Transient gene expression by nonintegrating lentiviral vectors.
Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.
Nightingale
Sarah J
SJ
Division of Research Immunology/Bone Marrow Transplant, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA 90027, USA.
Hollis
Roger P
RP
Pepper
Karen A
KA
Petersen
Denise
D
Yu
Xiao-Jin
XJ
Yang
Catherine
C
Bahner
Ingrid
I
Kohn
Donald B
DB
D018952:Antigens, CD34 / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D004351:Drug Resistance; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D019073:HT29 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D019868:Kanamycin Kinase / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D016662:Virus Integration
D018952:Antigens, CD34; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase; D009355:Neomycin