Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocy tes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a s urgical two-thirds partial hepatectomy, we have determined the proportion o f integrated and extrachromosomal rAAV genomes in mouse livers and their re lative contribution to stable gene expression in vivo. Plasma human coagula tion factor IX (hF.IX) levels in mice originating from a chromosomally inte grated hF.IX-expressing transposon vector remained unchanged with hepatecto my. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein i njection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10 (11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and th e average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of th ree mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless , in general, these results indicate that, in most cases, no more than simi lar to 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not int egrated, genomes are the major form of rAAV in the liver and are the primar y source of rAAV-mediated gene expression. This small fraction of integrate d genomes greatly decreases the potential risk of vector-related insertiona l mutagenesis associated with all integrating vectors but also raises uncer tainties as to whether rAAV-mediated hepatic gene expression can persist li felong after a single vector administration.