Abstract
BackgroundHematopoietic 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.Methodology/Principal FindingsUsing 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/SignificanceThis study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34+ cells.
Highlights
Gene therapy holds promise for the cure of various inherited and acquired diseases, as evidenced by the success in the treatment of X-linked severe combined immunodeficiency (SCID-X1) [1], adenosine deaminase (ADA) deficiency [2] and chronic granulomatous disease (CGD) [3]
Cryopreserved granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (PB) CD34+ cells were thawed and seeded at 1-26105cells/ml in various serum free media supplemented with a combination of cytokines, either as recommended by the manufacturer or a ‘‘standard’’ cytokine combination used in our laboratory (SCF, flt3/flk2 ligand (Flt3L) and TPO, each at 50 ng/ ml)
CD34+ cells were assessed via FACS analysis at day 4 and/or day 7 for cell expansion using counting beads and the differentiation status of the cells was determined by the expression of various primitive markers (CD34, CD38, CD133, CD90, CD135, CD117)
Summary
Gene therapy holds promise for the cure of various inherited and acquired diseases, as evidenced by the success in the treatment of X-linked severe combined immunodeficiency (SCID-X1) [1], adenosine deaminase (ADA) deficiency [2] and chronic granulomatous disease (CGD) [3]. Efficient gene delivery into target cells and the maintenance of the biological functions of these cells are crucial to the success of gene therapy. Various sources of HSCs have been used in research and clinical studies including cord blood (CB), adult bone marrow (BM) and mobilized peripheral blood (PB) HSCs. CB HSCs are relatively easier to manipulate in vitro, harbor a higher proliferative capacity and are more susceptible to gene transduction [5,6], adult HSCs are more therapeutically relevant for human gene therapy applications. Efficient gene delivery into these target cells without compromising self-renewal and multipotency 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. Sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin
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