Enhanced transgene expression in quiescent and activated human CD8+ T cells.
The level of expression of retroviral vector-encoded proteins in T cells, decreasing during periods of quiescence, could be an obstacle to their clinical utility. To identify promoter systems that could increase the strength and persistence of transgene expression in primary human CD8(+) T cells, we designed a panel of Moloney retroviral vectors to express a destabilized enhanced green fluorescent protein (d4EGFP) reporter protein (t(1/2) = 4 hr). We found that the promoters phosphoglycerate kinase (Pgk), beta-actin, and long terminal repeat (LTR) produced the highest levels of d4EGFP expression in proliferating T cells, but that expression dramatically declined in quiescent cells with all promoters. To improve gene expression, we examined the effect of the beta-interferon (IFN) scaffold attachment region (SAR). This SAR augmented expression from mammalian promoters in cycling T cells, but had a small effect on maintenance of expression in resting T cells. However, when the SAR was combined with the LTR promoter, it significantly enhanced expression in resting and cycling cells. These data support use of the IFN-beta SAR with the LTR in Moloney retroviral vectors to help sustain gene expression in resting primary human CD8(+) T cells and to enhance gene expression in activated T cells.
Cooper
Laurence J N
LJ
Pediatric Oncology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA. lcooper@coh.org
Topp
Max S
MS
Pinzon
Cris
C
Plavec
Ivan
I
Jensen
Michael C
MC
Riddell
Stanley R
SR
Greenberg
Philip D
PD
D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D045744:Cell Line, Tumor; D004351:Drug Resistance; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D005839:Gentamicins / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D016899:Interferon-beta / Q000235:genetics*; D008213:Lymphocyte Activation; D045170:Matrix Attachment Regions / Q000235:genetics*; D008979:Moloney murine leukemia virus / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020079:Terminal Repeat Sequences / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
D005839:Gentamicins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016899:Interferon-beta; C010680:antibiotic G 418