Suppression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insulator to a lentiviral vector.
Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin bar-rier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among indi-vidual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.
Evans-Galea
Marguerite V
MV
Department of Experimental Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Wielgosz
Matthew M
MM
Hanawa
Hideki
H
Srivastava
Deo Kumar
DK
Nienhuis
Arthur W
AW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D002877:Chromosomes, Human / Q000235:genetics / Q000821:virology; D002999:Clone Cells; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods; D005822:Genetic Vectors / Q000009:adverse effects; D005914:Globins / Q000235:genetics; D006801:Humans; D038101:Insulator Elements; D019169:Jurkat Cells; D016086:Lentivirus / Q000235:genetics*; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D012449:Safety; D016662:Virus Integration
D017931:DNA Primers; D005914:Globins