Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors.
Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.
Department of Human Genetics, University of Pittsburgh, Graduate School of Public Health, PA, USA.
J
J
Mannion-Henderson
A
A
Kemp
T
T
Mohney
M
M
Nimgaonkar
J
J
Lancia
M T
MT
Beeler
J
J
Mierski
A B
AB
Bahnson
E D
ED
Ball
J A
JA
Barranger
D018952:Antigens, CD34 / Q000032:analysis*; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005962:Glucosylceramidase / Q000235:genetics*; D006098:Granulocytes / Q000201:enzymology; D006412:Hematopoietic Stem Cells / Q000201:enzymology*; D006801:Humans; D008264:Macrophages / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*
D018952:Antigens, CD34; D004247:DNA; D005962:Glucosylceramidase