., 1973) and leads to progressive RD and vision impairment. MPS VII mice lack

β-glucuronidase activity due to a single base pair deletion in Gusb (Birkenmeier et al., 1989; Sands and Birkenmeier, 1993) and display progressive shortening of photoreceptor outer segments, apoptotic photoreceptor death, accumulation of lysosomal storage bodies in the RPE, and diminished ERG amplitudes (Birkenmeier et al., 1989; Lazarus et al., 1993; Ohlemiller et al., 2000; Birkenmeier et al., 1991; Stramm et al., 1990). AAV2 was used to deliver GUSB, driven by the CBA promoter, to the intravitreal space of 4-week- old MPS VII mice. At 16 weeks of age, vector-treated eyes had near normal levels of β-glucuronidase, preservation of cells in the outer nuclear layer, decreased RPE lysosomal storage, and significantly improved rod ERGs (Hennig et al., 2004). A similar vector administered systemically to newborn MPS VII mice prior to the initiation of disease resulted in almost complete ERG protection (Daly et al., 1999a, 1999b, 2001).

A second lysosomal storage disease having an ocular pathology is INCL, a neurodegenerative disorder described as the earliest onset form of Batten disease. INCL is caused by mutations in the palmitoyl protein thioesterase-1 gene (PPT1), a lysosomal hydrolase that is trafficked to the lysosome via the mannose-6-phosphate receptor (Vesa et al., 1995; Sleat et al., 1996). A Ppt1 knockout mouse model of INCL displays significant rod and cone ERG deficits as early as 2 months of age, as well as progressive RD relative to congenic controls (Gupta et al., 2001; Griffey et al., 2005). Intravitreal delivery of AAV2 carrying human PPT1 driven by the CMV promoter into P18–P21 PPT −/− mice resulted in increased PPT1 expression, significant improvements in rod and cone ERGs, and a slower rate of degeneration relative to that in untreated controls (Griffey et al., 2005). It is likely that use of an AAV5 vector or subretinal injection may increase the amount of PPT1 expression in photoreceptors and lead to improved histopathological protection.

Achromatopsia. Achromatopsia, also known as rod monochromatism, is an early-onset form of severe color blindness that results from defects in cone photoreceptors. A mouse model of recessive achromatopsia was found by ERG screening in the mouse strain collection at the Jackson Laboratory. The mutation, termed cpfl3, for cone photoreceptor function loss-3, was mapped to mouse chromosome 3, the same location as human GNAT2 (Chang et al., 2006). GNAT2 encodes the α subunit of cone transducin, the G protein involved in phototransduction, and, when mutated in the Cpfl3 mouse, renders the G protein nonfunctional. As a result, cone photoreceptors are unable to sustain the phototransduction cascade in response to light, leading to a very weak photopic cone response at 3 weeks of age that is lost by 9 weeks (Chang et al., 2006). Rod ERG responses are initially normal but also diminish somewhat with age. Cone transducin α immunolabeling was minimally detectable and

was accompanied by some photoreceptor outer segment disorganization. Subretinal injection of AAV5 containing Gnat2 cDNA driven by a cone-targeting PR2.1 promoter partially preserved retinal structure, completely restored photopic cone responses, and restored visual acuity to near normal (Alexander et al., 2007). These studies, which targeted transgene expression specifically to cones, hold promise for analogous cone therapy in other models of achromatopsia, as well as for other cone-targeted retinal diseases.

Neurotrophic factor gene therapy. Neurotrophic cell survival factors have shown promise in preserving photoreceptor cells from apoptosis, the common fate of photoreceptors in many RT diseases. In surveys of neurotrophic factors injected directly into the vitreous of various rodent models of photoreceptor degeneration, a number have been shown to promote photoreceptor survival (Faktorovich et al., 1990; LaVail et al., 1992, 1998). AAV-mediated delivery of cDNAs for such factors is an attractive option that would not require multiple injections and could supplement gene replacement therapies. AAV2-mediated delivery of ciliary neurotrophic factor (CNTF) has been evaluated in several mouse models of RD, including the rhodopsin knockout mouse (Rho−/−), the peripherin knockout (PrphRd2/Rd2) mouse, and a heterozygous Prph2 mouse with a transgenic copy of the P216L mutant Prph2 allele (rds+/−P216L) (Liang et al., 2001a, 2001b; Bok et al., 2002; Schlichenbrede et al., 2003; Buch et al., 2006). All studies showed that vectored CNTF expression promotes photoreceptor survival. Although not evaluated in the Rho−/− mouse (Liang et al., 2001a), in both Prph2Rd2/Rd2 and rds+/−P216L mice, photoreceptor function, as evaluated by ERG, was either not improved or reduced in vector injected eyes relative to untreated controls (Bok et al., 2002; Schlichenbrede et al., 2003; Buch et al., 2006). This treatment-related loss in photoreceptor function also appeared to be dose dependent and occurred in normal eyes as well (Bok et al., 2002; Buch et al., 2006). Additionally, in the Prph2Rd2/Rd2 mouse, when AAV-mediated CNTF expression was supplemented with either AAV2-Prph2 or AAV2-rhodopsin, there was still no improvement relative to untreated control eyes. Taken together, these results suggest that CNTF dosing may be crucial to avoid any negative effects of the neurotrophin (Schlichenbrede et al., 2003). Indeed, in a recent study, AAV-mediated CNTF expression was found to be deleterious to photoreceptor function in wild-type, C57BL6, mice (Rhee et al., 2007). What remains to be determined is the dosage of CNTF delivered in vivo by AAV vectors relative to that expected in a clinical trial, and to what extent toxicity seen in one species, the mouse, can be carried over into the human.

In contrast, AAV-mediated glial cell line–derived neurotrophic factor (GDNF) expression driven by the CBA promoter in the Prph2Rd2/Rd2 mouse slowed photoreceptor loss

and, more importantly, did not have the adverse effects on photoreceptor function that were seen with CNTF. In addition, when used in combination with the appropriate gene replacement therapy, enhanced photoreceptor function was observed (Buch et al., 2006).

Conclusions and future directions for adenoassociated virus gene therapy in the mouse retina

A large body of evidence supports the use of AAV as an effective vector for gene delivery to the mouse retina. Optimization of gene therapy strategies, as well as the development of new mouse models of human retinal disease, suggests that appropriately designed AAV-mediated gene therapy may eventually be applied broadly to human subjects. Current research is focusing on mouse models that are more “human-like” and therefore may address the species differences between man and mouse. One such study is the development of the neural retina leucine zipper knockout (Nrl −/−) mouse. As determined by structural and functional characteristics, the Nrl −/− mouse, in contrast to the normal roddominant mouse retina, contains an all cone-like retina (Daniele et al., 2005). This mouse may be a better rodent model of the cone-rich human macula, suggesting that subjecting the Nrl −/− mouse to ROP conditioning or laserinduced CNV, for example, may provide a better model for central field loss in neovascular disorders such as diabetic retinopathy and AMD. Additionally, crossing the Nrl −/− mouse with known cone degeneration models may aid in the development of more effective cone-targeted gene therapies.

Although this chapter has summarized gene replacement strategies in mouse models of RD that are predominantly inherited in an autosomal recessive fashion, progress has also been made toward addressing autosomal dominantly inherited RD. To date, most of this work has been carried out in the P23H rhodopsin rat, the primary current model for a common form of autosomal dominant retinitis pigmentosa (Dresner et al., 1998; Lewin et al., 1998; LaVail et al., 2000). A mouse model mimicking autosomal dominant macular dystrophy is the ELOVL4 mouse (Raz Praq et al., 2006; Vasireddy et al., 2006), which contains a heterozygous knock-in carrying the same 5 bp deletion in the ELOVL4 gene associated with dominant Stargardtlike macular degeneration (STGD3) in humans. The ELOVL4 mouse has a retinal phenotype similar to STGD3 patients (Vasireddy et al., 2006), and attempts are currently under way to mediate the mouse disease using AAV vectors.

Another recent advance in the field of AAV gene delivery is the development of self-complementary AAV vectors (scAAV), which package double-stranded DNA in the viral capsid, in contrast to single-stranded DNA in standard AAV vectors. This modification overcomes the rate-limiting step

for AAV-vectored transgene expression, the conversion of vector single-stranded DNA into double-stranded DNA by the host cell (McCarty et al., 2001, 2003). Studies have shown that scAAV mediates quicker and more efficient transgene expression than standard AAV vectors in mouse brain and retina (Fu et al., 2006; Hauswirth et al., 2006). scAAV-mediated gene therapy may therefore be of use in the treatment of disorders that require immediate, high-level intervention, particularly for some very rapidly degenerating mouse models of retinal disease.

In summary, based on a broad variety of successfully treated mouse models of retinal RD, the clinical future for AAV-vectored gene therapy seems bright.

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