Lentiviral and murine retroviral transduction of T cells for expression of human CD40 ligand.
Efficient transduction of primary human T cells is an important goal toward treating a number of genetic defects. Patient T cells could be harvested by leukapheresis, transduced, and returned to the donor. A wide range of secreted or cell surface therapeutic proteins may be delivered in this way. The ability to produce antibodies is the consequence of interactions between T cells and B cells and lack of expression of CD40 ligand (CD40L) on T cells causes X-linked hyper-IgM syndrome (XHIM). We are investigating delivery of a normal CD40 ligand to treat this disorder. We tested promoters driving the expression of either reporter genes such as enhanced green fluorescent protein (eGFP) or human CDC40L. Using murine retroviruses, the best able to drive gene expression in T cells was the cytomegalovirus (CMV) promoter enhancer element; however, transduction efficiency was low. To achieve efficient, high-level gene expression we tested lentiviral gene delivery vectors. At a low multiplicity of infection (MOI) (0.5-2) a large fraction of target cells was transduced by lentiviral vectors (40-93%), and the strength of gene expression was high, as determined by flow cytometric analysis. We monitored the expression of eGFP or human CD40L on T cell lines and untransformed primary human T cells from normal and CD40L-deficient patients. We achieved efficient gene expression without an extended exposure to virus, and without the need for selection. These results are encouraging for efficient lentivirus-mediated transduction of refractory human cells to achieve therapeutic gene delivery.
Barry
S C
SC
Department of Pediatrics, University of Washington, Seattle 98195, USA.
Seppen
J
J
Ramesh
N
N
Foster
J L
JL
Seyama
K
K
Ochs
H D
HD
Garcia
J V
JV
Osborne
W R
WR
D000818:Animals; D015139:Blotting, Southern; D023201:CD40 Ligand; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D016086:Lentivirus / Q000235:genetics*; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D051381:Rats; D011916:Rats, Inbred F344; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*; D020079:Terminal Repeat Sequences / Q000235:genetics; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase