RATIONALE:Mutations in the cardiac Ryanodine Receptor gene (RYR2) cause dominant Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), a leading cause of sudden death in apparently healthy ...individuals exposed to emotions or physical exercise.
OBJECTIVE:We investigated the efficacy of allele specific silencing by RNA interference to prevent CPVT phenotypical manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2.
METHODS AND RESULTS:We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2-R4496C mRNA over the corresponding wild-type (WT) allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 function following in vivo delivery by intraperitoneal injection in neonatal and adult RyR2 heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10 the ratio between WT and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2-R4496C mRNA, while protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively1) reduced isoproterenol-induced delayed after depolarizations and triggered activity in infected cells, 2) reduced adrenergically mediated ventricular tachycardia in treated mice, 3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and T-tubules and 4) attenuated mitochondrial abnormalities.
CONCLUSIONS:The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice suggesting that reduction of mutant RyR2 may be a novel therapeutic approach for CPVT.
Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are ...controlled by light is largely unknown. Here, we show that the light‐responsive miR‐211 controls lysosomal biogenesis at the beginning of light–dark transitions in the RPE by targeting Ezrin, a cytoskeleton‐associated protein essential for the regulation of calcium homeostasis. miR‐211‐mediated down‐regulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light‐mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR‐211−/− mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR‐211−/− phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.
Synopsis
MicroRNA‐204/211 (miR‐211) are expressed in murine retinal pigment epithelium (RPE) and regulate eye differentiation and function. Light‐activated miR‐211 targets the membrane/cytoskeleton‐crosslinking protein Ezrin to activate the calcineurin/TFEB pathway in the RPE, thereby inducing lysosomal biogenesis and the degradation of photoreceptor outer segments (POS).
Light‐responsive miR‐211 is required for daily lysosomal biogenesis and function in the RPE by targeting Ezrin.
miR‐211‐induced downregulation of Ezrin promotes the release of lysosomal Ca2+ through TRPML1, which triggers TFEB nuclear translocation and Calcineurin‐mediated activation of CLEAR network.
miR‐211−/− mice show impaired lysosomal degradation of POS and a progressive accumulation of lipofuscin in the RPE that resembles human age‐related macular degeneration (AMD) disease.
Pharmacological inhibition of Ezrin in miR‐211−/− mice rescues these defects, pointing to a new therapeutic target to counteract AMD onset and progression.
Downregulation of the membrane/cytoskeleton‐crosslinking protein Ezrin activates the calcineurin/TFEB pathway to promote lysosomal degradation of photoreceptor outer segments in murine retinal pigment epithelium cells.
Abstract
Retinal gene therapy has advanced considerably in the past three decades. Initial efforts have been devoted to comprehensively explore and optimize the transduction abilities of gene ...delivery vectors, define the appropriate intraocular administration routes and obtain evidence of efficacy in animal models of inherited retinal diseases (IRDs). Successful translation in clinical trials of the initial promising proof-of-concept studies led to the important milestone of the first approved product for retinal gene therapy in both US and Europe. The unprecedented clinical development observed during the last decade in the field is however highlighting new challenges that will need to be overcome to bring gene therapy to fruition to a larger patient population within and beyond the realm of IRDs.
Gyrate atrophy of choroid and retina (GACR) is a chorioretinal degeneration caused by pathogenic variants in the gene encoding ornithine aminotransferase (OAT), an enzyme mainly expressed in liver. ...Affected patients have increased ornithine concentrations in blood and other body fluids and develop progressive constriction of vision fields leading to blindness. Current therapies are unsatisfactory and better treatments are highly needed. In two mouse models of OAT deficiency that recapitulates biochemical and retinal changes of GACR, we investigated the efficacy of an intravenously injected serotype 8 adeno‐associated (AAV8) vector expressing OAT under the control of a hepatocyte‐specific promoter. Following injections, OAT‐deficient mice showed reductions of ornithine concentrations in blood and eye cups compared with control mice injected with a vector expressing green fluorescent protein. AAV‐injected mice showed improved electroretinogram response and partial restoration of retinal structure up to one‐year post‐injection. In summary, hepatic OAT expression by AAV8 vector was effective at correction of hyperornithinemia and improved function and structure of the retina. In conclusion, this study provides proof‐of‐concept of efficacy of liver‐directed AAV‐mediated gene therapy of GACR.
Synopsis
Deficiency of ornithine aminotransferase (OAT) results in increased plasma ornithine concentrations and retinal toxicity, especially in the retinal pigment epithelium (RPE). This study investigated liver‐directed gene transfer for OAT deficiency.
Intravenous injections of AAV8 vector delivering OAT gene to hepatocytes reduced ornithine concentrations in plasma and eye.
The treatment improved function and structure of the retina.
Deficiency of ornithine aminotransferase (OAT) in liver results in increased ornithine concentrations and retinal toxicity, especially the retinal pigment epithelium (RPE). This study investigated liver‐directed gene transfer for OAT deficiency.
Pompe disease (PD) is a metabolic myopathy due to acid alpha-glucosidase deficiency and characterized by extensive glycogen storage and impaired autophagy. We previously showed that modulation of ...autophagy and lysosomal exocytosis by overexpression of the transcription factor EB (TFEB) gene was effective in improving muscle pathology in PD mice injected intramuscularly with an AAV-TFEB vector. Here we have evaluated the effects of TFEB systemic delivery on muscle pathology and on functional performance, a primary measure of efficacy in a disorder like PD. We treated 1-month-old PD mice with an AAV2.9-MCK-TFEB vector. An animal cohort was analyzed at 3 months for muscle and heart pathology. A second cohort was followed at different timepoints for functional analysis. In muscles from TFEB-treated mice we observed reduced PAS staining and improved ultrastructure, with reduced number and increased translucency of lysosomes, while total glycogen content remained unchanged. We also observed statistically significant improvements in rotarod performance in treated animals compared to AAV2.9-MCK-eGFP-treated mice at 5 and 8 months. Cardiac echography showed significant reduction in left-ventricular diameters. These results show that TFEB overexpression and modulation of autophagy result in improvements of muscle pathology and of functional performance in the PD murine model, with delayed disease progression.
Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), ...whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.
As gene therapy begins to produce its first clinical successes, interest in ocular gene transfer has grown owing to the favorable safety and efficacy characteristics of the eye as a target organ for ...drug delivery. Important advances also include the availability of viral and non-viral vectors that are able to efficiently transduce various ocular cell types, the use of intraocular delivery routes and the development of transcriptional regulatory elements that allow sustained levels of gene transfer in small and large animal models after a single administration. Here, we review recent progress in the field of ocular gene therapy. The first experiments in humans with severe inherited forms of blindness seem to confirm the good safety and efficacy profiles observed in animal models and suggest that gene transfer has the potential to become a valuable therapeutic strategy for otherwise untreatable blinding diseases.
Enzyme replacement therapy (ERT) is the standard of care for several lysosomal storage diseases (LSDs). ERT, however, requires multiple and costly administrations and has limited efficacy. We ...recently showed that a single high dose administration of adeno-associated viral vector serotype 8 (AAV2/8) is at least as effective as weekly ERT in a mouse model of mucopolysaccharidosis type VI (MPS VI). However, systemic administration of high doses of AAV might result in both cell-mediated immune responses and insertional mutagenesis. Here we evaluated whether the combination of low doses of AAV2/8 with a less frequent (monthly) than canonical (weekly) ERT schedule may be as effective as the single treatments at high doses or frequent regimen. A greater reduction of both urinary glycosaminoglycans, considered a sensitive biomarker of therapeutic efficacy, and storage in the myocardium and heart valves was observed in mice receiving the combined than the single therapies. Importantly, these levels of correction were similar to those we obtained in a previous study following either high doses of AAV2/8 or weekly ERT. Our data show that low-dose gene therapy can be used as a means to rarify ERT administration, thus reducing both the risks and costs associated with either therapies.
Leber congenital amaurosis (LCA) is a rare degenerative eye disease, linked to mutations in at least 14 genes. A recent gene therapy trial in patients with LCA2, who have mutations in RPE65, ...demonstrated that subretinal injection of an adeno-associated virus (AAV) carrying the normal cDNA of that gene (AAV2-hRPE65v2) could markedly improve vision. However, it remains unclear how the visual cortex responds to recovery of retinal function after prolonged sensory deprivation. Here, 3 of the gene therapy trial subjects, treated at ages 8, 9, and 35 years, underwent functional MRI within 2 years of unilateral injection of AAV2-hRPE65v2. All subjects showed increased cortical activation in response to high- and medium-contrast stimuli after exposure to the treated compared with the untreated eye. Furthermore, we observed a correlation between the visual field maps and the distribution of cortical activations for the treated eyes. These data suggest that despite severe and long-term visual impairment, treated LCA2 patients have intact and responsive visual pathways. In addition, these data suggest that gene therapy resulted in not only sustained and improved visual ability, but also enhanced contrast sensitivity.
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