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.
Duan
D
D
Department of Anatomy and Cell Biology and Department of Internal Medicine, University of Iowa Medical Center, Iowa City, Iowa, USA.
Sharma
P
P
Yang
J
J
Yue
Y
Y
Dudus
L
L
Zhang
Y
Y
Fisher
K J
KJ
Engelhardt
J F
JF
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