TY - JOUR
T1 - Self-lnactivating lentiviral vectors with U3 and U5 modifications
AU - Iwakuma, Tomoo
AU - Cui, Yan
AU - Chang, Lung Ji
N1 - Funding Information:
We thank Dr. Zhong Chen Kou and Mr. Edward R. Mason for technical assistance. Lung-Ji Chang is a Markey Scholar of the Lucille P. Markey Charitable Trust. This work was supported by grants from the National Health and Research Development Program (NHRDP) in Canada and National Institutes of Health (HL-59412) in the United States.
PY - 1999/8/15
Y1 - 1999/8/15
N2 - Lentiviral vectors have gained much attention in recent years mainly because they integrate into nondividing host-cell genomes. For clinical applications, 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 includes a packaging vector pHP, a transducing vector pTV, and an envelope-encoding plasmid pHEF-VSVG. Cotransfection of these three plasmids into TE671 human rhabdomyosarcoma cells routinely yields 105-106 infectious units per milliliter in 24 h. Here we have extensively modified long terminal repeats (LTRs) of pTV to generate a safer lentiviral vector system. The 5' U3 was replaced with a truncated cytomegalovirus (CMV) immediate early (IE) enhancer/TATA promoter and the 3' U3 (except for the integration attachment site) was also deleted. These modifications resulted in a vector with 80% wild-type vector efficiency. Further deletion of 3' U5 impaired vector function; however, this problem was solved by replacing the 3' U5 with bovine growth hormone polyadenylation (bGHpA) sequence. The pTV vector containing all these modifications including the 5' promoter substitution, the 3' U3 deletion, and the substitution of 3' U5 with bGHpA exhibited a self- inactivating (SIN) phenotype after transduction, transduced both dividing and nondividing cells at similar efficiencies, and produced vector titers twice as high 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 deletion of 5' U5 impaired vector efficiency, suggesting that the 5' U5 has critical roles in vector function.
AB - Lentiviral vectors have gained much attention in recent years mainly because they integrate into nondividing host-cell genomes. For clinical applications, 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 includes a packaging vector pHP, a transducing vector pTV, and an envelope-encoding plasmid pHEF-VSVG. Cotransfection of these three plasmids into TE671 human rhabdomyosarcoma cells routinely yields 105-106 infectious units per milliliter in 24 h. Here we have extensively modified long terminal repeats (LTRs) of pTV to generate a safer lentiviral vector system. The 5' U3 was replaced with a truncated cytomegalovirus (CMV) immediate early (IE) enhancer/TATA promoter and the 3' U3 (except for the integration attachment site) was also deleted. These modifications resulted in a vector with 80% wild-type vector efficiency. Further deletion of 3' U5 impaired vector function; however, this problem was solved by replacing the 3' U5 with bovine growth hormone polyadenylation (bGHpA) sequence. The pTV vector containing all these modifications including the 5' promoter substitution, the 3' U3 deletion, and the substitution of 3' U5 with bGHpA exhibited a self- inactivating (SIN) phenotype after transduction, transduced both dividing and nondividing cells at similar efficiencies, and produced vector titers twice as high 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 deletion of 5' U5 impaired vector efficiency, suggesting that the 5' U5 has critical roles in vector function.
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U2 - 10.1006/viro.1999.9850
DO - 10.1006/viro.1999.9850
M3 - Article
C2 - 10441560
AN - SCOPUS:0033566372
SN - 0042-6822
VL - 261
SP - 120
EP - 132
JO - Virology
JF - Virology
IS - 1
ER -