DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Abstract

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased >300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.Adeno-associated virus (AAV) is a dependent human parvovirus capable of integration into host DNA (1). Transducing vectors based on AAV are able to transfer nonviral genes into mammalian cells and represent an alternative to the integrating retroviral vectors currently used in many gene therapy protocols. Up to 4.7 kb of foreign DNA can be packaged into stable AAV vector particles as single-stranded DNA molecules of either polarity (2). A wide variety of cell types can be transduced by AAV vectors, including cultured normal human cells (2-6). Although the wild-type provirus often integrates at a specific site on chromosome 19 (7-9), stable transductants usually contain randomly integrated vector sequences (3, 10).The transduction of nondividing cell populations such as hematopoietic stem cells, quiescent lymphocytes, neurons, and normal epithelial cells remains a major goal of gene therapy research. Retroviral vectors based on murine leukemia virus require mitosis for efficient transduction (11-13), limiting their usefulness in many gene therapy protocols. AAV vectors also require cell proliferation for efficient transduction. However, it is S phase of the cell cycle that is important. By using stationary human fibroblast cultures, we found that AAV vectors transduce S-phase cells at >200 times the frequency of non-S-phase cells (3). This preference for S-phase cells was thought to reflect the requirement for host polymerases in the conversion of single-stranded vector genomes to double-stranded transcription templates.Based on these results, we reasoned that the transduction of nondividing cells by AAV vectors might be increased by inducing unscheduled DNA repair synthesis and found that non-S-phase cells were transduced at up to 750-fold higher frequencies after treatment with DNA-damaging agents (14). These agents included y-and UV-irradiation and cisplatinum, all of which reduced the colony-forming ability of treated fibroblasts at the optimal concentrations required for increased transduction. In this report, we have tested otherThe publication costs of this article were defrayed in part by page charge payment....

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

“…13,14,19 The scAAV will suffer no such limitation and can be used with marker genes to directly determine whether a cell is permissive for rAAV transduction in all other steps irrespective of DNA synthesis. In light of the drastic treatments which have been proposed to promote rAAV transduction efficiency in vivo (ie gamma radiation, HU, topoisomerase inhibitors) 14,29 it will be prudent to evaluate whether such lengths would be either necessary or effectual using scAAV in preliminary studies. Regardless of the ability of the target cell to make the rAAV complementary strand, it is clear that these reagents provide an alternative AAV delivery system for genes that may require rapid onset.…”

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38− subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

“…Furthermore, others have demonstrated preferential transduction of cells in S-phase by AAV vectors. 47 We cannot, however, exclude the possibility of high-dose cytokines potentiating rAAVmediated transduction of CD34 + cells by other mechanisms such as up-regulation of heparan sulfate or ␣V␤5 integrin which mediate AAV entry into cells. 35,48 Indeed up-regulation of ␣v␤5 integrin on the surface of monocytes by GM-CSF facilitated adenovirus-mediated gene transfer.…”

Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters

“…Extrapolations from these numbers generate transduction efficiencies of approximately 15 infectious irradiation or adenovirus coinfection are not required to particles per positive cell for the NSE rAAV and 45 infectious achieve efficient transduction. [32][33][34][35] particles per positive cell for the PDGF rAAV.…”