Correct a Mouse Model of Methylmalonic Acidemia. (MMA). Jessica L. Schneller, Randy J. Chandler, Charles P. Venditti. National Institutes of Health, Bethesda, ...
Gene Therapy for CNS Diseases 359. Targeted Genome Editing Using TALENs to Correct a Mouse Model of Methylmalonic Acidemia (MMA) Jessica L. Schneller, Randy J. Chandler, Charles P. Venditti National Institutes of Health, Bethesda, MD
Methylmalonic acidemia (MMA) is an autosomal recessive metabolic disorder, in which the body is unable to oxidize valine and isoleucine as well as odd chain fatty acids. Approximately 60 percent of MMA cases are caused by mutations in methylmalonylCoA mutase (MUT), the enzyme that catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. Current treatments for MUT class MMA include dietary protein restriction and cofactor supplementation; however patients continue to exhibit severe morbidity and high rates of mortality. A murine model of MMA was created by knocking out exon 3 of the Mut gene and exhibits the severest clinical phenotype of MMA, neonatal lethality. Gene therapy treatments using recombinant adeno-associated viral vectors to treat mice with MMA have proven effective, however long-term follow-up of mice treated as neonates revealed genotoxicity caused by AAV integrations, leading the mice to develop hepatocellular carcinoma. Programmable nucleases such as TALENs should make it possible to correct at the MUT locus, ameliorate disease symptoms and reduce the effects of off-target integration by precise genome modification. Furthermore, AAV-mediated delivery of TALENs with an HR cassette carrying a rescue cDNA for the Mut gene in a disease model of MMA should enable in vivo correction in the liver, and ameliorate the MMA phenotype. We have designed TALENs that target intron 2 of the Mut gene and a partial rescue cassette that contains codon-optimized exons 3 to 13 of the Mut gene to restore Mut expression in the exon 3 knock out model of MMA after successful homologous recombination. The TALENs are expressed under the control of the liver-specific thyroxine binding globulin promoter to ensure cleavage in hepatocytes, and delivered using a hepatotropic AAV serotype 8 capsid. To restore expression and achieve targeted integration, the rescue cassette is preceded by a splice acceptor site and flanked by homology arms around the TALEN cleavage site. Integration of the rescue cassette will restore Mut expression at the locus, using the endogenous promoter and exons 1 and 2 of the Mut gene to reconstitute a functional Mut gene. AAV packaging limitations necessitate two AAV vectors for delivery of the TALENs, which are 4kb per pair, and required regulatory elements, as well as a third vector for delivery of the Mut homologous recombination rescue cassette. All three vectors will be delivered systemically in the neonatal period. We hypothesize that in vivo editing at the Mut locus with subsequent HR will provide sufficient Mut enzyme activity to improve the clinical and biochemical phenotypes of the MMA mice, with minimal off-target integration, thereby decreasing the risk of genotoxicity associated with AAV gene therapy.
360. Genotype/Phenotype Correlation of Cellular Function and AAV-Mediated Gene Delivery to Treat Xeroderma Pigmentosum - Cockayne Syndrome (XP - CS)
Skylar Rizzo1, Amy L. Donate1, Michael Jones1, Kelsey Manko1, Katherine E. Santostefano2, Nao Terada2, Fowzan S. Alkuraya3, Peter B. Kang1, Christina A. Pacak1 1 Pediatrics, University of Florida, Gainesville, FL, 2Pathology, University of Florida, Gainesville, FL, 3Genetics, Alfaisal University, Riyadh, Saudi Arabia
Cockayne Syndrome (CS) is a rare, autosomal recessive, neurodegenerative disorder characterized by deficiencies which all contribute to an overall phenotype of premature aging. The underlying cause is a defect in genes involving DNA repair mechanisms. S144
This includes CSA and CSB as well as several genes associated with Xeroderma Pigmentosum (XP). Specifically, Xeroderma Pigmentosum Group G (XPG) is a disorder with two distinct clinical presentations: photosensitivity alone (XP) and photosensitivity with neurodegeneration (XP-CS). XPG’s role as an endonuclease in nucleotide excision repair following UV exposure is well-described and explains the photosensitivity phenotype, yet a separate function for XPG that explains the neurological/early aging deficit that occurs in some patients remains obscure. Other CS proteins known to be involved in nuclear DNA repair have been shown to also function as free radical scavengers in mitochondria. As growing bodies of data illustrate the importance of mitochondria in aging, neuronal cell development and maintenance, and tissue repair, we hypothesize that XPG is trafficked to mitochondria where it could play an important role in mitochondrial function via free radical regulation. Such a deficiency could explain the neurodegeneration and multi-system early aging phenotype observed in CS patients. Our preliminary data (expansion rates, oxygen consumption, ATP generation, free radical sensitivity, Metronidazole [a drug that causes liver failure in CS patients] sensitivity) support this hypothesis through demonstration of decreased viability and mitochondrial function in fibroblasts derived from XPG patients displaying the XP-CS phenotype but not in those from patients displaying the XP phenotype alone. Mitochondrial isolations are being performed to confirm the presence of XPG in these organelles, as well as to better evaluate mitochondrial function. These characterizations will be used as outcome measures to determine the efficacy of AAV mediated gene therapy for XP-CS. A functional CMV-XPG plasmid has been cloned, and is being packaged into Adeno-associated virus (AAV). Further analyses in XP and XP-CS patient induced pluripotent stem cells differentiated into neurons and an XPG mouse model which closely replicates the human phenotype will yield useful information regarding the dual roles of this protein and provide data to support translation of gene therapy for CS. If successful, this will be the first therapeutic measure to demonstrate correction of the debilitating effects of XP-CS.
Gene Therapy for CNS Diseases 361. IV Gene Therapy Corrects Feline GM1 Gangliosidosis Long Term
Heather Edwards1, Ashley Randle1, Miguel Sena-Esteves2, Douglas Martin1 1 Auburn University, Auburn, AL, 2University of Massachusetts, Worcester, MA GM1 gangliosidosis is a fatal neurodegenerative disease that affects all ages. Intracranial injection of adeno-associated viral (AAV) gene therapy has resulted in a >6 fold increase in lifespan. While untreated GM1 cats live to 8.0±0.6 months, many treated cats are still alive at 5-6 years of age (Sci Transl Med 2014. 6, 231). To bypass the invasiveness of brain injection, AAV9 at 1.5e13 vg/kg was injected into the cephalic vein in two GM1 cats at one month of age. Currently over 2 years of age (Fig. 1A), IV treated cats have been followed with biomarkers derived from blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, abdominal ultrasound, 7T magnetic resonance imaging (MRI), and MR spectroscopy. Neurologic abnormalities are limited to mild hindlimb muscle atrophy and fine ear tremors, with remarkable preservation of brain architecture (Fig. 1B). Evaluation of CSF showed complete normalization (p
