Intraperitoneal chromophore injections delay early-onset and rapid retinal cone degeneration in a mouse model of Leber congenital amaurosis.

Highly expressed in the retinal pigment epithelium (RPE), the RPE-specific 65-kDa (RPE65) enzyme is indispensable to generate 11-cis-retinal (11cRAL), a chromophore for rhodopsin and cone photopigments. RPE65 deficiency can lead to Leber congenital amaurosis type 2 (LCA2), in which the isomerization of photobleached all-trans-retinal into photosensitive 11cRAL is blocked, ultimately causing severe retinal dysfunction and degeneration. The related mouse models, which are constructed through gene knockout or caused by spontaneous mutations, morphologically present with early-onset and rapid retinal cone cells degeneration, including loss of short-wavelength-sensitive cone opsins (S-opsins) and mislocalization of medium-wavelength-sensitive cone opsins (M-opsins). Studies have shown that routine Rpe65 gene replacement therapy, mediated by an adeno-associated virus (AAV) vector, can restore RPE65 protein. However, AAV transfection and Rpe65 transgene expression require at least one to two weeks, and the treatment cannot fully block the early-onset cone degeneration. To determine the feasibility of delaying cone degeneration before gene therapy, we investigated the impact of 11cRAL treatment in an early-age LCA2 retinal degeneration 12 (rd12) mouse model. Similar to human patients, the mouse model carries a spontaneous mutation in the Rpe65 gene, which results in disrupted endogenous 11cRAL regeneration. We found that RPE65 deficiency did not notably affect rodent retinal vessels. Under red light illumination, the rd12 mice were intraperitoneally injected with exogenous 11cRAL from postnatal day (P) 14 to P21. Three days after the last injection, a notable recovery of retinal function was observed using scotopic and photopic electroretinograms. Using optical coherence tomography and histological analyses of the deficient retinas, we found changes in the thickness of the photoreceptor outer segment (OS); this change could be rescued by early 11cRAL treatment. In addition, the treatment notably preserved M- and S-opsins, both of which maintained appropriate localization inside cone cells, as shown by the wild-type mice. In contrast, the age-matched untreated rd12 mice were characterized by retinal S-opsin loss and M-opsin mislocalization from the photoreceptor OS to the inner segment, outer nuclear layer, or outer plexiform layer. Notably, 11cRAL treatment could not maintain retinal function for a long time. Ten days after the last injection, the rod and M-cone electroretinograms significantly decreased, and S-cone responses almost extinguished. Our findings suggest that early 11cRAL treatment is useful for restoring retinal function and rescuing morphology in the rd12 mouse model, and the early-onset and rapid cone degeneration can be delayed before gene therapy.

Repurposing bortezomib for choroidal neovascularization treatment via antagonizing VEGF-A and PDGF-D mediated signaling.

Neovascular age-related macular degeneration (neoAMD) is the leading cause of blindness in AMD and manifests as choroidal neovascularization (CNV). Anti-vascular endothelial growth factor (VEGF) therapies are the mainstay treatments but with limited efficacy and cause detrimental effects on the retina after long-term application. These disadvantages warrant alternative strategy. Herein, we examined the effect on CNV by intravitreal injection of bortezomib, a reversible proteasome inhibitor, and further dissected the mechanism. Krypton red Laser was used to create CNV model in mice. The angiogenesis volume was assessed in choroidal flat-mount with isolectin GS-IB4 labeling and the leakage was examined with fluorescein fundus angiography. Injection of Borsub inhibited angiogenesis in the CNV model which was dose-dependent; the injection significantly inhibited leakage as well. Furthermore, Borsub injection reduced the contents of VEGF-A, macrophage chemotactic factor 1 (MCP-1), and platelet-derived growth factor (PDGF)-D but not PDGF-B, examined by enzyme-linked immunosorbent assay, in choroid/retinal pigment epithelium (RPE) tissue. These injections also reduced phospho-VEGFR-2 and phospho-PDGFRß in choroid/RPE tissue examined by immunoblotting. Moreover, Borsub inhibited the recruitment of mural cells or macrophages to laser-injured spots. Injection of Borsub indicated negative effect on scotopic and photopic responses recorded by electroretinogram. Altogether, intravitreal injection of Borsub significantly reduced CNV by antagonizing VEGF-A/Flk-1 and PDGF-D/PDGFRß pathways without impacting electroretinography parameters. Thus, Borsub may offer an invaluable therapy for the prevention and treatment of neoAMD.

Rodent retinal microcirculation and visual electrophysiology following simulated microgravity.

How the absence of gravity affects the physiology of human beings is generating global research interest as space exploration, including missions aboard the International Space Station, continues to push boundaries. Here, we examined changes in retinal microcirculation and visual electrophysiology in mice suspended by their tails to simulate the cephalad movement of blood that occurs under microgravity conditions. Tail suspension was performed with a head-down tilt with a recommended angle of 30°. Mice in the control groups were similarly attached to a tether but could maintain a normal position. Morphologically, the 15-day tail-suspended mice showed retinal microvascular dilation, tortuosity, and a relatively long fluorescence retention; however, the average diameter of the major retinal vessels was not notably changed. In addition, optical coherence tomography showed their optic nerve head had an increased diameter. However, the mice could adapt to the change, with microcirculation and the optic nerve head recovering following 30-day tail suspension. Expression of rhodopsin and cone-opsins was not notably changed, and no retinal apoptotic-positive cells were detected between 15- and 30-day tail suspensions. Moreover, the three experimental groups of suspended mice showed normal retinal layers and thickness. Functionally, following 15-day tail suspension, scotopic electroretinograms showed a decline in the oscillatory potentials (OPs), but not in the b wave; simultaneously, the peak time of flash visual evoked potential component N1 was delayed compared to its baseline and the time-matched control. Following 30-day tail suspension, the OPs (O2) amplitude recovered to approximately 97% of its baseline or 86% of the time-matched control level. By simulating cephalad shifting of blood, short-term tail suspension can affect rodent retinal microcirculation, the optic nerve head, and disturb visual electrophysiology. However, the change is reversible with no permanent injury observed in the retina. The mice could adapt to the short-term change of retinal microcirculation, indicating new conditions that could be combined with, or could enhance, simulated microgravity for further studying the impact of short- or long-term outer space conditions on the retina.

Photoreceptor degeneration in a new Cacna1f mutant mouse model.

The Cacna1f gene encodes the α1F subunit of an L-type voltage-gated calcium channel, Cav1.4. In photoreceptor synaptic terminals, Cav1.4 channels mediate glutamate release and postsynaptic responses associated with visual signal transmission. We have discovered a new Cacna1f mutation in nob9 mice, which display more severe phenotypes than do nob2 mice. To characterize the nob9 phenotype at different ages, we examined the murine fundus, applied retinal optical coherence tomography, measured flash electroretinograms (ERGs) in vivo, and analyzed the retinal histology in vitro. After identifying the X-linked recessive inheritance trait, we sequenced Cacna1f as the candidate gene. Mutations in this gene were detected by polymerase chain reaction (PCR) and confirmed by restriction fragment length polymorphism. Morphologically, an early-onset of retinal disorder was detected, and the degeneration of the outer plexiform layers progressed rapidly. Moreover, the mutant mice showed drastically reduced scotopic ERGs with increasing age. In 14-month-old nob9 retinas, immunostaining of cone opsins demonstrated a reduction in the number of short-wavelength opsins (S-opsins) to 54% of wild-type levels, and almost no middle-wavelength opsins (M-opsins) were observed. No cone ERGs could be detected from residual cones, in which S-opsins abnormally migrated to inner segments of the photoreceptors. The mutations of the Cacna1f gene in nob9 mice involved both a single nucleotide G to A transition and a 10-nucleotide insertion, the latter resulting in a frame-shift mutation in exon 14.

Long-term retinal cone rescue using a capsid mutant AAV8 vector in a mouse model of CNGA3-achromatopsia.

