Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo

Abstract

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steadystate levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.Recombinant adeno-associated virus (rAAV) vectors can safely transduce various tissues and result in persistent gene expression in vivo (16,17,27,31,(34)(35)(36). Determining the mechanism(s) and rate-limiting step(s) for transduction is of great importance in improving on these vectors' ability to transduce a larger proportion of cells in vivo. An unclarified issue is how the stable double-stranded (ds) rAAV genomes are formed from input single-stranded (ss) rAAV genomes.Following intraportal administration of vector into animals, rAAV-mediated gene expression in the liver slowly increases over a period of 4 to 6 weeks concomitant with a loss of ss rAAV genomes. During this period, some of the rAAV genomes become integrated into host chromosomal DNA as concatemers in a small fraction of the hepatocytes that take up the vector following its infusion into the mouse liver (25,26,28,35). The reason why only a small percentage of hepatocytes become stably transduced is not known, but it appears to be unrelated to the cell cycle status (25). There is increasing evidence that intermolecular recombination of rAAV genomes is involved in the formation of rAAV concatemers in the liver (25, 29), a process recently established in muscle tissue, where heteroconcatemers were shown to be formed (42).While these studies have provided important information, they do not completely resolve whether the stable ds rAAV genomes result from comple...

Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis

“…It has more recently been suggested that reannealing of complementary vDNA from separate infecting rAAV particles may be an important pathway for rAAV transduction. 16 The requirement for complementary-strand synthesis, or recruitment, is now considered to be a limiting factor in the efficiency of rAAV vectors. The transduction rate for rAAV in mouse liver has been estimated at approximately 5% of hepatocytes after portal vein infusion of 4.2 × 10 10 particles.…”

Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo

“…3 The requirement for complementary-strand synthesis, or recruitment by base pairing with a coinfecting complementary genome, is an important limiting step in rAAV transduction, which severely reduces its efficiency in many cell types. [4][5][6] Transduction by AAV-2 in mouse liver is only 1-5% at a dose where vector DNA can be detected in most of the hepatocytes. 7 This vector DNA can be activated by coinfection with the adenovirus, which promotes complementary-strand synthesis.…”

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

“…Since single-stranded viral genomes of either polarity are encapsidated with equal frequency, 1 it has been suggested that following infection, DNA strand-annealing renders the viral genomes transcriptionally active. 2,3 Others and we, on the other hand, have suggested that viral second-strand DNA synthesis is the rate-limiting step in AAV-mediated transgene expression. [4][5][6][7][8][9][10][11] We previously observed that following intravenous (i.v.)…”