436. Recombinant Adeno-Associated Virus Gene Therapy for Cob(I)Alamin Adenosyltransferase Deficiency

Abstract

Metabolism of inactive cobalamins, both in the recycling and assimilation from dietary sources, into the B12 coenzymes is vital for various metabolic pathways. Disruption of vitamin B12 coenzyme synthesis has been linked to chronic liver disease, cancer, elevated serum homocysteine (possible risk factor for heart disease), increased rate of DNA damage and impaired cognitive function. Vitamin B12 (cyano-cobalamin, CNCbl) is a precursor of two B12 coenzymes, adenosyl-cobalamin (AdoCbl) and methyl-cobalamin (CH3Cbl), which are instable due to a labile upper ligand to the cobalt atom. The assimilation and recycling pathway consists of a multi-enzyme pathway, the last step being the conversion of cob(I)alamin to AdoCbl by the ATP dependent cob(I)alamin adenosyltransferase (ATR). In humans, methylmalonyl-CoA mutase (MCM), responsible for the conversion of propionyl-CoA to the TCA cycle intermediate succinyl-CoA, is dependent on AdoCbl. Propionyl-CoA is produced by the catabolism of the amino acids valine, isoleucine, methionine, and threonine, as well as thymine, cholesterol and odd-chain fatty acids. The impaired activity of MCM, resulting from the lack of coenzyme B12 (AdoCbl), leads to methylmalonic aciduria (MMA), a disease that is often fatal in the first years of life with a frequency of 1/50,000 births. MMA is diagnosed primarily by a buildup of methylmalonyl-CoA in the serum and in the urine. We have obtained human fibroblast cell lines from patients with known ATR deficiencies. Identification of mutations, generally seen to be a single nucleotide or missense mutation, have been performed on most. The human ATR cDNA is 753 bp, the appropriate length for use in rAAV gene therapy vector. It has been cloned into our rAAV vector driving expression of hATR from the hybrid CMV enhancer chicken-beta actin promoter and pseudotyped in AAV 2 and 8 capsids. Next, cell lines were stably transfected with an ATR-neo plasmid and selected for using G418. Levels of methylmalonic acid in the tissue culture medium is measured using tandem mass spectroscopy and an enzyme assay for the conversion of cob(I)alamin to AdoCbl with the ATR enzyme. RESULTS: Overexpression of hATR message from the pCB-hATR vector in patient and control cell lines was confirmed by semi-quantitative RT-PCR. Transgene expression in neomycin resistant clones was 3 to 7 times greater than endogenous expression. Correction of deficient patient cell lines will be assayed at varying doses of rAAV virus. Further experiments will include testing vector expressed ATR mRNA and increased enzyme acitivity in the muscle and liver tissues of C57Bl/6 mice.