Chromosomal Integration and Homologous Gene Targetingby Replication-Incompetent Vectors Based on the Autonomous ParvovirusMinute Virus ofMice

Abstract

The molecular mechanisms responsible for random integration and gene targeting by recombinant adenoassociated virus (AAV) vectors are largely unknown, and whether vectors derived from autonomous parvoviruses transduce cells by similar pathways has not been investigated. In this report, we constructed vectors based on the autonomous parvovirus minute virus of mice (MVM) that were designed to introduce a neomycin resistance expression cassette (neo) into the X-linked human hypoxanthine phosphoribosyl transferase (HPRT) locus. High-titer, replication-incompetent MVM vector stocks were generated with a two-plasmid transfection system that preserved the wild-type characteristic of packaging only one DNA strand. Vectors with inserts in the forward or reverse orientations packaged noncoding or coding strands, respectively. In human HT-1080 cells, MVM vector random integration frequencies (neo ؉ colonies) were comparable to those obtained with AAV vectors, and no difference was observed for noncoding and coding strands. HPRT gene-targeting frequencies (HPRT mutant colonies) were lower with MVM vectors, and the noncoding strand frequency was threefold greater than that of the coding strand. Random integration and gene-targeting events were confirmed by Southern blot analysis of G418-and 6-thioguanine (6TG)-resistant clones. In separate experiments, correction of an alkaline phosphatase (AP) gene by gene targeting was nine times more effective with a coding strand vector. The data suggest that single-stranded parvoviral vector genomes are substrates for gene targeting and possibly for random integration as well.The Parvoviridae are nonenveloped viruses with an encapsidated, single-stranded, linear DNA genome of approximately 5 kb (3). Autonomous parvoviruses such as minute virus of mouse (MVM) are a diverse subgroup that differs from dependoviruses such as adeno-associated virus (AAV) in their ability to productively infect host cells in the absence of additional helper viruses (9). Unlike AAV, autonomous parvoviruses have not been shown to integrate into host chromosomes. Viral genomes were not detected in high-molecular-weight DNA during a lytic MVM infection (44) or in cells persistently infected with the immunosuppressive strain of MVM (45). Corsini et al. demonstrated that wild-type MVM can integrate into an episome containing the left terminal origin of replication in a reaction that depended on the viral nonstructural (NS) protein NS1 (7,8). This reaction is analogous to AAV integration at viral repeat sequences that bind the AAV Rep proteins (33, 52). While Rep-dependent site-specific integration of AAV can occur at a sequence in human chromosome 19 similar to the viral terminal repeats (32, 49), murine and human genomes do not contain sequences homologous to the MVM binding and nicking sites for NS1 (see Materials and Methods), which may account for the lack of detectable MVM integration.AAV vectors have been extensively developed for gene transfer applications (reviewed in references 6, 18, and 47) and are curre...

Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome

“…In MVM, it has been shown that specific elements within the REH between nt 4489 to 4636 and nt 4636 to 4695 are necessary for efficient replication of MVM duplex DNA (59). During the development of the recombinant MVMp vector (rMVMp), a large portion of cis element remained at the 3= end (nt 4631 to 5149) of the rMVMp genome (60). However, how these cis elements outside the hairpins facilitate viral DNA replication has not been studied.…”

AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the ProteasomeIn Vivo

“…The field of AAV gene therapy research continues to be dominated by gene addition approaches using cDNA cassettes driven by heterologous promoter and enhancer sequences to augment expression of a mutated disease gene. Yet, drawbacks to this methodology abound including unregulated and transient episomal expression (Garrick et al, 1998;Russell and Kay, 1999), random integration (Hendrie et al, 2003), increased oncogenic risks (Donsante et al, 2007), and the inability to treat genetically dominant disorders arising from misfolded or abnormally functioning proteins.…”

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

“…Like conventional gene targeting with plasmid DNA, increasing the length of homology between the AAV vector and the target locus (Hirata and Russell, 2000) and creating doublestrand breaks at the target locus (Miller et al, 2003;Porteus et al, 2003) increase the frequency of gene targeting. However, the active participant in AAV-mediated gene targeting appears to be the single-stranded AAV vector genome (Hirata and Russell, 2000;Hendrie et al, 2003). As microinjection of AAV vector genomes does not lead to efficient gene targeting (Liu et al, 2004), viral proteins or other components of vector virion particles must influence the reaction.…”