Adeno-associated virus (AAV) vectors are important gene delivery tools for the treatment of many recessively inherited retinal diseases. For example, a wild-type (WT) AAV5 vector can deliver a full-length Cnga3 (cyclic nucleotide-gated channel alpha-3) cDNA to target cells of the cone photoreceptor function loss 5 (cpfl5) mouse, a spontaneous animal model of achromatopsia with a Cnga3 mutation. Gene therapy restores cone-mediated function and blocks cone degeneration in the mice. However, since transgene expression delivered by an AAV vector shows relatively short-term effectiveness, this cannot be regarded as a very successful therapy. AAV2 and AAV8 vectors with capsid mutations have significantly enhanced transduction efficiency in retinas compared to WT AAV controls. In this study, AAV8 (Y447, 733F+T494V)-treated cpfl5 retinas showed greater preservation of short-term cone electroretinogram (ERG) responses than AAV8 (Y447, 733F)- or AAV2 (Y272, 444, 500, 730F+T491V)-mediated treatments. To explore the long-term rescue effect, AAV8 (Y447, 733F+T494V)-treated cpfl5 retinas were evaluated at 9 months following postnatal day 14 (P14) treatment. Rescued ERG responses in the cones of treated cpfl5 eyes decreased with increasing age, but still maintained more than 60% of the WT mouse responses at the oldest time point examined. Expression of CNGA3 and M/S-opsins was maintained in cone outer segments of the treated cpfl5 eyes and was equal to expression in age-matched WT retinas. Near-normal cone-mediated water maze behavior was observed in the treated cpfl5 mice. As these are the longest follow-up data reported thus far, AAV8 with capsid Y-F and T-V mutations may be one of the most effective AAV vectors for long-term treatment in a naturally occurring mouse model of CNGA3 achromatopsia.

The Degeneration and Apoptosis Patterns of Cone Photoreceptors in rd11 Mice.

The retinal degeneration 11 (rd11) mouse is a new animal model with rapid photoreceptor degeneration. The long-term efficacy of gene therapy has a direct relationship with the onset of photoreceptor degeneration or apoptosis, whereas the degeneration or apoptosis patterns of photoreceptors are still unclear in rd11 mice. The distribution patterns of cone function-related L- and S-opsin were examined by immunofluorescence staining, and the apoptosis was performed by TUNEL assay in rd11 mice. The expression pattern of L-opsin or S-opsin in rd11 retina at postnatal day (P) 14 was similar to the pattern observed in wildtype retina. With increasing age, the expression of L-opsin and S-opsin, especially S-opsin, decreased significantly in rd11 mice. The degeneration of L-opsin began around the optic nerve and expanded to the periphery of the retina, from the ventral/nasal to dorsal/temporal retina, whereas the expression of S-opsin gradually decreased from the dorsal/temporal to ventral/nasal retina. Apoptotic signal appeared at P14 and was strongest at P28 of rd11 mice. The key genes associated with apoptosis confirmed those changes. These indicated that the degeneration and apoptosis of cone photoreceptors began at P14 of rd11 mice, which was a key point for gene therapy.

Effects of Subretinal Gene Transfer at Different Time Points in a Mouse Model of Retinal Degeneration.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is necessary for photoreceptors to generate an important lipid component of their membranes. The absence of LPCAT1 results in early and rapid rod and cone degeneration. Retinal degeneration 11 (rd11) mice carry a mutation in the Lpcat1 gene, and are an excellent model of early-onset rapid retinal degeneration (RD). To date, no reports have documented gene therapy administration in the rd11 mouse model at different ages. In this study, the AAV8 (Y733F)-smCBA-Lpcat1 vector was subretinally injected at postnatal day (P) 10, 14, 18, or 22. Four months after injection, immunohistochemistry and analysis of retinal morphology showed that treatment at P10 rescued about 82% of the wild-type retinal thickness. However, the diffusion of the vector and the resulting rescue were limited to an area around the injection site that was only 31% of the total retinal area. Injection at P14 resulted in vector diffusion that covered approximately 84% of the retina, and we found that gene therapy was more effective against RD when exposure to light was limited before and after treatment. We observed long-term preservation of electroretinogram (ERG) responses, and preservation of retinal structure, indicating that early treatment followed by limited light exposure can improve gene therapy effectiveness for the eyes of rd11 mice. Importantly, delayed treatment still partially preserved M-cones, but not S-cones, and M-cones in the rd11 retina appeared to have a longer window of opportunity for effective preservation with gene therapy. These results provide important information regarding the effects of subretinal gene therapy in the mouse model of LPCAT1-deficiency.

Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina.

PURPOSE: To introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology. METHODS: After full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80-100% of the retina was filled with subretinally injected solution; in group 2, approximately 50-70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection. RESULTS: After a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50-70% or 80-100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80-100% coverage group. CONCLUSIONS: Trans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.

Histone Deacetylases Inhibitors in the Treatment of Retinal Degenerative Diseases: Overview and Perspectives.

Retinal degenerative diseases are one of the important refractory ophthalmic diseases, featured with apoptosis of photoreceptor cells. Histone acetylation and deacetylation can regulate chromosome assembly, gene transcription, and posttranslational modification, which are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The histone deacetylase inhibitors (HDACis) have the ability to cause hyperacetylation of histone and nonhistone proteins, resulting in a variety of effects on cell proliferation, differentiation, anti-inflammation, and anti-apoptosis. Several HDACis have been approved for clinical trials to treat cancer. Studies have shown that HDACis have neuroprotective effects in nervous system damage. In this paper, we will summarize the neuroprotective effects of common HDACis in retinal degenerative diseases and make a prospect to the applications of HDACis in the treatment of retinal degenerative diseases in the future.

Vitreal delivery of AAV vectored Cnga3 restores cone function in CNGA3-/-/Nrl-/- mice, an all-cone model of CNGA3 achromatopsia.

The CNGA3(-/-)/Nrl(-/-) mouse is a cone-dominant model with Cnga3 channel deficiency, which partially mimics the all cone foveal structure of human achromatopsia 2 with CNGA3 mutations. Although subretinal (SR) AAV vector administration can transfect retinal cells efficiently, the injection-induced retinal detachment can cause retinal damage, particularly when SR vector bleb includes the fovea. We therefore explored whether cone function-structure could be rescued in CNGA3(-/-)/Nrl(-/-) mice by intravitreal (IVit) delivery of tyrosine to phenylalanine (Y-F) capsid mutant AAV8. We find that AAV-mediated CNGA3 expression can restore cone function and rescue structure following IVit delivery of AAV8 (Y447, 733F) vector. Rescue was assessed by restoration of the cone-mediated electroretinogram (ERG), optomotor responses, and cone opsin immunohistochemistry. Demonstration of gene therapy in a cone-dominant mouse model by IVit delivery provides a potential alternative vector delivery mode for safely transducing foveal cones in achromatopsia patients and in other human retinal diseases affecting foveal function.

The frequency-response electroretinogram distinguishes cone and abnormal rod function in rd12 mice.

Early studies on Rpe65 knockout mice reported that remaining visual function was attributable to cone function. However, this finding has been challenged more and more as time has passed. Electroretinograms (ERGs) showed that rd12 mice, a spontaneous animal model of RPE65 Leber's congenital amaurosis, had sizeable photopic responses. Unfortunately, the recorded ERG waveform was difficult to interpret because of a remarkably delayed peak-time, which resembles a rod response more than a cone response. Here, we compare flicker ERGs in animals with normal rod and cone function (C57BL/6J mice), pure rod function (cpfl5 mice), and pure cone function (Rho(-/-) mice) under different adaptation levels and stimulus intensities. These responses were then compared with those obtained from rd12 mice. Our results showed that normal rods respond to low frequency flicker (5 and 15 Hz) and that normal cones respond to both low and high frequency flicker (5-35 Hz). As was seen in cpfl5 mice, rd12 mice had recordable responses to low frequency flicker (5 and 15Hz), but not to high frequency flicker (25 and 35 Hz). We hypothesize that abnormal rods may be the source of residual vision in rd12 mice, which is proved correct here with double mutant rd12mice. In this study, we show, for the first time, that frequency-response ERGs can effectively distinguish cone- and rod-driven responses in the rd12 mouse. It is another simple and valid method for evaluating the respective contributions of retinal rods and cones.

AAV-mediated lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene replacement therapy rescues retinal degeneration in rd11 mice.

PURPOSE: The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. The rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model. METHODS: The AAV8 (Y733F)-smCBA-Lpcat1 was delivered subretinally to postnatal day 14 (P14) rd11 mice in one eye only. At 10 weeks after injection, treated rd11 mice were examined by visually-guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT), and then killed for morphologic and biochemical examination. RESULTS: Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. The SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type LPCAT1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually-guided water maze behavioral performances were observed in treated rd11 mice. CONCLUSIONS: The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and, moreover, is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. To our knowledge, this is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation.

