Frederick A. Fletcher

Molecular cloning of a ligand for the flt3 flk-2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Expression of human adenosine deaminase in murine hematopoietic cells.

Abstract Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

Assignment of the gene (EPLG2) encoding a high-affinity binding protein for the receptor tyrosine kinase elk to a 200-kilobasepair region in human chromosome Xq12

Elk is a member of the eph family of receptor tyrosine kinases. Elk is expressed only in the brain and testes of the developing and adult rat, and the interaction of elk with its ligand(s) has been suggested to play a role in the development or maintenance of the nervous system. The mouse gene Eplg2 encodes a potential elk ligand that is highly conserved among rat, mouse, and human. Eplg2 has been mapped to the central portion of the mouse X chromosome, tightly linked to the androgen receptor (Ar) locus. Linkage conservation between the mouse and the human X chromosomes suggested that the human homologue (EPLG2) would map near human AR, in the interval Xq11-q12. In the present study, we have confirmed this prediction and have localized EPLG2 to a 200-kb interval in Xq12 by somatic cell hybrid analysis, two-color fluorescence in situ hybridization (FISH), and yeast artificial chromosome (YAC) hybridization. 12 refs., 1 fig.

Effects of leukemia inhibitory factor (LIF) on gene transfer efficiency into murine hematolymphoid progenitors.

We have investigated the effects of the cytokine leukemia inhibitory factor (LIF) on recovery and retroviral infection of murine hematopoietic stem cells maintained in short-term culture. Up to a two-fold increase in CFU-S13 recovery was observed, from 9.7 x 10(-5) cells in untreated controls to 17.6 x 10(-5) cells when 10U/ml LIF is added to the culture medium. Intermediate concentrations of LIF (.1U/ml and 1U/ml) were not significantly different from the control. Histological analysis of spleen colonies harvested thirteen days posttransplant demonstrated that LIF does not cause a detectable alteration in the differentiative potential of CFU-S13. The efficiency of retroviral-vector infection in CFU-S13 is also improved, from 15% (24/158) in untreated controls to 91% (116/127) at a LIF concentration of 10U/ml. LIF concentrations of .1U/ml and 1U/ml increased infection efficiency to 35% (14/40) and 71% (37/51), respectively. Analysis of proviral insertion sites in spleen colonies indicated that some CFU-S13 precursors were infected in the LIF-treated marrows, but no identical pairs were detected in the controls. Finally, long-term expression of provirally-encoded human adenosine deaminase (hADA) was measured in hematopoietic tissues of bone marrow transplant recipients six months posttransplant. In all tissues analyzed (spleen, thymus, bone marrow, splenic B cells, peritoneal macrophages, and blood) differentiated progeny of LIF-treated marrows had higher levels of hADA than untreated controls. Tenfold increases in levels of hADA are detected in some tissues, but levels were variable. These experiments demonstrate that LIF directly or indirectly enhances retroviral infection efficiency of hematopoietic stem cells, and might be used to improved existing gene transfer protocols.

Human Gene Expression in Murine Hemopoietic Cells In Vivo

Somatic gene transfer offers the possibility of a new approach in the treatment of human genetic disease. Defects affecting the blood and blood forming tissues are candidates for therapies involving transfer of new genetic information into hemopoietic stem cells. One such defect, adenosine deaminase (ADA) deficiency, is being used as a model in which hemopoietic gene transfer techniques can be developed and evaluated. In this model, gene transfer is mediated by a retroviral vector. Retroviral vectors have been used extensively to deliver information to hemopoietic cells1–14. We have previously reported delivery and expression of human ADA (hADA) sequences in murine hemopoietic progenitors in vitro15 and in vivo16, but were not able to demonstrate long term stability of expression. We describe here the construction and testing of four new vectors, representing the first demonstration of efficient transfer and long term in vivo expression of hADA in murine hemopoietic cells.