267. Phase I Gene Therapy Preliminary... (PDF Download Available)
Dec 18, 2017 - The RESCUE (NCT02652767) and REVERSE (NCT02652780) Phase III studies of GS010 have been initiated in the ...
Gene Therapy for Neurosensory Diseases PNAS, 2013; 110: 1732-7). With a goal to develop this approach for treatment of RP, we used adeno-associated virus (AAV)-delivered CRISPR/Cas9 for Nrl-knockdown in rod photoreceptors. AAV vectors were constructed to carry a photoreceptor-specific Cas9 nuclease expression cassette or a single-guided RNA (sgRNA) targeting Nrl or eGFP gene. The Cas9 and the sgRNA vectors were co-delivered into mice by subretinal administration. Potency of the AAV-CRISPR/Cas9 system was validated by EGFP knockdown in a mouse line with eGFP-labeled rods. Nrl knockdown was conducted in wild-type C57/Bl6 or Crxp-Nrl, a mouse line with rod-only photoreceptors. Molecular, histological and functional alterations were examined by next generation sequencing, immunoblot analysis, immunofluorescence, electron microscopy, and electroretinography (ERG). Our results showed that eGFP and Nrl were efficiently knocked down following AAV-CRISPR/Cas9 treatment. For Nrl knockdown, almost all insertions and deletions were detected in the targeted Nrl locus, and very few mutations were identified in ten potential off-target loci. A majority of the transduced rods acquired characteristics of cone photoreceptors following Nrl-CRISPR/Cas9 vector treatment, as demonstrated by reduced expression of rodspecific genes and enhanced expression of cone-specific genes, loss of the unique rod chromatin pattern, and diminished rod ERG response. Rescue of retinal degeneration was assessed in three mouse models harboring either recessive or dominant rod-specific mutations. In all three models, the Nrl-CRISPR/Cas9 vector treated eyes maintained significantly better photoreceptor viability and cone function than control eyes, as revealed by remarkably thicker photoreceptor layer, higher cone cell number, greater cone ERG amplitude and better optomotor behavior. In conclusion, AAV-CRISPR-mediated Nrl gene knockdown can efficiently reprogram rods into conelike photoreceptors and prevent secondary cone death in retinal degeneration, which could be developed into a viable treatment for RP in humans.
267. Phase I Gene Therapy Preliminary Clinical Results for Treatment of ND4 Leber Hereditary Optic Neuropathy with rAAV2-2-ND4
Scott Uretsky1, Catherine Vignal2, Samuel Bidot2, Serge Fitoussi1, Anne Galy1, Sandrine Meunier1, Roxane Noel1, Céline Bouquet1, Nitza Thomasson1, Jean-Philippe Combal1, José A. Sahel2 1 Gensight-Biologics, Paris, France, 2Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, Paris, France Introduction Leber Hereditary Optic Neuropathy (LHON) is a rare mitochondrial genetic disorder predominantly affecting young males. Affected patients experience bilateral severe central vision loss. Currently no therapy is approved in the United States to prevent, halt or reverse vision loss due to LHON. Preliminary safety and pharmacodynamic results of a first-in-man trial of GS010, a gene therapy candidate for patients with LHON carrying the ND4 mutation will be presented. Methods GS010 is a recombinant adenoassociated viral vector, serotype 2, carrying the wild-type ND4 gene (rAAV2/2-ND4) and is an experimental gene therapy for the treatment of LHON due to the G11778A ND4 mitochondrial mutation. GS010 has received orphan drug designation in EU & USA. GS010 contains a Mitochondrial Targeting Sequence (MTS) that allows localization of the wild-type protein to the mitochondrion, enabling restoration of mitochondrial function. An open-label Phase I/IIa safety study (NCT02064569) included patients with vision loss due to ND4 LHON and has completed recruitment. Four dose escalation cohorts and an extension cohort were comprised of 3 patients each. Patients received a single intra-vitreal injection of rAAV2/2-ND4 in their worse seeing eye. Primary outcome was the occurrence of adverse events (AE). Secondary outcomes included immune response to AAV2 and evaluation of visual function. Results Systemic safety was excellent as S106
no unexpected adverse events occurred. Ocular tolerability was good with mostly mild inflammation that were responsive to and resolve with standard therapies. Of the first 9 patients with 48 week follow up, preliminary results indicate that symptom duration could impact magnitude of treatment effect . Additionally, baseline vision status at time of treatment also indicate a relation with potential greater magnitude of effect which was noted with relatively shorter disease duration (< 2 years). These data confirm the importance of treating early from onset of vision loss. The RESCUE (NCT02652767) and REVERSE (NCT02652780) Phase III studies of GS010 have been initiated in the United States and some European Union countries. Both studies are randomized, double-masked, sham-controlled trials and will specifically include patients up to 6 months and one year after the onset of vision loss.
268. Optogenetic Engineering of Retinal Ganglion Cells with AAV2.7m8-ChrimsonRtdTomato (GS030-DP) Is Well Tolerated and Induces Functional Responses to Light in NonHuman Primates
Anne M. Douar1, Celine Bouquet1, Didier Pruneau1, Joel Chavas1, Deniz Dalkara2, Jens Duebel2, Ryad Benosman2, Guillaume Chenegros2, Serge Picaud2, José Sahel2, Nitza Thomasson1 1 GenSight Biologics, Paris, France, 2Vision Institute, Paris, France Introduction: Expression of a light-sensitive opsin in retinal ganglion cells (RGCs) is an attractive strategy to restore vision. We evaluated the ability of ChrimsonR-tdTomato (ChrR-tdT), derived from the algal light-gated cation channel ChrimsonR (Ed Boyden, MIT), to convert light insensitive RGCs into photoactivatable cells in normal macaques. A photostimulation device (GS030MD) is developed in parallel to complement the biologics. This GS030 combination treatment is intended to treat blindness caused by retinal degenerative diseases such as retinitis pigmentosa. Methods: Cynomolgus macaques were injected intravitreally with the AAV2.7m8 vector encoding ChrR-tdT under the control of the CAG promoter (GS030-DP; 5×1011 vg/eye). Electrophysiological measurements by microelectrode array (MEA) and patch clamp as well as expression of the ChrR-tdT protein by immunofluorescence were assessed on explanted retinas 2 months after injection. Local tolerance was evaluated by ophthalmic examination and histology at 2 and 6 months post administration. Results: ChR-tdT was essentially expressed in RGCs and its expression restricted to the perifoveal area. MEA recordings showed light responses in all treated retinas, with 3 out of 4 retinas displaying high amplitudes of electrical responses to light stimulation (up to 360 Hz). One retina was less responsive (50 Hz). In patch clamp experiments, conducted by targeting tdTexpressing RGCs, large photocurrents were recorded in 3 out of 4 retinas in response to illumination, and according to the expected action spectrum for ChR-tdT. An exploratory study was conducted in parallel in monkeys, which received a single bilateral intravitreal administration of GS030-DP (5×1011 vg/eye) to assess local tolerance, systemic toxicity and immunogenicity at 2 and 6 months. No clinical signs indicative of systemic toxicity or local intolerance were observed. No adverse effects were seen by ophthalmic or histological examinations, especially no retinal structural modifications, inflammation or necrosis. Anti-AAV2 neutralizing antibodies (NAbs) measured in serum at 2, 3 and 6 months slightly increased at month 2 (≤ 1:100) and then returned to baseline levels at month 6. No NAbs were detected in aqueous humor at necropsy (at 2 or 6 months). In parallel, in preparation of a first-in-man clinical trial, a complete prototype of the photostimulation device (“goggles”) was developed with a full functional optical chain, an electronic subsystem, and firmware and software architecture. These goggles (GS030-MD) capture external scenes through an event-based camera and deliver Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy