title: A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.
abstract: Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.
authors.affiliation: Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA 19140, USA.
authors.forename: Biao
authors.initials: B
authors.lastname: Dong
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Moore
authors.forename: Jihong
authors.initials: J
authors.lastname: Dai
authors.forename: Sean
authors.initials: S
authors.lastname: Roberts
authors.forename: Kirk
authors.initials: K
authors.lastname: Chu
authors.forename: Philipp
authors.initials: P
authors.lastname: Kapranov
authors.forename: Bernard
authors.initials: B
authors.lastname: Moss
authors.forename: Weidong
authors.initials: W
authors.lastname: Xiao
mesh_terms: D003593:Cytoplasm / Q000821:virology; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D016679:Genome, Viral; D057809:HEK293 Cells; D006367:HeLa Cells; D006378:Helper Viruses / Q000235:genetics; D006801:Humans; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics