Advances in Mitochondrial Disease Darius J. Adams, M.D. Genetics and Metabolism

Diagnostics and Therapeutics

Mitochondrial Function 1200-1500 genes involved in mitochondrial function Combination of: Nuclear DNA Mitochondrial DNA 37 genes Reference: http://www.kathleensworld.com/mitochon.html

Diagnostics Critical to potential future therapies Nuclear Gene discovery Autosomal recessive mitochondrial genes Autosomal dominant mitochondrial genes X-linked

Next Generation Genomic Sequencing Can analyze the coding regions 20,000 genes with one blood test The targeted panels are based on clinical findings Mito panels now with over 1,200 genes

Goals of Treatment Slow or arrest progression of symptoms Increase mitochondrial ATP production Support electron transfer Inhibit free radicals Stabilize OXPHOS complexes Avoid drugs capable of affecting the respiratory complexes

Current Cofactor Treatment of Mitochondrial Disorders L-Carnitine Coenzyme Q10/Ubiqinone/CytoQ L-Arginine Alpha Lipoic Acid Dietary manipulation (low carbohydrate diet) Creatine (high energy phosphate bond) Vit. C up to 4g/day Vit. E 10 U/Kg/day Vit. B1 (PDH), B2(CI&II), Nicotinamide/Niacin Vit. B6, B12 and Biotin Vit. K Menadione and Phylloquinone Exercise

Experimental Cofactor Treatment of Mitochondrial disorders Idebenone 90 mg/day similar to CoQ10 (experimental) Succinate 6 g/day in MELAS and Complex I deficiency Uridine (support general enzyme function) Dichloroacetate (found to be harmful) Organ transplantation Physical therapy

Medications That Impact Mitochondria AZT (Inhibitor to gamma polymerase) Fialuridine antiviral agent for Treatment of Hepatitis B Valproate, aspirin due to (effect on FAO or CoA sequestration) Nucleoside analogues: didanosine, zalcitabine Lamivudine and famciclovir are permitted Gentamicin and Tetracyclines

Gene Therapy

Gene Therapy Challenges and Successes Targeting gene insertion Successfully inserting plasmids Immune reactions Use of Adeno-associated viral (AAV) vectors minimizes immune response Differential tissue targeting AAV vectors can target specific tissues like brain and liver

Gene Therapy Success demonstrated in individuals with severe combined immunodeficiency (SCID) Exploring direct injection vs. IV infusion of gene therapy Advances in DNA diagnostics for mitochondrial disease allowing of possible gene correction

Lysosome

Active Trials in Gene Therapy University of North Carolina, Dr. Steven Gray and Thomas Jefferson, Dr. David Wenger Using AAV9 to transfer corrective gene for Krabbe disease (lysosomal storage disorder) Discovered that may need a combination of a bone marrow transplant, intrathecal gene therapy and blood gene therapy

Going Forward Now that we are able to obtain more precise diagnostics, targeting specific mutations associated with mitochondrial disorders can be explored Start to prepare for human phase I trials

Personalized Treatment It is likely, given the variety of mutations, gene therapy treatment will need to be customized to the individual Will need specific replacement gene and to be placed in the transport vector

Model Vector Cassette ITR RBE sequence Transgene - ssDNA Rep 68/78 mRNA Promoter PolyA Rep 68/78 mRNA needed to integrate into chromosome 19 region that is devoid of active genes for permanent correction

Model of Gene Correction

Plasmids

How Do We Get There? Likely will need proof of concept to be done at Academic Medical Centers BioTech will likely be needed for clinical trial work as this will require vast resources to prove safety and efficacy However, making advances on multiple fronts

Conclusion Advancing our diagnostic abilities will allow for the implementation of targeted therapies Nutritional and cofactor interventions continue to be refined Genetic therapeutics are now much closer with advances in vector technology