In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8

• Published in: The journal of gene medicine 2007-06, Vol.9 (6), p.462-469
• Main Authors: Erger, Kirsten E., Conlon, Thomas J., Leal, Nicole A., Zori, Robert, Bobik, Thomas A., Flotte, Terence R.
• Format: Article
• Language: English
• Subjects: AAV pseudotypes; Adeno-associated virus; Alkyl and Aryl Transferases - genetics; Alkyl and Aryl Transferases - metabolism; Animals; Blotting, Western; Cytomegalovirus; Dependovirus - classification; Dependovirus - genetics; Female; Gene Expression Regulation, Enzymologic; Gene therapy; Genetic Vectors; Genome, Viral - genetics; Humans; Liver - cytology; Liver - metabolism; liver gene transfer; metabolic disorders; methylmalonic aciduria; Mice; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism; Transduction, Genetic; Vitamin B 12 - metabolism
• ISSN: 1099-498X; 1521-2254
• DOI: 10.1002/jgm.1040

Background Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000‐fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B12, adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl‐CoA mutase (MCM). Methods The human ATR cDNA was cloned into a recombinant adeno‐associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 × 1010 and 1 × 1011 particles. Eight weeks post‐injection RNA, genomic DNA and protein were then extracted and analyzed. Results Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken β‐actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3‐ to 5‐fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. Conclusions These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments. Copyright © 2007 John Wiley & Sons, Ltd.