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 neocontaining 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.

Engraftment of Gene-Marked Hematopoietic Progenitors in Myeloma Patients after Transplant of Autologous Long-term Marrow Cultures

“…Our preclinical studies of ex vivo gene transfer in Dexter-type LTMCs supported further clinical evaluation (Carter et al, 1992;Bienzle et al, 1994;Dubé et al, 1996;Stewart et al, 1996;Lutzko et al, 1999). These LTMCs are 1999.10:1953-1964. hem atopoietic microenvironm ents in which hematopoiesis may be maintained ex vivo for several weeks (Dexter et al, 1977).…”

“…It was therefore postulated that appropriate manipulation of stromal-based culture systems might promote HSC activation into cell cycle (Cashm an et al, 1990;Bienzle et al, 1994) and, simultaneously, facilitate gene transfer into appropriate target cells. Preclinical studies indicated that multiple exposures of long-term, stroma-supporte d marrow cultures to retroviral vectors resulted in high-efficiency gene transfer into hem atopoietic progenitor cells with capacities for long-term in vivo proliferation (Carter et al, 1992;Bienzle et al, 1994;Dubé et al, 1996;Lutzko et al, 1999).…”

Effects of Megakaryocyte Growth and Development Factor on Survival and Retroviral Transduction of T Lymphoid Progenitor Cells

“…To this end, we used LTMC cells, which contain long-term culmre-initiating cells (LTC-IC). LTC-IC, which represent the most primitive progenitors detectable in vitro, can be transduced with retroviral vectors (Dube et al, 1996). Transduced LTC-IC are capable of reconstituting long-term hematopoiesis in large animals (Bienzle et al, 1994;Kiem et al, 1996).…”

Preferential sequestration in vitro of BCR/ABL negative hematopoietic progenitor cells among cytokine nonresponsive CML marrow CD34+ cells

“…Information demonstrating the presence of normal stem cells in the marproduction in short-and long-term hematopoietic cultures were significantly altered by the addition of MIP1-␣. Lack row of many CML patients [10][11][12]15,29 has become vital for the development of procedures which may allow for their of detectable MIP1-␣ growth inhibitory or stimulatory effects in our studies may be due to the concentration of differential manipulation. Various in vitro methodologies have been devised to purge leukemic marrow in order to MIP1-␣ used and to the frequency of its addition to the culture.…”