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In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno‐associated virus (rAAV) serotypes 2 and 8
Author(s) -
Erger Kirsten E.,
Conlon Thomas J.,
Leal Nicole A.,
Zori Robert,
Bobik Thomas A.,
Flotte Terence R.
Publication year - 2007
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1040
Subject(s) - methylmalonic acidemia , biology , adeno associated virus , microbiology and biotechnology , mutase , virology , methylmalonic acid , recombinant dna , biochemistry , enzyme , endocrinology , vitamin b12 , vector (molecular biology) , gene
Abstract 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 B 12 , 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 × 10 10 and 1 × 10 11 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.