title: In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8.
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 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.
authors.affiliation: Department of Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Kirsten E
authors.initials: KE
authors.lastname: Erger
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Conlon
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Leal
authors.forename: Robert
authors.initials: R
authors.lastname: Zori
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Bobik
authors.forename: Terence R
authors.initials: TR
authors.lastname: Flotte
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D000229:Dependovirus / Q000145:classification* / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D019883:Alkyl and Aryl Transferases; C019920:cob(I)alamin adenosyltransferase; D014805:Vitamin B 12