115. In Vivo Expression of Human Cob(I)alamin Adenosyltransferase Using rAAV Serotypes 1,2 and 8, an Approach to Gene Therapy for Methylmalonic Aciduria (MMA)

Abstract

Top of pageAbstract Methylmalonic aciduria (MMA) is an autosomal recessive disease with an incidence of approximately 1 in 50,000 people in the United States. Symptoms include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000 fold greater then normal. Currently, eight different complementation groups (cblA-H) for MMA have been described. CblB MMA is caused by a deficiency in the enzyme Cob(I)alamin adenosyltransferase (ATR). ATR functions within the mitochondria matrix in the final conversion of inactive vitamin B12 to coenzyme B12, adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). AdoCbl and MCM catalyze methylmalonyl-CoA to succinyl-CoA, which is further metabolized in the TCA cycle. The human ATR cDNA was cloned into an rAAV vector containing the hybrid cytomegalovirus (CMV) enhancer with the chicken beta-actin promoter and AAV2-inverted terminal repeats and pseudotyped in AAV1, 2 or 8 capsids. AAV serotypes 2 and 8 were delivered by portal vein injection to six week old female C57/Bl6 mice at a low dose of 1|[times]|1010 particles and a high dose of 1|[times]|1011 particles. Eight-weeks post injection, these animals were sacrificed and their livers harvested. RNA, genomic DNA and protein were then extracted from these tissues and used for reverse transcriptase PCR, real time PCR, western blotting and ATR enzyme assays. Portions of the liver were also reserved for immunostaining. AAV1 and 2 were delivered intramuscularly to the tibialis anterior of C57/Bl6 at a low dose of 1|[times]|1010 and a high dose of 1|[times]|1011 particles. These animals were sacrificed at ten weeks and the injected muscles were harvested. RT-PCR was used to confirm transcript levels, and immunostaining was performed for tissue biodistribution. RESULTS: Using primer pairs specific to the CMV enhancer/chicken beta-actin promoter of rAAV vectors, genome copies/cell in the liver were found to 0.03, 2.03 and 0.10 for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Using primer pairs specific for the vector genome, a substantial increase of message was seen with AAV8 high dose over AAV2 dose. Similar mRNA expression was seen between the AAV8 low dose and AAV2 high dose animal tissues. Western blotting performed on mitochondrial protein extracts demonstrated the presence of pre-ATR and post-translocated protein in all three dose groups. Levels were comparable to control levels in the AAV8 low dose and AAV2 high dose animals. Levels three to five fold higher than control levels were observed in AAV8 high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the AAV8 high dose animals, compared to 9% and 5% transduction in AAV2 high dose and AAV8 low dose animals, respectively. In muscle, an increase of rAAV ATR message was seen in AAV1 high dose animal muscles as compared to AAV2 high dose animals.