Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning

Abstract

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

Genetic protection of repopulating hematopoietic cells with an improved MDR1‐retrovirus allows administration of intensified chemotherapy following stem cell transplantation in mice

“…32 The results shown here suggest that a limited gene transfer efficiency in the engineered graft may be sufficient to achieve protection from lethal hematotoxicity in clinical trials. Similar, if not increased, gene transfer efficiency compared to that seen in the present study has recently been reported from primate models of retroviral gene transfer, 33,34 as well as in repopulating human BM cells that have been analyzed as xenografts in immunodeficient mice, 28,35,36 and also in a clinical study. 20 The moderate transduction rate obtained in the present mouse model therefore indicates that SF1m might also be effective in human gene therapy.…”

Non-human primate dendritic cells

“…The greater effect of Flt3-L on the mDC population has also been observed in rhesus macaques, where a seven-to 57-fold increase in mDC versus a 4.7-to fivefold increase in pDC has been reported [15,17]. Rhesus monkeys are of sufficient weight and size to enable leukapheresis procedures to be performed, and growth factor-mobilized DC or DC precursors, including CD34 ϩ stem cells, can be enriched in this manner [49,50,52,69,85]. Flt3-L also mobilizes immature DC into parenchymal organs in rhesus monkeys, including liver and kidney [84].…”

Impact of irradiation and immunosuppressive agents on immune system homeostasis in rhesus macaques

“…A polystyrene spoiler was used to ensure adequate superficial dose. An irradiation dose of 2-4 Gy is within a range used in non-ablative allogeneic transplantation protocols [50][51][52][53]. [19,23,24].…”