Self-inactivating lentiviral vectors with U3 and U5 modifications

Lentiviral vectors have gained much attention in recent years mainly becaus e they integrate into nondividing host-cell genomes. For clinical applicati ons, a safe and efficient lentiviral vector system is required. Previously, we have established a human immunodeficiency virus type 1 (HIV-1)-derived three-plasmid lentiviral vector system for viral vector production which in cludes a packaging vector pHP, a transducing vector pTV, and an envelope-en coding plasmid pHEF-VSVG. Cotransfection of these three plasmids into TE671 human rhabdomyosarcoma cells routinely yields 10(6)-10(6) infectious units per milliliter in 24 h. Here we have extensively modified long terminal re peats (LTRs) of pTV to generate a safer lentiviral vector system. The 5' U3 was replaced with a truncated cytomegalovirus (CMV) immediate early (IE) e nhancer/TATA promoter and the 3' U3 (except for the integration attachment site) was also deleted. These modifications resulted in a vector with 80% w ild-type vector efficiency. Further deletion of 3' U5 impaired vector funct ion; however, this problem was solved by replacing the 3' U5 with bovine gr owth hormone polyadenylation (bGHpA) sequence. The pTV vector containing al l these modifications including the 5' promoter substitution, the 3' U3 del etion, and the substitution of 3' U5 with bGHpA exhibited a self-inactivati ng (SIN) phenotype after transduction, transduced both dividing and nondivi ding cells at similar efficiencies, and produced vector titers twice as hig h as that of the wild-type construct. Thus, both safety and efficacy of the HP/TV vector have been improved by these LTR modifications, Further deleti on of 5' U5 impaired vector efficiency, suggesting that the 5' U5 has criti cal roles in vector function. (C) 1999 Academic Press.