Pathways of removal of free DNA vector ends in normal and DNA-PKcs-deficient SCID mouse hepatocytes transduced with rAAV vectors.
Elucidation of the mechanisms of transformation of single-stranded (ss) recombinant adeno-associated virus (rAAV) vector genomes into a variety of stable double-stranded (ds) forms is key to a complete understanding of rAAV vector transduction in vivo. Ds monomer genome formation and cellular ds DNA break (DSB) repair pathways that remove free vector ends toxic to cells, presumably play a central role in this process. By delivering rAAV and naked ds linear DNA vectors into livers of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-deficient severe combined immunodeficiency (SCID) and wild-type mice, we demonstrate the presence of three major pathways for free ds vector end removal: (1) DNA-PKcs-dependent self-circularization, (2) DNA-PKcs-independent self-circularization, and (3) DNA-PKcs-independent concatemerization. By using the DNA-PKcs-independent pathways, mouse hepatocytes efficiently removed free ds rAAV vector ends even in the absence of DNA-PKcs. Our studies suggest a hierarchical organization of these processes; self-circularization is the preferred pathway over concatemerization, although the former has a limited capacity to remove free vector ends. These studies shed new light on the molecular mechanisms of rAAV vector transduction in vivo.
Nakai
Hiroyuki
H
Departments of Pediatrics and Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
Storm
Theresa A
TA
Fuess
Sally
S
Kay
Mark A
MA
D020413:3' Untranslated Regions / Q000378:metabolism; D000818:Animals; D004270:DNA, Circular / Q000032:analysis; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D000229:Dependovirus / Q000235:genetics*; D005164:Factor IX / Q000235:genetics; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D009154:Mutation; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020079:Terminal Repeat Sequences / Q000235:genetics; D014161:Transduction, Genetic
D020413:3' Untranslated Regions; D004270:DNA, Circular; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D005164:Factor IX; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases