Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle

The skeletal muscle provides a very permissive physiological environment fo r adeno-associated virus (AAV) type 2-mediated gene transfer. We have studi ed the early steps leading to the establishment of permanent transgene expr ession, after injection of recombinant AAV (rAAV) particles in the quadrice ps muscle of mice. The animals received an rAAV encoding a secreted protein , murine erythropoietin (mEpo), under the control of the human cytomegalovi rus major immediate-early promoter and were sacrificed between 1 and 60 day s after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 w eeks, followed by a stabilization at maximal plateau values. The rAAV seque nces were analyzed by Southern blotting following neutral or alkaline gel e lectrophoresis of total DNA from injected muscles. While a high number of r AAV sequences were detected during the first 5 days following the injection , only a few percent of these sequences was retained in the animals analyze d after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stabl e gene expression at later time points are low in copy number and structure d as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests tha t the molecular transformations involved in the formation of stable concate mers may involve a rolling circle type of DNA replication.