Plerixafor+G-CSF-Mobilized Blood From Thalassemic Mice Provides High Yields Of Long-Term Repopulating Cells With Enhanced Engrafting Capacity : Implications For Human Stem Cell Gene Therapy

A durable therapeutic effect of gene therapy in β-thalassemia largely depends on the infusion of high numbers of genetically-modified hematopoietic stem cells (HSCs) with an enhanced engrafting potential.These requirements are necessary to compensate for the lack of a selective advantage of the thalassemic HSCs over the endogenous unmodified stem cells, especially if a nonmyeloablative conditioning is used. The higher HSC yields acquired by the mobilized peripheral blood and their faster time to engraftment as compared to bone marrow,qualifies mobilized blood cells as the preferable HSC source for genetic modification.

To determine the optimal mobilization strategy for thalassemia, thalassemic (Hbb^th345.2⁺) mice were mobilized with G-CSF or Plerixafor or Plerixafor+G-CSF and the yields of hematopoietic stem (Lin^-sca-1⁺ckit⁺ cells-LSK cells) and progenitor cells (CFU-GM) were assayed by flow cytometry (FCM) and methylcellulose cultures and calculated in absolute numbers. C-kit+ progenitors mobilized by all the above approaches, were compared in terms of migration capacity towards SDF-1a, cell cycle status and CXCR4 and CD26 expression. To investigate the engrafting and long-term repopulation capacity of cells mobilized by different modes, whole blood from mobilized thalassemic 45.2⁺mice was infused into lethally irradiated PepBoy (45.1⁺) mice at a 3:1 (donors:recipient) ratio or as equal number of LSK cells, along with a fixed number of competitor 45.1+ cells (0.5x10⁶). Donor chimerism was tested monthly by the frequency of 45.2+ cells in the recipients’ blood by FCM analysis. Six months after primary transplantation, 2x10⁶bm cells from the competitively engrafted mice were injected into lethally-irradiated secondary recipients in a noncompetitive manner.

Plerixafor+G-CSF yielded significantly higher numbers of LSKs and CFU-GM over G-CSF-alone or Plerixafor-alone (p≤0.004) while Plerixafor-alone didn’t further impove mobilization over G-CSF-alone. After competitive transplantation, Plerixafor+G-CSF-mobilized cells achieved earlier hematological reconstitution and increased donor chimerism over G-CSF- or Plerixafor-alone-mobilized cells (p≤0.05). To test whether the higher levels ofengraftment simply reflect the presence of higher numbers of stem/progenitor cells in the Plerixafor+G-CSF-mobilized graft (p<0.05) rather than an inherent qualitative difference of these cells, equal numbers (1X10⁴) of LSK cells from each mobilization group was transplanted in a competitive manner. Plerixafor+G-CSF-mobilized LSKs showed again significantly higher engraftment levels than the single-agent mobilized LSKs (p<0.05), thus implying a qualitative benefit of this type of graft. This benefit was also evident after secondary transplantation, as the Plerixafor+G-CSF-cell secondary recipients exhibited the same or even higher levels of engraftment compared to primary engraftment, in contrast to the reduced secondary donor chimerism of mice that underwent transplantation from Plerixafor- or G-CSF- bone marrow primary chimeras, respectively. Plerixafor-mobilized cells showed decreased competiveness over G-CSF- or Plerixafor+G-CSF cells both in primary and secondary transplantations, a finding which could be attributed to the mobilization by Plerixafor of more actively proliferating cells compared to G-CSF or the Plerixafor+G-CSF combination (S/G2/M:G0+G1 ratio, p≤0.03) as well as to the higher CD26 expression in c-kit+ Plerixafor-mobilized progenitors (p=0.04).

In conclusion, Plerixafor+G-CSF-mobilized peripheral blood represents an optimal graft for gene therapy of β-thalassemia because of the higher HSC-yields, the faster hematological recovery and the superiority in long-term engraftment over single-agent-mobilized blood cells. The therapeutic implications however, are not restricted to thalassemia but are of obvious relevance to all other stem cell gene therapy applications.