5. Differential Expression of PIT1 and PIT2 after G-CSF Mobilization Is Associated with Efficient Gene Transfer Using GALV-Pseudotyped Gammaretroviral Vectors

Abstract

The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF), however, SCF is unavailable for clinical use. A recent study of a competitive repopulation assay in the rhesus macaque model showed very inefficient engraftment of transduced G-CSF-mobilized peripheral blood (G-PBSC) CD34+ cells relative to G-CSF- and SCF-mobilized cells using amphotropic pseudotypes (Hematti P et al Blood 101, 2003, 2199|[ndash]|2205). Because G-PBSC would be the preferred target cell population for most clinical stem cell gene therapy applications, we asked whether we could achieve efficient engraftment of transduced G-PBSC in our baboon model using Phoenix-GALV pseudotype vectors. In order to better compare these results to those from previous experiments utilizing G-CSF- and SCF-primed bone marrow (G&S-BM) as a stem cell source, the first two baboons also received G-CSF-primed BM (G-BM) in a competitive repopulation. We transplanted three baboons with G-CSF-mobilized CD34+ cells transduced with GALV pseudotyped retroviral vectors. We observed high-level, persistent engraftment of transduced G-PBSC in all three animals with gene marking in granulocytes in one animal up to 60%. To determine retrovirus integration pattern of clones that contributed to long-term hematopoiesis linear amplification mediated (LAM)-PCR was used for all animal samples studied. In all animals analyzed multiple clones could be detected and in animals that received both G-PBSC and G-BM multiple clones contributed from both transduced cell populations. These in vivo marking results were not in agreement with the report by Hematti et al so we sought to determine whether the different results might be explained by the fact that different combinations of cytokines (G-CSF and SCF) for mobilization modulate receptor expression of the amphotropic (PIT2) and GALV (PIT1). Analysis of receptor expression in baboon CD34+ cells after administration of different combinations of cytokines for mobilization found receptor expression was dramatically affected by different mobilization regimens with G-CSF and SCF increasing PIT2 expression and G-CSF alone increasing PIT1 expression. The low in vivo gene marking in previous reports using amphotropic pseudotyped vectors and G-CSF alone for mobilization could be explained because in animals treated with G-CSF alone, at least in baboons, the amphotropic receptor expression was minimal while including SCF in the mobilization regimen substantially increased amphotropic receptor expression. In contrast to other studies, our high in vivo marking in G-CSF-mobilized baboon peripheral blood CD34+ cells demonstrates the feasibility of efficiently transducing G-PBSC in clinical gene therapy trials and verifies that there is no inherent stem cell deficiency in G-PBSC mobilized cells.