Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells
View Patent ↗The invention provides a novel retroviral packaging system, in which retroviral packaging plasmids and packagable vector transcripts are produced from high expression plasmids after stable or transient transfection in mammalian cells. High titers of recombinant retrovirus are produced in these transfected mammalian cells and can then transduce a mammalian target cell by cocultivation or supernatant infection. The methods of the invention include the use of the novel retroviral packaging plasmids and vectors to transduce primary human cells, including T cells and human hematopoietic stem cells, with foreign genes by cocultivation or supernatant infection at high efficiencies. The invention is useful for the rapid production of high titer viral supernatants, and to transduce with high efficiency cells that are refractory to transduction by conventional means.
1. A retroviral packaging plasmid for the production of high titers of recombinant retrovirus in human cells comprising one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5′LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site,
wherein said helper DNA sequence codes for ecotropic MMLV gag and pol and an envelope protein selected from a xenotropic, amphotropic, ecotropic or polytropic envelope protein.