A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication

Abstract

A recombinant plasmid carrying an infectious adeno-associated viral genome was constructed that differs in several key respects from previously described recombinants. First, the vector is pEMBL8(+), which allows isolation of viral plus and minus strands. Second, the inserted viral sequences contain two XbaI cleavage sites that flank the viral coding domain. These inserts do not affect replication of the virus, and they allow nonviral sequences to be easily inserted between the cis-acting terminal repeats of adeno-associated virus. Third, the viral genome is flanked by PvuII cleavage sites that allow the entire, infectious viral chromosome to be excised from plasmid sequences in vitro. Viral DNA was replicated more efficiently within adenovirus-infected 293 cells if it was excised from the vector with PvuII before transfection. Presumably, the increased efficiency reflects bypass of the excision step which must normally precede replication when a recombinant plasmid enters the nucleus. The ability to bypass the excision step was exploited to search for a viral function required specifically for excision of the viral genome from the integrated state. None of the mutants tested identified a gene product required for excision that was not also essential for replication. The ability to produce pure populations of viral plus and minus strands was used to demonstrate that both strands are infectious.

Suppression of human alpha-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors.

“…Plasmids, Viruses, and Cells. Recombinant clones of AAV plasmids have been described (15,16), and were obtained from Dr. K. J. Samulski (University of North Carolina, Chapel Hill, NC). Two recombinant AAV plasmids containing the neo R gene driven by either the SV40 promoter (pAAV-Neo), or the TK promoter (pWP-19), have also been described (17,18).…”

Baculovirus-Derived Vectors for Immunization and Therapeutic Applications

“…The classic technique for the production of AAVs involves HEK293, A549, or HeLa cells cotransfected with two plasmids, one containing the recombinant DNA (recombinant adeno-associated virus plasmid) and the other the rep and cap genes (pHelper). The cells are then infected with a helper virus such as an adenovirus or a herpes simplex virus to allow the replication of the recombinant AAV (Samulski et al, 1987). The downside to this methodology is the possibility of undesired recombination events between plasmids and that purification of the helper virus is time consuming, and the scale-up process is difficult to achieve.…”

Light‐activated gene transduction enhances adeno‐associated virus vector–mediated gene expression in human articular chondrocytes

“…Previous studies have indicated that naked DNA, retroviral, adenoviral, and herpesvirus‐based vectors are, in their present forms, less than ideal for use in human gene therapy (17, 19). The deficiencies of these vectors have led us and others to test the recombinant adeno‐associated virus (rAAV) vector, which is derived from an endemic, nonpathogenic, parvovirus (20–24), for its potential use in gene therapy for joint diseases (25–31). Indeed, rAAV vectors have several properties that make them attractive for use in musculoskeletal diseases (24).…”