Safety and Vision Outcomes of Subretinal Gene Therapy Targeting Cone Photoreceptors in Achromatopsia: A Nonrandomized Controlled Trial.

Importance: Achromatopsia linked to variations in the CNGA3 gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available. Objective: To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encoding CNGA3 (AAV8.CNGA3) in patients with CNGA3-linked achromatopsia. Design, Setting, and Participants: This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants in CNGA3 were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018. Intervention: Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011 total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017). Main Outcomes and Measures: Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment. Results: Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor-mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13; P = .006, 2-sided t test paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log; P = .003, 2-sided t test paired samples). Conclusions and Relevance: Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains. Trial Registration: ClinicalTrials.gov Identifier: NCT02610582.

Cystoid edema, neovascularization and inflammatory processes in the murine Norrin-deficient retina.

Mutations in the Norrin (NDP) gene cause severe developmental blood vessel defects in the retina leading to congenital blindness. In the retina of Ndph-knockout mice only the superficial capillary network develops. Here, a detailed characterization of this mouse model at late stages of the disease using in vivo retinal imaging revealed cystoid structures that closely resemble the ovoid cysts in the inner nuclear layer of the human retina with cystoid macular edema (CME). In human CME an involvement of Müller glia cells is hypothesized. In Ndph-knockout retinae we could demonstrate that activated Müller cells were located around and within these cystoid spaces. In addition, we observed extensive activation of retinal microglia and development of neovascularization. Furthermore, ex vivo analyses detected extravasation of monocytic cells suggesting a breakdown of the blood retina barrier. Thus, we could demonstrate that also in the developmental retinal vascular pathology present in the Ndph-knockout mouse inflammatory processes are active and may contribute to further retinal degeneration. This observation delivers a new perspective for curative treatments of retinal vasculopathies. Modulation of inflammatory responses might reduce the symptoms and improve visual acuity in these diseases.

Gene Therapy Successfully Delays Degeneration in a Mouse Model of PDE6A-Linked Retinitis Pigmentosa (RP43).

Retinitis pigmentosa type 43 (RP43) is a blinding disease caused by mutations in the gene for rod phosphodiesterase 6 alpha (PDE6A). The disease process begins with a dysfunction of rod photoreceptors, subsequently followed by a currently untreatable progressive degeneration of the entire outer retina. Aiming at a curative approach via PDE6A gene supplementation, a novel adeno-associated viral (AAV) vector was developed for expression of the human PDE6A cDNA under control of the human rhodopsin promotor (rAAV8.PDE6A). This study assessed the therapeutic efficacy of rAAV8.PDE6A in the Pde6anmf363/nmf363-mutant mouse model of RP43. All mice included in this study were treated with sub-retinal injections of the vector at 2 weeks after birth. The therapeutic effect was monitored at 1 month and 6 months post injection. Biological function of the transgene was assessed in vivo by means of electroretinography. The degree of morphological rescue was investigated both in vivo using optical coherence tomography and ex vivo by immunohistological staining. It was found that the novel rAAV8.PDE6A vector resulted in a stable and efficient expression of PDE6A protein in rod photoreceptors of Pde6anmf363/nmf363 mice following treatment at both the short- and long-term time points. The treatment led to a substantial morphological preservation of outer nuclear layer thickness, rod outer segment structure, and prolonged survival of cone photoreceptors for at least 6 months. Additionally, the ERG analysis confirmed a restoration of retinal function in a group of treated mice. Taken together, this study provides successful proof-of-concept for the cross-species efficacy of the rAAV8.PDE6A vector developed for use in human patients. Importantly, the data show stable expression and rescue effects for a prolonged period of time, raising hope for future translational studies based on this approach.

AAV-Mediated Gene Supplementation Therapy in Achromatopsia Type 2: Preclinical Data on Therapeutic Time Window and Long-Term Effects.

Achromatopsia type 2 (ACHM2) is a severe, inherited eye disease caused by mutations in the CNGA3 gene encoding the α subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Patients suffer from strongly impaired daylight vision, photophobia, nystagmus, and lack of color discrimination. We have previously shown in the Cnga3 knockout (KO) mouse model of ACHM2 that gene supplementation therapy is effective in rescuing cone function and morphology and delaying cone degeneration. In our preclinical approach, we use recombinant adeno-associated virus (AAV) vector-mediated gene transfer to express the murine Cnga3 gene under control of the mouse blue opsin promoter. Here, we provide novel data on the efficiency and permanence of such gene supplementation therapy in Cnga3 KO mice. Specifically, we compare the influence of two different AAV vector capsids, AAV2/5 (Y719F) and AAV2/8 (Y733F), on restoration of cone function, and assess the effect of age at time of treatment on the long-term outcome. The evaluation included in vivo analysis of retinal function using electroretinography (ERG) and immunohistochemical analysis of vector-driven Cnga3 transgene expression. We found that both vector capsid serotypes led to a comparable rescue of cone function over the observation period between 4 weeks and 3 months post treatment. In addition, a clear therapeutic effect was present in mice treated at 2 weeks of age as well as in mice treated at 3 months of age at the first assessment at 4 weeks after treatment. Importantly, the effect extended in both cases over the entire observation period of 12 months post treatment. However, the average ERG amplitude levels differed between the two groups, suggesting a role of the absolute age, or possibly, the associated state of the degeneration, on the achievable outcome. In summary, we found that the therapeutic time window of opportunity for AAV-mediated Cnga3 gene supplementation therapy in the Cnga3 KO mouse model extends at least to an age of 3 months, but is presumably limited by the condition, number and topographical distribution of remaining cones at the time of treatment. No impact of the choice of capsid on the therapeutic success was detected.

VEGF Mediates ApoE4-Induced Neovascularization and Synaptic Pathology in the Choroid and Retina.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. Recent findings suggest that retina of apoE4 mice have synaptic and functional impairments. We presently investigated the effects of apoE4 on retinal and choroidal vasculature and the possible role of VEGF in these effects. There were no histological differences between the retinal and choroidal vasculatures of naïve apoE3 and apoE4 mice. In contrast, laserdriven choroidal injury induced higher levels of choroidal neovascularization (CNV) in apoE4 than in apoE3 mice. These effects were associated with an inflammatory response and with activation of the Muller cells and asrocytic markers gluthatione synthetase and GFAP, all of which were more pronounced in the apoE4 mice. CNV also induced a transient increase in the levels of the synaptic markers synaptophysin and PSD95 which were however similar in the apoE4 and apoE3 naive mice. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury.

Elk3 deficiency causes transient impairment in post-natal retinal vascular development and formation of tortuous arteries in adult murine retinae.

Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(-/-) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a severe retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. Here, we used CNGB1-deficient (CNGB1(-/-)) mice, a mouse model for autosomal recessive RP, to evaluate the efficacy of adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of RP. The treatment restored normal expression of rod CNG channels and rod-driven light responses in the CNGB1(-/-) retina. This led to a substantial delay of retinal degeneration and long-term preservation of retinal morphology. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this study holds promise for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.

Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa.

In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.

Targeted ablation of CRB1 and CRB2 in retinal progenitor cells mimics Leber congenital amaurosis.

Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.

Myosin7a deficiency results in reduced retinal activity which is improved by gene therapy.

Mutations in MYO7A cause autosomal recessive Usher syndrome type IB (USH1B), one of the most frequent conditions that combine severe congenital hearing impairment and retinitis pigmentosa. A promising therapeutic strategy for retinitis pigmentosa is gene therapy, however its pre-clinical development is limited by the mild retinal phenotype of the shaker1 (sh1(-/-)) murine model of USH1B which lacks both retinal functional abnormalities and degeneration. Here we report a significant, early-onset delay of sh1(-/-) photoreceptor ability to recover from light desensitization as well as a progressive reduction of both b-wave electroretinogram amplitude and light sensitivity, in the absence of significant loss of photoreceptors up to 12 months of age. We additionally show that subretinal delivery to the sh1(-/-) retina of AAV vectors encoding the large MYO7A protein results in significant improvement of sh1(-/-) photoreceptor and retinal pigment epithelium ultrastructural anomalies which is associated with improvement of recovery from light desensitization. These findings provide new tools to evaluate the efficacy of experimental therapies for USH1B. In addition, although AAV vectors expressing large genes might have limited clinical applications due to their genome heterogeneity, our data show that AAV-mediated MYO7A gene transfer to the sh1(-/-) retina is effective.

Optimized technique for subretinal injections in mice.

Subretinal injections in mice become increasingly important. Currently, the most prominent application is in gene therapy of inherited eye diseases by means of viral vector delivery to photoreceptors or the retinal pigment epithelium (RPE). Since there are no large animal models for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The protocol described here concerns a transscleral pars plana subretinal injection in small eyes, and may be used for but not limited to virus-mediated gene transfer.

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.