INTRABODY-MEDIATED KNOCKOUT OF THE HIGH-AFFINITY IL-2 RECEPTOR IN PRIMARY HUMAN T-CELLS USING A BICISTRONIC LENTIVIRUS VECTOR

A bicistronic human immunodeficiency virus type 1 (HIV-1)-based vector is described in which the expression of a selectable marker and a sec ond gene of interest are for coupled by means of an internal ribosome entry site. The vector provides high-level expression of We coselected gene in approximately 90% of transduced cells and has been used to ex press an endoplasmic reticulum-targeted antibody (intrabody) directed against a subunit receptor, IL-2R alpha. In the established T cell lin e and also in primary human T cells stably transduced with the intrabo dy vector, the cell surface expression of IL-2R alpha could be reduced to a low or undetectable level. Responsiveness to IL-2 was reduced 10 -fold in We IL-2R alpha-negative cells, consistent with a lack of high -affinity IL-2 receptors. Pseudotyping of the HIV-I core with the vesi cular stomatitis virus G protein improved particle stability by two- t o three-fold and enhanced vector entry into established T cell lines u p to 230-fold. Vector entry into primary human T cells was most effici ent when the amphotropic murine leukemia virus envelope was used. The forced, high-expression capability of the bicistronic vector, together with the capacity of HIV-1 vectors to infect nondividing cells, make this an attractive tool for the genetic manipulation of primary cell t ypes.