Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells.
BACKGROUND
Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34(+) HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.


RESULTS
Using commercially available G-CSF mobilized peripheral blood (PB) CD34(+) cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.


CONCLUSIONS
This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+) cells.
Millington
Michelle
M
Johnson and Johnson Research Pty Ltd., Eveleigh, New South Wales, Australia.
Arndt
Allison
A
Boyd
Maureen
M
Applegate
Tanya
T
Shen
Sylvie
S
D018952:Antigens, CD34 / Q000276:immunology*; D001769:Blood; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor / Q000008:administration & dosage; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D014161:Transduction, Genetic
D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D016179:Granulocyte Colony-Stimulating Factor