Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors.
Three different second-generation lentiviral self-inactivating vectors containing CMV, EF1alpha and PGK promoter, respectively, and all carrying the exogenous GFP gene, were compared for expression in human B-cell precursor ALL blasts. At a comparable percentage of transduction and vector DNA copy number, CMV clearly showed better efficiency of transcription. Human bone marrow stromal cells were favored compared to the MRC-5 cell line, as support for cell viability during infection. Cells were infected and analyzed after variable culture times ranging from 4 to 12 days, to reduce the possibility of pseudotransduction. In 10/14 samples, we detected more than 20% GFP-positive cells after exposure to high-titer viral supernatants. We then tested a similar vector carrying the human CD40L cDNA and, in similar infection conditions, obtained more than 20% transduction in 6/6 samples. The levels of transduction obtained were sufficient to induce the upregulation of CD83 molecule in cocultured immature dendritic cells.
Bonamino
M
M
Centro M Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Ospedale San Gerardo, Monza, Italy.
Serafini
M
M
D'Amico
G
G
Gaipa
G
G
Todisco
E
E
Bernasconi
S
S
Golay
J
J
Biondi
A
A
Introna
M
M
D023201:CD40 Ligand / Q000235:genetics*; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000628:therapy*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014161:Transduction, Genetic / Q000379:methods
D008164:Luminescent Proteins; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins