Expression of red/green-cone opsin mutants K82E, P187S, M273K result in unique pathobiological perturbations to cone structure and function.

Long-and middle-wavelength cone photoreceptors, which are responsible for our visual acuity and color vision, comprise ~95% of our total cone population and are concentrated in the fovea of our retina. Previously, we characterized the disease mechanisms of the L/M-cone opsin missense mutations N94K, W177R, P307L, R330Q and G338E, all of which are associated with congenital blue cone monochromacy (BCM) or color-vision deficiency. Here, we used a similar viral vector-based gene delivery approach in M-opsin knockout mice to investigate the pathogenic consequences of the BCM or color-vision deficient associated L-cone opsin (OPN1LW) mutants K82E, P187S, and M273K. We investigated their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. K82E mutants were detected predominately in cone outer segments, and its expression partially restored expression and correct localization of cone PDE6α' and cone transducin γ. As a result, K82E also demonstrated the ability to mediate cone light responses. In contrast, expression of P187S was minimally detected by either western blot or by immunohistochemistry, probably due to efficient degradation of the mutant protein. M273K cone opsin appeared to be misfolded as it was primarily localized to the cone inner segment and endoplasmic reticulum. Additionally, M273K did not restore the expression of cone PDE6α' and cone transducin γ in dorsal cone OS, presumably by its inability to bind 11-cis retinal. Consistent with the observed expression pattern, P187S and M273K cone opsin mutants were unable to mediate light responses. Moreover, expression of K82E, P187S, and M273K mutants reduced cone viability. Due to the distinct expression patterns and phenotypic differences of these mutants observed in vivo, we suggest that the pathobiological mechanisms of these mutants are distinct.

Structural and functional rescue of cones carrying the most common cone opsin C203R missense mutation.

An arginine to cysteine substitution at amino acid position 203 (C203R) is the most common missense mutation in human cone opsin. Linked to color blindness and blue cone monochromacy (BCM), C203 is involved in a crucial disulfide bond required for proper folding. It has previously been postulated that expression of mutant C203R cone opsin exerts a toxic effect on cone photoreceptors, similar to some well-characterized missense mutations in rhodopsin that lead to protein misfolding. In this study, we generated and characterized a BCM mouse model carrying the equivalent C203R mutation (Opn1mwC198R Opn1sw-/-) to investigate the disease mechanism and develop a gene therapy approach for this disorder. Untreated Opn1mwC198R Opn1sw-/- cones phenocopied affected cones in human patients with the equivalent mutation, exhibiting shortened or absent cone outer segments and loss of function. We determined that gene augmentation targeting cones specifically yielded robust rescue of cone function and structure when Opn1mwC198R Opn1sw-/- mice were treated at early ages. Importantly, treated cones displayed elaborated outer segments and replenished expression of crucial cone phototransduction proteins. Interestingly, we were unable to detect OPN1MWC198R mutant opsin at any age. We believe this is the first proof-of-concept study exploring the efficacy of gene therapy in BCM associated with a C203R mutation.

A clinically viable approach to restoring visual function using optogenetic gene therapy.

Optogenetic gene therapies offer a promising strategy for restoring vision to patients with retinal degenerative diseases, such as retinitis pigmentosa (RP). Several clinical trials have begun in this area using different vectors and optogenetic proteins (Clinical Identifiers: NCT02556736, NCT03326336, NCT04945772, and NCT04278131). Here we present preclinical efficacy and safety data for the NCT04278131 trial, which uses an AAV2 vector and Chronos as the optogenetic protein. Efficacy was assessed in mice in a dose-dependent manner using electroretinograms (ERGs). Safety was assessed in rats, nonhuman primates, and mice, using several tests, including immunohistochemical analyses and cell counts (rats), electroretinograms (nonhuman primates), and ocular toxicology assays (mice). The results showed that Chronos-expressing vectors were efficacious over a broad range of vector doses and stimulating light intensities, and were well tolerated: no test article-related findings were observed in the anatomical and electrophysiological assays performed.

Blue cone monochromacy and gene therapy.

Blue cone monochromacy (BCM) is a congenital vision disorder characterized by complete loss or severely reduced long- and middle-wavelength cone function, caused by mutations in the OPN1LW/OPN1MW gene cluster on the X-chromosome. BCM patients typically suffer from poor visual acuity, severely impaired color discrimination, myopia, and nystagmus. In this review, we cover the genetic causes of BCM, clinical features of BCM patients, genetic testing, and clinical outcome measurements for future BCM clinical trials. However, our emphasis is on detailing the animal models for BCM and gene therapy using adeno-associated vectors (AAV). We describe two mouse models resembling the two most common causes of BCM, current progress in proof-of-concept studies to treat BCM with deletion mutations, the challenges we face, and future directions.

Gene Therapy in Opn1mw-/-/Opn1sw-/- Mice and Implications for Blue Cone Monochromacy Patients with Deletion Mutations.

Blue cone monochromacy (BCM) is a congenital vision disorder affecting both middle-wavelength (M) and long-wavelength (L) cone photoreceptors of the human retina. BCM results from abolished expression of green and red light-sensitive visual pigments expressed in M- and L-cones, respectively. Previously, we showed that gene augmentation therapy to deliver either human L- or M-opsin rescues dorsal M-opsin dominant cone photoreceptors structurally and functionally in treated M-opsin knockout (Opn1mw-/-) mice. Although Opn1mw-/- mice represent a disease model for BCM patients with deletion mutations, at the cellular level, dorsal cones of Opn1mw-/- mice still express low levels of S-opsin, which are different from L- and M-cones of BCM patients carrying a congenital opsin deletion. To determine whether BCM cones lacking complete opsin expression from birth would benefit from AAV-mediated gene therapy, we evaluated the outcome of gene therapy, and determined the therapeutic window and longevity of rescue in a mouse model lacking both M- and S-opsin (Opn1mw-/-/Opn1sw-/-). Our data show that cones of Opn1mw-/-/Opn1sw-/- mice are viable at younger ages but undergo rapid degeneration. AAV-mediated expression of human L-opsin promoted cone outer segment regeneration and rescued cone-mediated function when mice were injected subretinally at 2 months of age or younger. Cone-mediated function and visually guided behavior were maintained for at least 8 months post-treatment. However, when mice were treated at 5 and 7 months of age, the chance and effectiveness of rescue was significantly reduced, although cones were still present in the retina. Crossing Opn1mw-/-/Opn1sw-/- mice with proteasomal activity reporter mice (UbG76V-GFP) did not reveal GFP accumulation in Opn1mw-/-/Opn1sw-/- cones eliminating impaired degradation of ubiquitinated proteins as stress factor contributing to cone loss. Our results demonstrate that AAV-mediated gene augmentation therapy can rescue cone structure and function in a mouse model with a congenital opsin deletion, but also emphasize the importance that early intervention is crucial for successful therapy.

Disease mechanisms of X-linked cone dystrophy caused by missense mutations in the red and green cone opsins.

Cone photoreceptors are responsible for the visual acuity and color vision of the human eye. Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q, and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in vitro, and the reported impairments were not always consistent. Here we expressed these mutants via AAV specifically in vivo in M-opsin knockout mouse cones to investigate their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. We show that these mutations alter the M-opsin structure, function, and localization. N94K and W177R mutants appeared to be misfolded since they localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q, and G338E mutants were detected predominately in cone outer segments. Expression of R330Q and G338E, but not P307L opsins, also partially restored expression and correct localization of cone PDE6α' and cone transducin γ and resulted in partial rescue of M-cone-mediated light responses. Expression of W177R and P307L mutants significantly reduced cone viability, whereas N94K, R330Q, and G338E were only modestly toxic. We propose that although the underlying biochemical and cellular defects caused by these mutants are distinct, they all seem to exhibit a dominant phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations. The understanding of the molecular mechanisms associated with these cone opsin mutants is fundamental to developing targeted therapies for cone dystrophy/dysfunction.

Incidence and risk factors of postoperative ileus after hysterectomy for benign indications.

PURPOSE: Postoperative ileus (POI) after abdominal surgery is associated with prolonged hospital stay and increased costs. The aim of this study is to investigate the incidence of, risk factors for, and outcomes associated with POI in patients undergoing hysterectomy for benign indications. METHODS: A retrospective review of 1017 consecutive patients undergoing benign hysterectomy over the period 2012-2017 in a single center was performed. POI was predefined as absence of flatus and defecation for more than 2 days with the presence of one or more of the following symptoms: nausea, vomiting, and abdominal distention. The association between perioperative variables and the risk of POI was evaluated by univariate analysis. Independent risk factors were identified by multivariate logistic regression analysis. RESULTS: Overall incidence of POI was 9.2%. Incidence of POI did not differ significantly among three different surgical approaches (abdominal hysterectomy, 10.6%; laparoscopic hysterectomy, 7.8%; vaginal hysterectomy, 11.3%; P = 0.279). Independent risk factors of POI identified by multivariate analysis included anesthesia technique (odds ratio [OR] 2.662, 95% interval [CI] 1.533-4.622, P = 0.001), adhesiolysis (odds ratio [OR] 1.818, 95% interval [CI] 1.533-4.622, P = 0.011), duration of operation (odds ratio [OR] 1.005, 95% interval [CI] 0.942-6.190, P = 0.029), previous cancer (odds ratio [OR] 4.789, 95% interval [CI] 1.232-18.626, P = 0.024), and dysmenorrhea (odds ratio [OR] 1.859, 95% interval [CI] 1.182-2.925, P = 0.007). CONCLUSION: POI is a common complication after hysterectomy. This study identified risk factors of POI specifically for gynecologic patients. Patients exposed to these factors should be monitored closely for the development POI.

Rescue of M-cone Function in Aged Opn1mw-/- Mice, a Model for Late-Stage Blue Cone Monochromacy.

Purpose: Previously we showed that AAV5-mediated expression of either human M- or L-opsin promoted regrowth of cone outer segments and rescued M-cone function in the treated M-opsin knockout (Opn1mw-/-) dorsal retina. In this study, we determined cone viability and window of treatability in aged Opn1mw-/- mice. Methods: Cone viability was assessed with antibody against cone arrestin and peanut agglutinin (PNA) staining. The rate of cone degeneration in Opn1mw-/- mice was quantified by PNA staining. AAV5 vector expressing human L-opsin was injected subretinally into one eye of Opn1mw-/- mice at 1, 7, and 15 months old, while the contralateral eyes served as controls. M-cone-mediated retinal function was analyzed 2 and 13 months postinjection by full-field ERG. L-opsin transgene expression and cone outer segment structure were examined by immunohistochemistry. Results: We showed that dorsal M-opsin dominant cones exhibit outer segment degeneration at an early age in Opn1mw-/- mice, whereas ventral S-opsin dominant cones were normal. The remaining M-opsin dominant cones remained viable for at least 15 months, albeit having shortened or no outer segments. We also showed that AAV5-mediated expression of human L-opsin was still able to rescue function and outer segment structure in the remaining M-opsin dominant cones when treatment was initiated at 15 months of age. Conclusions: Our results showing that the remaining M-opsin dominant cones in aged Opn1mw-/- mice can still be rescued by gene therapy is helpful for establishing the window of treatability in future blue cone monochromacy clinical trials.

Toxicology and Pharmacology of an AAV Vector Expressing Codon-Optimized RPGR in RPGR-Deficient Rd9 Mice.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 108 or 4 × 109 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.

Cone Phosphodiesterase-6γ' Subunit Augments Cone PDE6 Holoenzyme Assembly and Stability in a Mouse Model Lacking Both Rod and Cone PDE6 Catalytic Subunits.

Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6α and PDE6ß and two identical inhibitory PDE6γ subunits, while cone PDE6 is composed of two identical PDE6α' catalytic subunits and two identical cone-specific PDE6γ' inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones. In this study, we generated an rd10/cpfl1 mouse model lacking rod PDE6ß and cone PDE6α' subunits. Both rod and cone photoreceptor cells are degenerated with age and all PDE6 subunits degrade in rd10/cpfl1 mice. We expressed cone PDE6α' in both rods and cones of rd10/cpfl1 mice by adeno-associated virus (AAV)-mediated delivery driven by the ubiquitous, constitutive small chicken ß-actin promoter. We show that expression of PDE6α' rescues rod function in rd10/cpfl1 mice, and the restoration of rod light sensitivity is attained through restoration of endogenous rod PDE6γ and formation of a functional PDE6α'γ complex. However, improved photopic cone responses were achieved only after supplementation of both cone PDE6α' and PDE6γ' subunits but not by PDE6α' treatment alone. We observed a two fold increase of PDE6α' levels in the eyes injected with both PDE6α' plus PDE6γ' relative to eyes receiving PDE6α' alone. Despite the presence of both PDE6γ' and PDE6γ, the majority of PDE6α' formed functional complexes with PDE6γ', suggesting that PDE6α' has a higher association affinity for PDE6γ' than for PDE6γ. These results suggest that the presence of PDE6γ' augments cone PDE6 assembly and enhances its stability. Our finding has important implication for gene therapy of PDE6α'-associated achromatopsia.

Publisher Correction: Gene-based Therapy in a Mouse Model of Blue Cone Monochromacy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Human L- and M-opsins restore M-cone function in a mouse model for human blue cone monochromacy.

Purpose: Blue cone monochromacy (BCM) is an X-linked congenital vision disorder characterized by complete loss or severely reduced L- and M-cone function. Patients with BCM display poor visual acuity, severely impaired color discrimination, myopia, nystagmus, and minimally detectable cone-mediated electroretinogram. Recent studies of patients with BCM with adaptive optics scanning laser ophthalmoscopy (AOSLO) showed that they have a disrupted cone mosaic with reduced numbers of cones in the fovea that is normally dominated by L- and M-cones. The remaining cones in the fovea have significantly shortened outer segments but retain sufficient structural integrity to serve as potential gene therapy targets. In this study, we tested whether exogenously expressed human L- and M-opsins can rescue M-cone function in an M-opsin knockout (Opn1mw-/- ) mouse model for BCM. Methods: Adeno-associated virus type 5 (AAV5) vectors expressing OPN1LW, OPN1MW, or C-terminal tagged OPN1LW-Myc, or OPN1MW-HA driven by a cone-specific promoter were injected subretinally into one eye of Opn1mw-/- mice, while the contralateral eye served as the uninjected control. Expression of cone pigments was determined with western blotting and their cellular localization identified with immunohistochemistry. M-cone function was analyzed with electroretinogram (ERG). Antibodies against cone phototransduction proteins were used to study cone outer segment (OS) morphology in untreated and treated Opn1mw-/- eyes. Results: We showed that cones in the dorsal retina of the Opn1mw-/- mouse do not form outer segments, resembling cones that lack outer segments in the human BCM fovea. We further showed that AAV5-mediated expression of either human M- or L-opsin individually or combined promotes regrowth of cone outer segments and rescues M-cone function in the treated Opn1mw-/- dorsal retina. Conclusions: Exogenously expressed human opsins can regenerate cone outer segments and rescue M-cone function in Opn1mw-/- mice, thus providing a proof-of-concept gene therapy in an animal model of BCM.

Risk factors for postoperative acute mesenteric ischemia among adult patients undergoing cardiac surgery: A systematic review and meta-analysis.

PURPOSE: We aimed to seek risk factors for AMI among adult patients undergoing cardiac surgery. MATERIALS AND METHODS: We searched electronic bibliographic databases for studies reporting risk factors for AMI among adult patients undergoing cardiac surgery. Pooled odds ratios (OR) and standard mean differences (SMD or MD) for risk factors between AMI and control group were estimated. RESULTS: 11 studies with 67,195 patients met the inclusion criteria. 14 risk factors were found to be statistically significant: preoperative factors including age (MD 4.62years, 95% CI (1.97,7.27)), cardiac shock (OR 5.17, (1.17,22.81)), peripheral vascular disease (OR 3.53, (2.05,6.09)), need for intra-aortic balloon pump (IABP) (OR 5.89, (3.26,10.65)), emergency surgery (OR 3.75, (1.69,8.33)), and postoperative factors including atrial fibrillation (OR 2.41, (1.79,3.24)), CK-MB level (SMD 1.06, (0.62 to 1.50)), serum creatinine >200µmol/L (OR 23.39, (11.61,47.12)), blood loss (MD 358.32mL, (53.56,663.07)), prolonged ventilation (OR 9.04, (5.24,15.62)), need for IABP (OR 6.32, (3.19,12.54)), inotropic treatment (OR 8.40, (3.19,22.14)), blood transfusion (OR 9.15, (4.79,17.48)), reoperation (OR 3.30, (1.55,7.04)). CONCLUSIONS: 14 risk factors were associated with an increased risk of AMI, which indicated that AMI might occur via stenosis or occlusion of mesenteric vessels, reduced blood volume or maldistribution of blood flow.

Gene-based Therapy in a Mouse Model of Blue Cone Monochromacy.

Cones are responsible for daylight, central, high acuity and color vision. Three proteins found in human cones, i.e. long-wavelength (L)-, middle-wavelength (M)-, and short-wavelength sensitive (S)-opsins, are responsible for red, green and blue color recognition, respectively. Human blue cone monochromacy (BCM) is characterized by functional loss of both L- and M-cone opsins due to mutations in the OPN1LW/OPN1MW gene cluster on the X chromosome. BCM patients, who rely on their vision from only S-cones and rods, suffer severely reduced visual acuity and impaired color vision. Recent studies show that there is sufficient cone structure remaining in the central fovea of BCM patients to consider AAV-mediated gene augmentation therapy. In contrast, mouse retina has only two opsins, S-opsin and M-opsin, but no L-opsin. We generated an M-opsin knockout mouse (Opn1mw -/-) expressing only S-opsin as a model for human BCM. We show that recombinant M-opsin delivered by AAV5 vectors rescues M-cone function in Opn1mw -/- mice. We also show that AAV delivered M-opsin localizes in the dorsal cone outer segments, and co-localizes with S-opsin in the ventral retina. Our study demonstrates that cones without M-opsin remain viable and respond to gene augmentation therapy, thereby providing proof-of-concept for cone function restoration in BCM patients.

Prophylaxis Against Atrial Fibrillation After General Thoracic Surgery: Trial Sequential Analysis and Network Meta-Analysis.

BACKGROUND: Postoperative atrial fibrillation/flutter (POAF) is associated with significant morbidity and mortality after general thoracic surgery, but the need for and the best agent for prophylaxis remains obscure. METHODS: A systematic literature search was performed to identify randomized controlled trials that compared regimens for POAF prophylaxis after general thoracic surgery. Random-effects meta-analyses with trial sequential analyses were performed to compare the effects of medical prophylaxis vs placebo/usual care. The risk of POAF among patients receiving various prophylactic regimens was subjected to Bayesian network meta-analysis. RESULTS: Twenty-two trials (2,891 patients and 11 regimens) were included. Overall, medical prophylaxis reduced the incidence of POAF (OR, 0.33; 95% CI, 0.22-0.49) but not short-term mortality (OR, 0.85; 95% CI, 0.41-1.73). There was no significant difference in patient withdrawal due to adverse events (OR, 1.67; 95% CI, 0.67-4.16). Trial sequential analysis showed that as of 2012, sufficient evidence had accrued in support of the effectiveness of medical prophylaxis in reducing POAF after general thoracic surgery. In network meta-analysis, ß-blockers, angiotensin-converting enzyme inhibitors, amiodarone, magnesium, and calcium channel blockers significantly reduced the risk of POAF compared with placebo/usual care. ß-Blockers had the highest probability of being the most effective agents (OR, 0.12; 95% credible interval [CrI], 0.05-0.27; probability of being best, 77.7%; number needed to treat, 5.2). CONCLUSIONS: The current literature supports the effectiveness and tolerability of medical prophylaxis and the superiority of ß-blockers in preventing POAF after general thoracic surgery. ß-Blockers are recommended, taking into consideration the status of the bronchopulmonary system.

AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy.

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken ß-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.

Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial.

MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed filamentary keratitis, and two patients developed progressive cataract. Of these two patients, one also developed transient subfoveal fluid after the injection as well as monocular oscillopsia. Two patients developed a rise in AAV antibodies, but neither patient was positive for rAAV vector genomes via PCR. Three patients also displayed measurable improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.

Gene Therapy for MERTK-Associated Retinal Degenerations.

MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.

Pathological Effects of Mutant C1QTNF5 (S163R) Expression in Murine Retinal Pigment Epithelium.

PURPOSE: The mutation S163R in complement C1q tumor necrosis factor-related protein-5 (C1QTNF5) causes an autosomal dominant disorder known as late-onset retinal degeneration (L-ORD). In this study, our goal is to evaluate the consequences of mutant S163R C1QTNF5 expression in mouse RPE following its delivery using an adeno-associated viral (AAV) vector. METHODS: We generated AAV vectors containing either human wild-type C1QTNF5 or mutant S163R C1QTNF5 driven by an RPE-specific BEST1 promoter, and delivered them subretinally into one eye of adult C57BL/6 mice. Transgene expression was detected by immunohistochemistry. Retinal function was assessed by full-field ERG. Pathological changes were further examined by digital fundus imaging and spectral-domain optical coherence tomography (SD-OCT). RESULTS: We show that the AAV-expressed mutant S163R leads to pathological effects similar to some of those found in patients with advanced L-ORD, including RPE thinning, RPE cell loss, and retinal degeneration. In addition, we provide in vivo evidence that mutant S163R C1QTNF5 can form large, transparent, spherical intracellular aggregates throughout the RPE, which are detectable by light microscopy. In contrast to AAV-expressed wild-type C1QTNF5, which is secreted apically from the RPE toward the photoreceptor cells and the outer limiting membrane, the S163R mutant is primarily routed toward the basal side of RPE, where it forms thick, extracellular deposits over time. CONCLUSIONS: Adeno-associated viral-targeted expression of mutant S163R in the RPE represents a useful approach for quickly generating animal models that mimic pathological features of L-ORD and offers the potential to understand disease mechanisms and develop therapeutic strategies.