Use of SV40-based vectors to transduce foreign genes to normal human peripheral blood mononuclear cells.
Stable, efficient gene transfer to normal human peripheral blood mononuclear cells (PBMC) is a prerequisite for therapy of a number of diseases, both hereditary and acquired, affecting these cells. Current approaches to gene transfer to PBMC entail ex vivo mitogenic stimulation and multiple transduction steps followed by selection, usually of progenitor populations. Thus, the ability to transfer gene expression to normal, resting PBMC could complement gene transfer strategies that target dividing precursor cells. We report successful short-term transduction of human PBMC using two different SV40-derived viral vectors SV40-derivative viruses were constructed by cloning cDNAs for firefly luciferase (luc), or hepatitis B surface antigen (HBSAg), into shuttle plasmids to create the SV40 derivative viruses SVluc and SV(HBS) respectively. Both genes were cloned downstream from SV40 early promoter. Normal, resting, human PBMC were exposed to these viruses, and unselected cultured cells were assayed 24 to 48 h later for expression of transduced genes by immunochemistry and Northern blot analysis. Expression of both luciferase and HBSAg was detected using both approaches. Levels of expression of luciferase were slightly higher in PBMC which were stimulated with concanavalin A (con A). Conversely, expression of HBSAg was less in con A-stimulation did not alter infectivity of PBMC by SV40-derivative virus. While longevity and stability of expression in vitro are as yet unknown, this demonstration of successful gene transfer to resting, normal human PBMC, assayed on unselected cells, suggests that SV40-based transduction systems may be potential candidates for use in transient gene transfer to mononuclear blood cells.
Strayer
D S
DS
Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Kondo
R
R
Milano
J
J
Duan
L X
LX
D015152:Blotting, Northern; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D018076:DNA, Complementary; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008156:Luciferases / Q000032:analysis / Q000235:genetics*; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D013268:Stimulation, Chemical
D018076:DNA, Complementary; D006514:Hepatitis B Surface Antigens; D003208:Concanavalin A; D008156:Luciferases