Review: the history and role of naturally occurring mouse models with Pde6b mutations.

Mouse models are useful tools for developing potential therapies for human inherited retinal diseases, such as retinitis pigmentosa (RP), since more strains are being identified with the same mutant genes and phenotypes as humans with corresponding retinal degenerative diseases. Mutations in the beta subunit of the human rod phosphodiesterase (PDE6B) gene are a common cause of autosomal recessive RP (arRP). This article focuses on two well-established naturally occurring mouse models of arRP caused by spontaneous mutations in Pde6b, their discovery, phenotype, mechanism of degeneration, strengths and limitations, and therapeutic approaches to restore vision and delay disease progression. Viral vector, especially adeno-associated viral vector (AAV) -mediated gene replacement therapy, pharmacological treatment, cell-based therapy and other approaches that extend the therapeutic window of treatment, is a potentially promising strategy for improving photoreceptor function and significantly slowing the process of retinal degeneration.

[Effect of voltage-dependent calcium channel α1F subunit deficiency on the structure and function of the murine retina].

OBJECTIVE: To investigate the distribution and biological roles of voltage-dependent calcium channel (VDCC) α1F subunit in murine retina. METHODS: Experimental study.α1F(-/-) (homozygous mutant) mice (n = 35) and α1F(+/+) (wild type) mice (n = 35) were used in this study. Immunohistochemistry was performed to determine the expression of VDCC α1F subunit in the mouse retina. Retinae in α1F(-/-) mice and age-matched control mice at 3, 6, 9, 14-day and 3-month after birth were paraffin embedded, sectioned and HE stained, and full-field electroretinogram (ERG) were also recorded at these time points.Statistics were based on independent samples t-test. RESULTS: (1) α1F subunit was absent in α1F(-/-) mice retina. But in α1F(+/+) mice retina, α1F subunit was expressed most strongly in the outer plexiform layer (OPL), less in the inner plexiform layer (IPL) and ganglion cell layer (GCL). (2) OPL thickness in the subunit deficient mice gradually reduced after birth and lost at adult age. (3) In dark-adapted ERGs,standard response showed that the b-wave amplitude of α1F(-/-) mice [(163.8 ± 26.7) µV] significantly decreased compared with that of α1F(+/+) mice [(408.4 ± 54.5) µV] (t = -9.017, P = 0.000), whereas the a-wave amplitude of α1F(-/-) group [(208.2 ± 27.3) µV] was similar to that of control group [(196.0 ± 24.2) µV] (t = 0.748, P = 0.476). CONCLUSION: This study demonstrates that the lack of VDCC α1F subunit affect the structure and function in the OPL of the murine retina.

[Progress on study of achromatopsia and targeted gene therapy].

Achromatopsia is an early onset retinal dystrophy that causes severe visual impairment. To date, four genes have been found to be implicated in achromatopsia-associated mutations: guanine nucleotide-binding protein (GNAT2), cyclic nucleotide-gated channel alpha-3 (CNGA3), cyclic nucleotide-gated channel beta-3 (CNGB3) and phosphodiesterase 6C (PDE6C). Even with early onset, the slow progress and the good responses to gene therapy in animal models render achromatopsia a very attractive candidate for human gene therapy after the successful of the Phase I clinical trials of Leber's congenital amaurosis. With the development of molecular genetics and the therapeutic gene replacement technology, the adeno-associated viral (AAV) vector-mediated gene therapy for achromatopsia in the preclinical animal experiments achieved encouraging progress in the past years. This article briefly reviews the recent research achievements of achromatopsia with gene therapy.

Differential proteomics and functional research following gene therapy in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.

AAV-mediated cone rescue in a naturally occurring mouse model of CNGA3-achromatopsia.

