Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue.
Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.
Department of Anatomy and Cell Biology and Department of Internal Medicine, University of Iowa Medical Center, Iowa City, Iowa, USA.
D
D
Duan
P
P
Sharma
J
J
Yang
Y
Y
Yue
L
L
Dudus
Y
Y
Zhang
K J
KJ
Fisher
J F
JF
Engelhardt
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000821:virology*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D020079:Terminal Repeat Sequences; D013997:Time Factors; D014162:Transfection
D017931:DNA Primers; D004279:DNA, Viral; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins