HIGH-TITER HUMAN-IMMUNODEFICIENCY-VIRUS TYPE 1-BASED VECTOR SYSTEMS FOR GENE DELIVERY INTO NONDIVIDING CELLS

Previously we designed novel pseudotyped high-titer replication defect ive human immunodeficiency virus type 1 (HIV-1) vectors to deliver gen es into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then we have made several improvements wi th respect to the safety, flexibility, and efficiency of the vector sy stem. A three-plasmid expression system is used to generate pseudotype d HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harborin g a reporter gene such as neo, ShlacZ (encoding a phleomycin resistanc e/beta-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The p ackaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5' and 3' long terminal repeats, the Nef function, and the presumed packaging signal . Using G418 selection, we routinely obtained vector particles pseudot yped with the vesicular stomatitis virus G glycoprotein (VSV-G) with t iters of up to 8 x 10(7) CFU/mu g of p24, provided that a functional T at coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 x 10(6) to 8 x 10( 6) CFU/mu g of p24. Packaging constructs with a mutation within the in tegrase (IN) core domain profoundly affected colony formation and expr ession of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of o ther Env proteins to allow formation of pseudotyped HIV-1 particles, T he rabies virus and Mokola virus G proteins yielded high-titer infecti ous pseudotypes, while the human foamy virus Env protein did not. Usin g the improved vector system, we successfully transduced contact-inhib ited primary human skin fibroblasts and postmitotic rat cerebellar neu rons and cardiac myocytes, a process not affected by the lack of the a ccessory proteins.