1020. AAV Delivery of Suppression and Replacement Constructs for Rhodopsin-Linked Autosomal Dominant Retinitis Pigmentosa
Authors: Naomi Chadderton, Arpad Palfi, Mary O'Reilly, Sophia Millington-Ward, Marius Ader, Markus Hildinger, Alberto Auricchio, Gearoid Tuohy, Peter Humphries, Paul F. Kenna, G. Jane Farrar
Journal: Molecular Therapy (2006)

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder that can be inherited in an autosomal dominant, autosomal recessive, x-linked, digenic or mitochondrial fashion. One form of autosomal dominantly inherited RP (adRP) is due to mutations in the rhodopsin gene (RHO) which encodes the light sensitive pigment rhodopsin, the first component in the visual transduction cascade in rod photoreceptor cells. Over one hundred mutations have been characterised in this gene to date. Given the immense mutational heterogeneity present in RHO-linked RP, a therapeutic strategy termed suppression and replacement focused on correcting the primary genetic defect while circumventing the mutational heterogeneity present in rhodopsin-linked adRP has been proposed. The approach involves suppression of mutant and wild type RHO mRNA, together with provision of a replacement gene, which is altered in sequence using the degeneracy of the genetic code, such that transcripts from the replacement gene are protected from suppression but encode wild type RHO protein. Suppression and replacement constructs for RHO have been generated in recombinant adeno associated virus (rAAV2/5). The suppression aspect of the therapeutic is provided by short hairpin RNAs (shRNAs) targeting RHO which have previously been shown to be functional in cell culture and retinal explants. The replacement aspects are provided by RHO cDNAs with degenerate nucleotide changes over the target sites for RNAi suppression. rAAV2/5 engineered to contain shRNAs targeting human Rho together with a RHO replacement gene have been subretinally injected into transgenic mice expressing a mutant RHO transgene (Pro23His mice). Pro23His mice present with a severe retinal degeneration, the majority of photoreceptors being devastated by post natal day 14 (P14). Detailed retinal histology has been undertaken on retinas from Pro23His mice at P10, P13, P14, and P15 subsequent to a single subretinal injection of rAAV2/5 at P0. Eyes from Pro23His mice subretinally injected with a control virus were compared to those injected with the AAV2/5 therapeutic. Results from histological evaluation of Pro23His mice will be presented. Suppression and replacement technology is subject to patents and patent applications licensed to Genable, Technologies Limited, Dublin, Ireland. Retinitis pigmentosa (RP) is a genetically heterogeneous disorder that can be inherited in an autosomal dominant, autosomal recessive, x-linked, digenic or mitochondrial fashion. One form of autosomal dominantly inherited RP (adRP) is due to mutations in the rhodopsin gene (RHO) which encodes the light sensitive pigment rhodopsin, the first component in the visual transduction cascade in rod photoreceptor cells. Over one hundred mutations have been characterised in this gene to date. Given the immense mutational heterogeneity present in RHO-linked RP, a therapeutic strategy termed suppression and replacement focused on correcting the primary genetic defect while circumventing the mutational heterogeneity present in rhodopsin-linked adRP has been proposed. The approach involves suppression of mutant and wild type RHO mRNA, together with provision of a replacement gene, which is altered in sequence using the degeneracy of the genetic code, such that transcripts from the replacement gene are protected from suppression but encode wild type RHO protein. Suppression and replacement constructs for RHO have been generated in recombinant adeno associated virus (rAAV2/5). The suppression aspect of the therapeutic is provided by short hairpin RNAs (shRNAs) targeting RHO which have previously been shown to be functional in cell culture and retinal explants. The replacement aspects are provided by RHO cDNAs with degenerate nucleotide changes over the target sites for RNAi suppression. rAAV2/5 engineered to contain shRNAs targeting human Rho together with a RHO replacement gene have been subretinally injected into transgenic mice expressing a mutant RHO transgene (Pro23His mice). Pro23His mice present with a severe retinal degeneration, the majority of photoreceptors being devastated by post natal day 14 (P14). Detailed retinal histology has been undertaken on retinas from Pro23His mice at P10, P13, P14, and P15 subsequent to a single subretinal injection of rAAV2/5 at P0. Eyes from Pro23His mice subretinally injected with a control virus were compared to those injected with the AAV2/5 therapeutic. Results from histological evaluation of Pro23His mice will be presented. Suppression and replacement technology is subject to patents and patent applications licensed to Genable, Technologies Limited, Dublin, Ireland.