Preclinical Assessment of Human Hematopoietic Progenitor Cell Transduction in Long-Term Marrow Cultures

Abstract

Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1,427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neo^r-containing vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4–5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer. In nonmurine model systems, significant in vivo proliferation of genetically modified hematopoietic stem cells remains a desirable but elusive objective. One limiting factor to successful long-term gene transfer is the relative quiescence of hematopoietic stem cells (HSCs), which makes them resistant to retrovirus-mediated gene transfer. Even if stable gene transfer were achieved ex vivo, the genetically modified HSCs infused in most applications would not likely be among the small minority of HSCs that contribute actively to hematopoiesis in vivo at any given time point, particularly in the absence of in vivo selection. In the work reported here, an approach to stem cell activation and transduction, previously shown to be promising in a canine model, is evaluated using human hematopoietic cells and a spectrum of Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors. The results of these studies further support the use of in vitro hematopoietic microemironments to facilitate hematopoietic cell gene transfer.