Merck
  • Home
  • Search Results
  • Stability and Safety of an AAV Vector for Treating RPGR-ORF15 X-Linked Retinitis Pigmentosa.

Stability and Safety of an AAV Vector for Treating RPGR-ORF15 X-Linked Retinitis Pigmentosa.

Human gene therapy (2015-06-17)
Wen-Tao Deng, Frank M Dyka, Astra Dinculescu, Jie Li, Ping Zhu, Vince A Chiodo, Sanford L Boye, Thomas J Conlon, Kirsten Erger, Travis Cossette, William W Hauswirth
ABSTRACT

Our collaborative successful gene replacement therapy using AAV vectors expressing a variant of human RPGR-ORF15 in two canine models provided therapeutic proof of concept for translation into human treatment. The ORF15 sequence contained within this AAV vector, however, has ORF15 DNA sequence variations compared to the published sequence that are likely due to its unusual composition of repetitive purine nucleotides. This mutability is a concern for AAV vector production and safety when contemplating a human trial. In this study, we establish the safety profile of AAV-hIRBP-hRPGR and AAV-hGRK1-hRPGR vectors used in the initial canine proof-of-principle experiments by demonstrating hRPGR-ORF15 sequence stability during all phases of manipulation, from plasmid propagation to vector production to its stability in vivo after subretinal administration to animals. We also evaluate potential toxicity in vivo by investigating protein expression, retinal structure and function, and vector biodistribution. Expression of hRPGR is detected in the inner segments and synaptic terminals of photoreceptors and is restricted to the connecting cilium when the vector is further diluted. Treated eyes exhibit no toxicity as assessed by retinal histopathology, immunocytochemistry, optical coherence tomography, fundoscopy, electroretinogram, and vector biodistribution. Therefore, the hRPGR-ORF15 variant in our AAV vectors appears to be a more stable form than the endogenous hRPGR cDNA when propagated in vitro. Its safety profile presented here in combination with its proven efficacy supports future gene therapy clinical trials.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Dimethyl sulfoxide, meets EP testing specifications, meets USP testing specifications
Sigma-Aldrich
Dimethyl sulfoxide, PCR Reagent
Sigma-Aldrich
Betaine, ≥98% (perchloric acid titration)
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma
Sigma-Aldrich
Dimethyl sulfoxide, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Dimethyl sulfoxide, anhydrous, ≥99.9%
Sigma-Aldrich
8-Octanoyloxypyrene-1,3,6-trisulfonic acid trisodium salt, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Betaine, BioUltra, ≥99.0% (NT)
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.6%, ReagentPlus®
Sigma-Aldrich
Betaine solution, 5 M, PCR Reagent
Sigma-Aldrich
Anti-RPGR antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution