title: Stability and Safety of an AAV Vector for Treating RPGR-ORF15 X-Linked Retinitis Pigmentosa.
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.
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: Wen-Tao
authors.initials: WT
authors.lastname: Deng
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: Frank M
authors.initials: FM
authors.lastname: Dyka
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: Astra
authors.initials: A
authors.lastname: Dinculescu
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: Vince A
authors.initials: VA
authors.lastname: Chiodo
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: Sanford L
authors.initials: SL
authors.lastname: Boye
authors.affiliation: 2 Powell Gene Therapy Center, Department of Pediatrics, University of Florida , Gainesville, Florida.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Conlon
authors.affiliation: 2 Powell Gene Therapy Center, Department of Pediatrics, University of Florida , Gainesville, Florida.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Erger
authors.affiliation: 2 Powell Gene Therapy Center, Department of Pediatrics, University of Florida , Gainesville, Florida.
authors.forename: Travis
authors.initials: T
authors.lastname: Cossette
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, University of Florida , Gainesville, Florida.
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000229:Dependovirus / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012160:Retina / Q000473:pathology
chemical_list: D005136:Eye Proteins; C099929:RPGR protein, human