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Rajeshwari D. Koilkonda, William W. Hauswirth, Vince Chiodo, Sanford L. Boye, John Guy; Efficient Transduction of Tyrosine-to-Phenylalanine Mutated AAV2 vectors carrying Human ND4 gene and Biodistribution following Intravitreal Delivery in a Rodent Model- a Gene Therapy for Leber Hereditary Optic Neuropathy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1885.
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In this study, we evaluated the ocular transduction efficiency and biodistribution of self complimentary (sc) AAV2 (Y444+500+730)-vector genomes carrying the human ND4 gene after intravitreal administration.
Eight week old Sprague Dawley rats (n=8; male=4, female=4) were injected with scAAV containing wild type (wt)-human (h) P1ND4v2 (without FLAG tag) packaged in triple Y-F (Y444+500+730) mutated capsids into the vitreous cavity of right eyes (OD) (2X109vg in 4µl). Contralateral eyes (OS) remained un-injected. The animals were sacrificed 1m post injections (PI) and the following organs were harvested for DNA extraction: blood, eyes, optic nerves, right and left brain, heart, liver, skeletal muscle and gonads. Peripheral blood was also analyzed at day 1 and day 8 PI. In order to better characterize the results for the novel AAV2-triple Y-F capsid mutant, 2 rats (m=1, f=1) received scAAV2-GFP vector (2X109vg in 4µl) and served as AAV2 controls. To gauge the expression of human ND4 in retinal ganglion cells (RGCs), another group of mice (n=5) were injected with sc-AAV2-hP1ND4V2 (Triple Y-F) in both eyes, control group remained un-injected. Confocal microscopy was performed on retinal whole mounts at 10d PI by immunoflorescence.
Expression-Confocal microscopy using hND4 antibody on retinal whole mounts revealed punctuate and perinuclear expression of hND4 at 10d PI, which co-localised with Thy1.2, indicating expression in RGCs. hND4 expression was absent in un-injected controls. Biodistribution- The vector genome copy number was represented as average of overall tissue score (AOTS) for each tissue. AOTS for ocular tissues was, optic nerves, un-injected (OS): 0.83, injected (OD): 1.62, eyes, un-injected (OS): 0.6, injected (OD): 3.28. None of the other organs such as brain (left and right), liver, skeletal muscle, and gonads had the AAV genomes (AOTS: 0), indicating AAV2 displayed narrow tropism. Peripheral blood showed vector genome copies at day 1 (AOTS: 2.16) which however, dropped down to minimum levels at day 8 (AOTS: 0.25). In addition, male and female animals had similar AOT scores indicating AAV mediated transduction and biodistribution is not gender specific. The vector genome copy number was almost similar using either scAAV2-triple Y-F (Y444+500+730)-wt-hP1ND4v2 or scAAV2-GFP vectors at 1m (AOTS: 1.45 and 1.5).
Biodistribution of sc-triple Y-F mutated AAV2 carrying the human ND4 gene displayed a narrower tropism of ocular tissue, indicating safety in using these vectors for future gene therapy on LHON patients with optic neuropathy.
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