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. INTRODUCTIONRetinitis pigmentosa (RP) is a common form of genetically heterogeneous, inherited retinal diseases characterized by progressive degeneration of rod and cone photoreceptor cells and affects 1 in 4000 individuals.1,2 The X-linked form of RP (XLRP), comprising an estimated 15% of total RP cases, is among the most severe forms.3 Mutations in the gene coding for RP GTPase regulator (RPGR) cause XLRP and account for over 70% of cases.

Successful arrest of photoreceptor and vision loss expands the therapeutic window of retinal gene therapy to later stages of disease

“…2C) as previously reported when using retinal tissues fixed in paraformaldehyde for >1 h (30,31). Immunolabeling of the RPGR transgene product has been shown to be restricted to the connecting cilium only when using a modified short (1-2 min) paraformaldehyde fixation protocol (31,34). Importantly, control injections (n = 7) performed with balanced salt solution (BSS) in XLPRA2 eyes at different ages resulted in no measurable deviation of ONL thickness from the expected natural history (Fig.…”

“…RPGR mutant (XLPRA2) (28,29) and WT dogs were used to characterize the natural course of ONL thinning as a function of age (16) and the response to subretinal injections with an AAV2/5 vector (64) carrying a stabilized human RPGR 1-ORF15 cDNA under the control of a human IRBP promoter (30,65). The stabilized human RPGR 1-ORF15 cDNA (stable hRPGR) contained seven in-frame deletions, one 3-bp insertion, and 65-bp substitutions spread throughout the AG-rich region of exon ORF15 compared with the published (GenBank: NM_001034853) human RPGR 1-ORF15 sequence (34). Stable hRPGR was the same sequence as used in our previous RPGR gene augmentation study in XLPRA1 and XLPRA2 dogs (30).…”

Loss of RPGR glutamylation underlies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations

“…The ORF15 exon is a mutation hotspot in RPGR, with mutations identified in this region in up to 60% of patients with X-linked RP (8,13,14). Notably, in-frame deletions or insertions that alter the length of this region are well tolerated, as are missense changes (2,(15)(16)(17)(18)(19). However, frame shift mutations that lead to loss of the C-terminal basic domain are always disease causing, underscoring the functional importance of this domain (7).…”

Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa

“…coRPGR ORF15 seems unlikely in view of the persistent effects in the dark-adapted luminance series. Also, previous studies have indicated that RPGR expression in Rpgr −/y mice leads to sustained treatment effects 8, 9. The discrepancy between the unilateral, open-label experiments and the bilateral, sham-controlled experiments may indicate the possibility that the sham treatment also had a mild effect on ERG.…”

“…More worryingly, spontaneous mutations in the putative therapeutic transgene could potentially lead to a dominant-negative effect and accelerate disease progression 11 . To date, the majority of research groups working in the field of RPGR gene therapy have independently seen inadvertent sequence modifications in the transgene during vector development 5, 8, 9, 10…”