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 second gene of interest are forcibly coupled by means of an internal ribosome entry site. The vector provides high-level expression of the coselected gene in approximately 90% of transduced cells and has been used to express an endoplasmic reticulum-targeted single-chain antibody (intrabody) directed against a subunit of the interleukin-2 receptor, IL-2R alpha. In the established T cell line Kit225 and also in primary human T cells stably transduced with the intrabody 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 the IL-2R alpha-negative cells, consistent with a lack of high-affinity IL-2 receptors. Pseudotyping of the HIV-1 core with the vesicular stomatitis virus G protein improved particle stability by two- to three-fold and enhanced vector entry into established T cell lines up to 230-fold. Vector entry into primary human T cells was most efficient 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 types.
Richardson
J H
JH
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Hofmann
W
W
Sodroski
J G
JG
Marasco
W A
WA
D000818:Animals; D018920:Coculture Techniques; D015536:Down-Regulation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D015375:Receptors, Interleukin-2 / Q000276:immunology* / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism* / Q000821:virology; D014161:Transduction, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics
D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D014759:Viral Envelope Proteins