Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow.

BACKGROUND: Pericytes and vascular smooth muscle cells, collectively known as mural cells, are recruited through PDGFB (platelet-derived growth factor B)-PDGFRB (platelet-derived growth factor receptor beta) signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Here, we characterize the role of the transcription factor SRF (serum response factor) in MCs and study its function in developmental and pathological contexts. METHODS: We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis using RNA-sequencing, immunohistology, in vivo live imaging, and in vitro techniques. RESULTS: By postnatal day 6, pericytes lacking SRF were morphologically abnormal and failed to properly comigrate with angiogenic sprouts. As a consequence, pericyte-deficient vessels at the retinal sprouting front became dilated and leaky. By postnatal day 12, also the vascular smooth muscle cells had lost SRF, which coincided with the formation of pathological arteriovenous shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF (myocardin-related transcription factor) cofactors. We further show that MRTF-SRF signaling promotes pathological pericyte activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging, and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. CONCLUSIONS: SRF is crucial for distinct functions in pericytes and vascular smooth muscle cells. SRF directs pericyte migration downstream of PDGFRB signaling and mediates pathological pericyte activation during ischemic retinopathy. In vascular smooth muscle cells, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of arteriovenous shunts. These essential roles in physiological and pathological contexts provide a rationale for novel therapeutic approaches through targeting SRF activity in MCs.

Mural Serum Response Factor (SRF) Deficiency Provides Insights into Retinal Vascular Functionality and Development.

Serum response factor (SRF) controls the expression of muscle contraction and motility genes in mural cells (MCs) of the vasculature. In the retina, MC-SRF is important for correct angiogenesis during development and the continuing maintenance of the vascular tone. The purpose of this study was to provide further insights into the effects of MC SRF deficiency on the vasculature and function of the mature retina in SrfiMCKO mice that carry a MC-specific deletion of Srf. Retinal morphology and vascular integrity were analyzed in vivo via scanning laser ophthalmoscopy (SLO), angiography, and optical coherence tomography (OCT). Retinal function was evaluated with full-field electroretinography (ERG). We found that retinal blood vessels of these mutants exhibited different degrees of morphological and functional alterations. With increasing severity, we found vascular bulging, the formation of arteriovenous (AV) anastomoses, and ultimately, a retinal detachment (RD). The associated irregular retinal blood pressure and flow distribution eventually induced hypoxia, indicated by a negative ERG waveform shape. Further, the high frequency of interocular differences in the phenotype of individual SrfiMCKO mice points to a secondary nature of these developments far downstream of the genetic defect and rather dependent on the local retinal context.

Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia.

Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.

Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype.

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.

CRB2 acts as a modifying factor of CRB1-related retinal dystrophies in mice.

Mutations in the CRB1 gene lead to retinal dystrophies ranging from Leber congenital amaurosis (LCA) to early-onset retinitis pigmentosa (RP), due to developmental defects or loss of adhesion between photoreceptors and Müller glia cells, respectively. Whereas over 150 mutations have been found, no clear genotype-phenotype correlation has been established. Mouse Crb1 knockout retinas show a mild phenotype limited to the inferior quadrant, whereas Crb2 knockout retinas display a severe degeneration throughout the retina mimicking the phenotype observed in RP patients associated with CRB1 mutations. Crb1Crb2 double mutant retinas have severe developmental defects similar to the phenotype observed in LCA patients associated with CRB1 mutations. Therefore, CRB2 is a candidate modifying gene of human CRB1-related retinal dystrophy. In this study, we studied the cellular localization of CRB1 and CRB2 in human retina and tested the influence of the Crb2 gene allele on Crb1-retinal dystrophies in mice. We found that in contrast to mice, in the human retina CRB1 protein was expressed at the subapical region in photoreceptors and Müller glia cells, and CRB2 only in Müller glia cells. Genetic ablation of one allele of Crb2 in heterozygote Crb1(+/-) retinas induced a mild retinal phenotype, but in homozygote Crb1 knockout mice lead to an early and severe phenotype limited to the entire inferior retina. Our data provide mechanistic insight for CRB1-related LCA and RP.

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.

Mosaic synaptopathy and functional defects in Cav1.4 heterozygous mice and human carriers of CSNB2.

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

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.

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.

Auditory event-related signals in mouse ERG recordings.

PURPOSE: In murine disease models, particularly in cases when retinal electrical activity is reduced, an event-related component becomes apparent that does not change with the stimulus intensity in electroretinogram (ERG) recordings. In this work, we show that this electric component is evoked by the sound of the flash discharge rather than the light flash itself. METHODS: Wild-type mice (C57BL/6), mice with rod function only (Cnga3 (-/-)), mice lacking any photoreceptor function (Cnga3 (-/-) rho (-/-)), and mice with no auditory function (Cdh23 (vAlb/vAlb) ) were examined with Xenon flash ERG systems. An acoustic noise generator was used to mask discharge sounds. RESULTS: ERG recording modalities were identified where usually no discernible response can be elicited. These include photopic conditions in Cnga3 (-/-) mice, photopic conditions together with very low stimulus intensities in C57BL/6 mice, and both scotopic and photopic conditions in Cnga3 (-/-) rho (-/-) mice. However, in all of these cases, small signals, featuring an initial a-wave like deflection at about 20 ms and a subsequent b-wave like deflection peaking at about 40 ms after the flash, were detected. In contrast, such signals could not be detected in deaf Cdh23 (vAlb/vAlb) mice. Furthermore, masking the Xenon discharge sound by continuous acoustic noise led to a loss of the event-related signals in a reversible manner. CONCLUSIONS: We could identify an auditory event-related component, presumably resembling auditory evoked potentials, as a major source of ERG signals of non-visual origin in mice. This finding may be of particular importance for the analysis and interpretation of ERG data in mice with reduced visual responses.

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.

BK channels mediate pathway-specific modulation of visual signals in the in vivo mouse retina.

The modulatory role of large-conductance Ca(2+)-activated K(+) (BK) channels in the nervous system has been extensively studied. In the retina, it has been shown that BK channels play a pivotal role in modulating feedback from A17 amacrine cells to rod bipolar cells (RBCs). Here, we used electroretinography to examine the functional role of BK channels for rod and cone vision in the retina in vivo using a genetically engineered mouse lacking functional BK channels (Bk(-/-)). Under dark-adapted and light-adapted conditions, the lack of BK channels had no effect on photoreceptor activity, suggesting that these ion channels do not modulate photoreceptor responses. At the bipolar cell level, the ERG signals attributed to RBCs in Bk(-/-) mice were not different from those in wild-type mice at low scotopic stimulus intensities. However, at high scotopic and low mesopic stimulus intensities, close to RBC saturation, a significant reduction of ERG signals reflecting RBC activity was present in the Bk(-/-) retina. At higher mesopic stimulus intensities activating both RBCs and cone bipolar cells (CBCs), no difference in ERG signals between Bk(-/-) and wild-type mice was found. In photopic stimulus paradigms, activity of ON- and OFF-CBCs in Bk(-/-) and wild-type retinae was indistinguishable. These findings demonstrate that BK channels modulate visual responses in vivo at the bipolar cell level at intermediate stimulus conditions.

MPP3 regulates levels of PALS1 and adhesion between photoreceptors and Müller cells.

MPP3 and CRB1 both interact directly with PALS1/MPP5 and through this scaffold protein may form a large protein complex. To investigate the role of MPP3 in the retina we have analyzed conditional mutant Mpp3 knockout mice. Ultrastructural localization studies revealed that MPP3 is predominantly localized in apical villi of Müller glia cells. Retinas lacking MPP3 developed late onset retinal degeneration, with sporadic foci of rosette formation in the central part of the retina. Retinal degeneration in Mpp3 cKO mice was accelerated by exposure to moderate levels of white light. Electroretinography recordings in aging mice under both scotopic and photopic conditions ranged from normal to mildly subnormal, while the magnitude correlated with the strength and extent of morphological alterations. Loss of MPP3 resulted in significant loss of PALS1 at the subapical region adjacent to adherens junctions, and loss of MPP3 in Pals1 conditional knockdown retinas significantly accelerated the onset of retinal degeneration. These data suggest that MPP3 is required for maintaining proper levels of PALS1 at the subapical region, and indicate that the MPP3 gene is a candidate modulator of the Crumbs complex.

Mice lacking Period 1 and Period 2 circadian clock genes exhibit blue cone photoreceptor defects.

Many aspects of retinal physiology are modulated by circadian clocks, but it is unclear whether clock malfunction impinges directly on photoreceptor survival, differentiation or function. Eyes from wild-type (WT) and Period1 (Per1) and Period2 (Per2) mutant mice (Per1(Brdm1) Per2(Brdm1) ) were examined for structural (histology, in vivo imaging), phenotypical (RNA expression, immunohistochemistry) and functional characteristics. Transcriptional levels of selected cone genes [red/green opsin (Opn1mw), blue cone opsin (Opn1sw) and cone arrestin (Arr3)] and one circadian clock gene (RORb) were quantified by real-time polymerase chain reaction. Although there were no changes in general retinal histology or visual responses (electroretinograms) between WT and Per1(Brdm1) Per2(Brdm1) mice, compared with age-matched controls, Per1(Brdm1) Per2(Brdm1) mice showed scattered retinal deformations by fundus inspection. Also, mRNA expression levels and immunostaining of blue cone opsin were significantly reduced in mutant mice. Especially, there was an alteration in the dorsal-ventral patterning of blue cones. Decreased blue cone opsin immunoreactivity was present by early postnatal stages, and remained throughout maturation. General photoreceptor differentiation was retarded in young mutant mice. In conclusion, deletion of both Per1 and Per2 clock genes leads to multiple discrete changes in retina, notably patchy tissue disorganization, reductions in cone opsin mRNA and protein levels, and altered distribution. These data represent the first direct link between Per1 and Per2 clock genes, and cone photoreceptor differentiation and function.

Alterations of the tunica vasculosa lentis in the rat model of retinopathy of prematurity.

PURPOSE: To study the relationship between retinal and tunica vasculosa lentis (TVL) disease in retinopathy of prematurity (ROP). Although the clinical hallmark of ROP is abnormal retinal blood vessels, the vessels of the anterior segment, including the TVL, are also altered. METHODS: ROP was induced in Long-Evans pigmented and Sprague Dawley albino rats; room-air-reared (RAR) rats served as controls. Then, fluorescein angiographic images of the TVL and retinal vessels were serially obtained with a scanning laser ophthalmoscope near the height of retinal vascular disease, ~20 days of age, and again at 30 and 64 days of age. Additionally, electroretinograms (ERGs) were obtained prior to the first imaging session. The TVL images were analyzed for percent coverage of the posterior lens. The tortuosity of the retinal arterioles was determined using Retinal Image multiScale Analysis (Gelman et al. in Invest Ophthalmol Vis Sci 46:4734-4738, 2005). RESULTS: In the youngest ROP rats, the TVL was dense, while in RAR rats, it was relatively sparse. By 30 days, the TVL in RAR rats had almost fully regressed, while in ROP rats, it was still pronounced. By the final test age, the TVL had completely regressed in both ROP and RAR rats. In parallel, the tortuous retinal arterioles in ROP rats resolved with increasing age. ERG components indicating postreceptoral dysfunction, the b-wave, and oscillatory potentials were attenuated in ROP rats. CONCLUSIONS: These findings underscore the retinal vascular abnormalities and, for the first time, show abnormal anterior segment vasculature in the rat model of ROP. There is delayed regression of the TVL in the rat model of ROP. This demonstrates that ROP is a disease of the whole eye.

Three-year results of phase I retinal gene therapy trial for CNGA3-mutated achromatopsia: results of a non randomised controlled trial.

AIMS: To determine long-term safety and efficacy outcomes of a subretinal gene therapy for CNGA3-associated achromatopsia. We present data from an open-label, nonrandomised controlled trial (NCT02610582). METHODS: Details of the study design have been previously described. Briefly, nine patients were treated in three escalating dose groups with subretinal AAV8.CNGA3 gene therapy between November 2015 and October 2016. After the first year, patients were seen on a yearly basis. Safety assessment constituted the primary endpoint. On a secondary level, multiple functional tests were carried out to determine efficacy of the therapy. RESULTS: No adverse or serious adverse events deemed related to the study drug occurred after year 1. Safety of the therapy, as the primary endpoint of this trial, can, therefore, be confirmed. The functional benefits that were noted in the treated eye at year 1 were persistent throughout the following visits at years 2 and 3. While functional improvement in the treated eye reached statistical significance for some secondary endpoints, for most endpoints, this was not the case when the treated eye was compared with the untreated fellow eye. CONCLUSION: The results demonstrate a very good safety profile of the therapy even at the highest dose administered. The small sample size limits the statistical power of efficacy analyses. However, trial results inform on the most promising design and endpoints for future clinical trials. Such trials have to determine whether treatment of younger patients results in greater functional gains by avoiding amblyopia as a potential limiting factor.

Candidate chemosensory cells in the stomach mucosa of young postnatal mice during the phases of dietary changes.

The complex physiology of the gastrointestinal (GI) tract must permanently be adjusted according to the composition of ingested food, which requires continuous monitoring by appropriate sensory systems. Sensing the dietary constituents is thought to be mediated by chemosensory cells residing in the mucosa of the GI tract. We have examined the appearance and differentiation of candidate chemosensory cells at distinct postnatal stages and visualized cells that express gustducin or TRPM5. Two critical stages have been considered: the suckling period when the neonates are nourished exclusively on milk and the weaning period when the diet gradually changes to solid food. At early postnatal stages, only a few gustducin- or TRPM5-expressing cells have been found; they display an immature morphology. At the time of weaning, numerous gustducin- or TRPM5-positive cells are present in the gastric mucosa and are isomorphic to adult candidate chemosensory cells. The typical accumulation of gustducin and TRPM5 cells at the border between the forestomach and corpus region and the characteristic tissue fold or "limiting ridge" have not been observed at early postnatal stages but are complete at the time of weaning. The appearance of candidate chemosensory cells at the strategic position occurs within the last few days before weaning but after the formation of the limiting ridge. Thus, both the topographic arrangement of the cells and the limiting ridge seem to be important features for the processing of solid food in the mouse stomach.

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.

Optimized Subretinal Injection Technique for Gene Therapy Approaches.

Gene therapy for inherited eye diseases requires local viral vector delivery by intraocular injection. Since large animal models are lacking 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 method described here aims to provide the details relevant to perform a transscleral pars plana virus-mediated gene transfer to achieve an optimized therapeutic effect in the small mouse eye.