Achromatopsia is a rare autosomal recessive disorder which shows color blindness, severely impaired visual acuity, and extreme sensitivity to bright light. Mutations in the alpha subunits of the cone cyclic nucleotide-gated channels (CNGA3) are responsible for about 1/4 of achromatopsia in the U.S. and Europe. Here, we test whether gene replacement therapy using an AAV5 vector could restore cone-mediated function and arrest cone degeneration in the cpfl5 mouse, a naturally occurring mouse model of achromatopsia with a CNGA3 mutation. We show that gene therapy leads to significant rescue of cone-mediated ERGs, normal visual acuities and contrast sensitivities. Normal expression and outer segment localization of both M- and S-opsins were maintained in treated retinas. The therapeutic effect of treatment lasted for at least 5 months post-injection. This study is the first demonstration of substantial, relatively long-term restoration of cone-mediated light responsiveness and visual behavior in a naturally occurring mouse model of CNGA3 achromatopsia. The results provide the foundation for development of an AAV5-based gene therapy trial for human CNGA3 achromatopsia.

Gene therapy rescues cone structure and function in the 3-month-old rd12 mouse: a model for midcourse RPE65 leber congenital amaurosis.

PURPOSE: RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued. METHODS: Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin-specific antibodies. RESULTS: Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin-positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin-positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths. CONCLUSIONS: The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials.

Long-term retinal function and structure rescue using capsid mutant AAV8 vector in the rd10 mouse, a model of recessive retinitis pigmentosa.

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the ß subunit of rod cGMP-phosphodiesterase (PDEß). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEß was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEß expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEß-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEß-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.

Self-complementary AAV5 vector facilitates quicker transgene expression in photoreceptor and retinal pigment epithelial cells of normal mouse.

To clarify whether transduction efficiency and cell type specificity of self-complementary (sc) AAV5 vectors are similar to those of standard, single-stranded AAV5 vectors in normal retina, one micro liter of scAAV5-smCBA-GFP vector (1 x 10(12) genome-containing particles/ml) and AAV5-smCBA-GFP vector (1 x 10(12) genome-containing particles/ml) were subretinally or intravitreally (in both cases through the cornea) injected into the right and left eyes of adult C57BL/6J mice, respectively. On post-injection day (PID) 1, 2, 5, 7, 10, 14, 21, 28 and 35, eyes were enucleated; retinal pigment epithelium (RPE) wholemounts, neuroretinal wholemounts and eyecup sections were prepared to evaluate green fluorescent protein (GFP) expression by fluorescent microscopy. GFP expression following trans-cornea subretinal injection of scAAV5-smCBA-GFP vector was first detected in RPE wholemounts around PID 1 and in neuroretinal wholemounts between PID 2 and 5; GFP expression peaked and stabilized between PID 10-14 in RPE wholemounts and between P14 and P21 in neuroretinal wholemounts with strong, homogeneous green fluorescence covering the entire wholemounts. The frozen sections supported the following findings from the wholemounts: GFP expression appeared first in RPE around PID 1-2 and soon spread to photoreceptors (PR) cells; by PID 7, moderate GFP expression was found mainly in PR and RPE layers; between PID 14 and 21, strong and homogenous GFP expression was observed in RPE and PR cells. GFP expression following subretinal injection of AAV5-smCBA-GFP was first detected in RPE wholemounts around PID 5-7 and in neuroretinal wholemounts around PID 7-10; ssAAV5-mediated GFP expression peaked at PID 21 in RPE wholemounts and around PID 28 in neuroretinal wholemounts; sections from AAV5 treated eyes also supported findings obtained from wholemounts: GFP expression was first detected in RPE and then spread to the PR cells. Peak GFP expression in RPE mediated by scAAV5 was similar to that mediated by AAV5. However, peak GFP expression mediated by scAAV5 in PR cells was stronger than that mediated by AAV5. No GFP fluorescence was detected in any retinal cells (RPE wholemounts, neuroretinal wholemounts and retinal sections) after trans-cornea intravitreal delivery of either scAAV5-GFP or AAV5-GFP. Neither scAAV5 nor AAV5 can transduce retinal cells following trans-cornea intravitreal injection. The scAAV5 vector used in this study directs an earlier onset of transgene expression than the matched AAV5 vector, and has stronger transgene expression in PR cells following subretinal injection. Our data confirm the previous reports that scAAV vectors have an earlier onset than the standard, single strand AAV vectors (Natkunarajah et al., 2008; Yokoi et al., 2007). scAAV5 vectors may be more useful than standard, single-stranded AAV vector when addressing certain RPE and/or PR cell-related models of retinal dystrophy, particularly for mouse models of human retinitis pigmentosa that require rapid and robust transgene expression to prevent early degeneration in PR cells.