In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8
Authors
Erger, Kirsten E.Conlon, Thomas J.
Leal, Nicole A.
Zori, Robert
Bobik, Thomas A.
Flotte, Terence R.
Document Type
Journal ArticlePublication Date
2007-06-02Keywords
Alkyl and Aryl TransferasesAnimals
Blotting, Western
Dependovirus
Female
Gene Expression Regulation, Enzymologic
Genetic Vectors
Genome, Viral
Humans
Liver
Mice
Mice, Inbred C57BL
RNA, Messenger
Reverse Transcriptase Polymerase Chain Reaction
Transduction, Genetic
Vitamin B 12
Allergy and Immunology
Genetics and Genomics
Pediatrics
Metadata
Show full item recordAbstract
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 x 10(10) and 1 x 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 beta-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.Source
J Gene Med. 2007 Jun;9(6):462-9. Link to article on publisher's siteDOI
10.1002/jgm.1040Permanent Link to this Item
http://hdl.handle.net/20.500.14038/43802PubMed ID
17471589Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1002/jgm.1040