Gene Targeting by Adeno-Associated Virus Vectors Is Cell-Cycle Dependent

Abstract

Adeno-associated virus (AAV) vectors can be used to introduce site-specific mutations into homologous chromosomal sequences. There are many potential applications of this technique, but the process of AAV-mediated gene targeting and factors that influence targeting efficiency are not completely understood. We investigated the dependence of AAV-mediated gene targeting on the host cell-cycle status. The frequency of gene targeting by AAV vectors was compared in dividing and serum-arrested normal human fibroblast cultures. Gene targeting occurred in arrested fibroblast cultures at 0.15 to 1.1% the frequency of dividing cultures, and only took place in cells that had undergone DNA synthesis. Gene targeting was also reduced when DNA synthesis was inhibited by hydroxyurea.

The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

“…Importantly, MSCs, which bear differentiation potential to multiple lineages, such as bone, neurons, and cardiac tissues, have been effectively transduced with AAV serotype 2 vectors (Kumar et al, 2004;Chng et al, 2007). In concordance with the differential availability of the cellular DNA repair factors that mediate HDR or nonhomologous end-joining (NHEJ) throughout the cell cycle, AAV-mediated gene targeting has been shown to be dependent on the cell-cycle phase (Trobridge et al, 2005). Consequently, it has been speculated that transient cell-cycle arrest can affect the efficiency of ZFN-and AAV-mediated genome editing.…”

Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

“…Liu et al (2004) showed that, by enriching the targeted cells at G1/S phase of the cell cycle, the rAAV-mediated gene-targeting frequency was increased (w2 folds) in 293 cells. This strategy also works in normal human fibroblast cultures; the rAAV-mediated gene targeting was w100 folds higher in dividing cells than in arrested cells, and only occurred in cells undergoing DNA synthesis (Trobridge et al, 2005).…”

The Role of DNA Repair Pathways in Adeno-Associated Virus Infection and Viral Genome Replication / Recombination / Integration

“…Genotoxic treatment, which significantly augments rAAV genome integrations, does not affect gene targeting efficiency (Hirata & Russell, 2000). In addition, rAAV gene targeting occurs preferentially in S-phase cells and does not take place at an appreciable level in terminally differentiated murine skeletal muscle fibers (Liu et al, 2004;Trobridge et al, 2005). Moreover, the cell cycle dependence has not clearly been demonstrated in rAAV integration and a study has demonstrated a readily appreciable level of rAAV integration in terminally differentiated cardiomyocytes and skeletal myofibers (Inagaki et al, 2007a).…”