Patent ID: 6218187
Filing Date: 2001-04-17
Classification: A61K,C07K,C12N

Abstract:
A method to transduce mammalian hematopoietic stem cells with retroviral supernatants produced by transient transfection comprising the steps ofA) transient cotransfection of a first population of mammalian cells that can produce virus with:(i) 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 for packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral 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; and(ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;B) separation of said first population of mammalian cells from cell supernatant;C) adding adhesion molecules or antibodies to adhesion molecules to culture plates;D) growing a second population of mammalian hematopoietic stem cells on said culture plates; andE) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian hematopoietic stem cells, to transduce said second population of cells with said foreign gene,whereby target cells transduced with said foreign gene are obtained.