r AAVMediated PEX 1 Gene Delivery Improves Retinal

r. AAV-Mediated PEX 1 Gene Delivery Improves Retinal Functions and Peroxisome Metabolites in a Mouse Model for Zellweger Spectrum Disorder Catherine Argyriou 1, Ji Yun Song 2, Anna Polosa 3, Devin Mc. Dougald 2, Bradford Steele 4, Ning Huang 4, Bruno Cecyre 5, Erminia Di Pietro 1, Frederic Fournelle 6, Pierre Chaurand 6, Jean-Francois Bouchard 5, Joseph Hacia 4, Pierre Lachapelle 3, Jean Bennett 2, Nancy Braverman 1 1 Human Genetics, Mc. Gill University, Montreal, QC, Canada, 2 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 3 Ophtalmology & Visual Sciences, Mc. Gill University, 4 Keck School of Medicine, University of Southern California, Los Angeles, CA, 5 School of Optometry, Universite de Montreal, QC, Canada, 6 Department of Chemistry, Universite de Montreal INTRODUCTION: Zellweger Spectrum Disorder (ZSD) is a peroxisome biogenesis disease caused by mutations in PEX genes 1. We used here a mouse model bearing the murine equivalent to a common human ZSD allele (PEX 1 -G 844 D), that recapitulates several ZSD phenotypes, including retinopathy 2, 3 marked by cone cell anomalies, gradual reduction in electroretinogram (ERG) response, and diminished visual acuity (optomotor reflex, OMR). We present here the results of a proof-of-concept study for PEX 1 retinal gene augmentation therapy using a CMV promoter and AAV 8 capsid, as well as our observations of ERG, OMR, and peroxisome metabolites after Hs. PEX 1 gene delivery for two cohorts representing: (1) prevention of disease, and (2) recovery (or amelioration) of existing disease. Endpoint ERG (retinal function) and OMR (visual acuity) tests Vector design and validation - AAV 8. h. PEX 1. HA experimental vector and AAV 8. e. GFP control vector were produced. ; CMV promoter for maximum expression in photoreceptor cells. - PEX 1 protein expression from the vector was confirmed in 84 -31 cells - Recovery of peroxisome functions was confirmed in PEX 1 deficient cells Each point represents 1 animal; *=P<0. 05; **=P<0. 01 by t-test Non-injected age-matched PEX 1 -G 844 D mice were used as controls. - In both cohorts, scotopic and photopic responses were significantly higher in the left (experimental vector treated) compared to right (control treated) eyes of injected mutants. - Injection had no effect on ERG response in wild-type controls (not-shown). Peroxisome metabolites in retina after gene augmentation Experimental design - C 26: 0 lyso-PCs (a measure of very long chain fatty-acids) are increased in PEX 1 -G 844 D retinas due to peroxisome dysfunction - PEX 1 gene augmentation normalizes this in the ‘prevention’ cohort (1. 03 E+10 vg/µl) Each point represents 1 animal; *=P<0. 05; **=P<0. 01 by t-test Histological changes after gene augmentation Retinal immunohistochemistry shows successful (though variable) expression of AAV 8 vectors, with Hs. PEX 1 -HA localized to endogenous Mm. PEX 1 expression zones (IS/OS and OPL layers). No alterations in thickness of cell layers due to transgene expression or exposure to AAV 8; No infiltrate. - Non-injected mutants were used as controls for effect of injection - Injected and non-injected wild-type littermate controls were also included Preliminary ERG (retinal function) and OMR (visual acuity) tests ‘Validation ‘cohort 8 -11 wks post subretinal injection Development of MALDI-Imaging mass spec for mouse retinal sections - We will use this method in future studies to detect localized retinal lipid normalization after gene delivery (removing IS/OS contamination by nontransduced tissue). Combined with Hs. PEX 1 m. RNA or protein quantification techniques, this allows for correlation b/w vector expression and biochemical recovery. ‘Prevention’ and ‘recovery’ cohorts at 25 wks-old (16 or 20 wks post injection) Each point represents 1 animal. *=P<0. 05 ; **=P<0. 01 by t-test - Conclusions and Future Studies ZSD includes progressive visual loss that reduces quality, of life and retinal gene augmentation has great potential for therapy to preserve or improve vision 4. AAV 8. CMV. Hs. PEX 1. HA was subretinally delivered into the left eye of 5 and 9 -week-old PEX 1 G 844 D mice: Successful expression of the protein with no negative effected was observed. From 2 to 6 months after gene delivery, therapeutic vector-treated eyes showed improved ERG compared to control eyes, on average, in both the prevention and recovery cohorts. This implies clinical potential of gene delivery to improve vision at both earlier and later disease stages. Hs. PEX 1 gene delivery decreased very long chain fatty-acids in PEX 1 -G 844 D retinas, suggesting improved peroxisome function. The addition of more functional vision endpoints and correlation of phenotypic improvement to Hs. PEX 1 expression could strengthen the support for this experimental therapy References: 1. S Hiebler et al. , Mol Genet Metab 111(2014) 522 -532; 2. SJ Steinberg et al. , ]; Gene. Reviews 2012; 3. C Argyriou et al. , Exp Eye Res 2019 Sep; 186: 107713 4. J Bennett, Mol Ther 2017 3; 25(5): 1076 -1094