Abstract Details

Title Mitochondrial Gene Delivery of Wild-Type ATP6 Mediated by a Mitochondrial Targeting Sequence Modification to AAV Capsid VP2 Rescues Cells with Mutated T8993G MtDNA Responsible for Maternally Inherited Leigh Syndrome and Neurogenic Ataxia and Retinitis Pigmentosa

Topic Muscle Disease/Neuromuscular Junction

Presentation(s) P07 - (-)

Poster/Presentation
Number 023

Objective

Background BACKGROUND: Half of the MILS children die by age three, the rest by age 10.

Design/Methods DESIGN/METHODS: The human ATP6 gene with added FLAG epitope under control of a mitochondrial promoter was inserted into a self-complementary (sc) AAV plasmid. Sc-HSP-ATP6 was packaged with mito-targeted (COX8) VP2 capsid into AAV2 or AAV9 virions. Cybrid cells with 100% mutated T8993G mtDNA cultured in high glucose media were infected at MOI of 0, 500, 1000, 5000, 10000 and 50000. After 48 hrs, media was exchanged with galactose without glucose for 5 days. Then media was changed back to the high glucose. After 72 hrs, cell survival was assessed using activated MTT and absorbance with a spectrophotometer. Transcription and expression were assessed by qRT-PCR, sequencing and immunohistochemistry. The rate of ATP synthesis was measured using a luciferin-luciferase assay.

Results RESULTS: Cell survival. Following scAAV-HSP-ATP6 infection, cell survival increased in a dose-dependent matter with an absorbance 0.372, 0.405, 0.4055, 0.43554 for MOI of 500, 1000, 5000, 10000 and 50000 respectively. Relative to no infection (MOI= 0) differences were statistically significant with p = 0.02 (MOI = 500), 0.04 (MOI = 1000), 0.05 (MOI = 10000) and 0.08 (MOI = 50000). RT-PCR. Relative to unaffected, infected T8993G cybrids ATP6 transcripts were elevated 9 fold (p= 0.02, AAV2) and 34 fold (p = 0.01, AAV9). Immunohistochemistry. FLAG tagged ATP6 surrounded DAPI stained nuclei and exhibited the punctate perinuclear distribution of mitochondrial expression with colocalization to MitoTracker Green. Respiration. Relative to uninfected T8993G cybrids the rate of ATP synthesis increased 41% (p= 0.002).

Conclusions CONCLUSIONS: After intravenous injection, AAV9 crosses the blood-brain barrier as well as goes to lung, liver, heart and muscle, so our mitotargeted construct should be able to save the lives of children with MILS.

Authors/Disclosures
John R. Guy, MD (Bascom Palmer Eye Institute) PRESENTER	No disclosure on file
	No disclosure on file
Douglas L. Arnold, MD, FAAN (Montreal Neurological Institute, McGill Univ)	Dr. Arnold has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Biogen. Dr. Arnold has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for BMS. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Frequency Therapeutics. Dr. Arnold has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Merck. Dr. Arnold has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Novartis. Dr. Arnold has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Roche. Dr. Arnold has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Sanofi. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Shionogi. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Xfacto communications. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Eli Lilly. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for EMD Serono. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biohaven. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Find therapeutics. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for GSK. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Idorsia. Dr. Arnold has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Kiniksa. Dr. Arnold has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Clario.

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