The research goal of the Hematopoiesis Section is to develop strategies to introduce new genetic material into the pluripotent hematopoietic stem cell (PHSC). The PHSC is the ultimate progenitor of all mature cells found in the circulating blood. The PHSC is capable of extensive self renewal without losing the capacity to differentiate into cells with functions as diverse as red blood cells or T-lymphocytes. This self renewal allows small numbers of PHSC to repopulate the entire hematopoietic system of bone marrow transplant recipients. The clinical relevance of this research is in the field of gene therapy, in which a patient with an inherited genetic disorder of the hematopoietic system is treated by the introduction of the therapeutic gene, for example the adenosine deaminase (ADA) gene for patients with ADA deficients severe combined immunodeficiency (SCID) or the beta-globin gene for patients with sickle cell disease. Finally, the basic biology of hematopoiesis can be investigated by inserting genes for transcription factors thought to be involved in hematopoietic differentiation into normal PHSC. This approach can identify the role each transcription factor plays and the stage of hematopoiesis at which the transcription factor acts. Previously we have shown that retrovirus mediated gene transfer can deliver new genetic material to approximately 25% of mouse PHSC but to only 1-2% of primate PHSC. We are investigating several strategies to augment gene transfer efficiency. Previously we showed that treatment of mice with the cytokines Granulocyte Colony Stimulating Factor (G-CSF) and Stem Cell Factor (SCF) mobilized PHSC which could be efficiently transduced with retrovirus particles. We have found that following the cessation of cytokine treatment, the repopulating ability of the bone marrow is transiently increased by 20 fold. Since retrovirus integration depends on DNA replication in the host cell, we tested this cell population for gene transfer efficiency in both mouse and Rhesus monkey models. In mouse models, post cytokine marrow cells transduced as efficiently as bone marrow cells from 5-FU treated donors, our previous ideal condition. The efficiency of gene transfer into the cytokine treated Rhesus PHSC was 10 to 100 fold higher than that observed in human patients with the same virus vectors. We feel that cytokine treated bone marrow and peripheral blood cells may represent an excellent alternative population of PHSC for clinical gene therapy protocols. Gene expression in the PHSC can be studied using our previously described strategy for enriching mouse PHSC by elutriation followed by selection foe cells expressing a high level of the c-kit receptor. RNA extracted from these cells can be analyzed for the expression of genes for which we would like to eventually perform gene therapy. One example of this is the IL-2 receptor g chain (g common) which is mutated in human X-linked SCIDS. In collaboration with the Immunogenetics Section (J. Puck) we have shown that g common is expressed in all hematopoietic cells, and that it is the expression of its partners IL-2Ra and IL-7R which is tightly regulated. Thus, regulated expression of gcommon will not be necessary in gene transfer protocols. For the transduction of mouse PHSC, ecotropic virus particles are used, while for the transduction of primate PHSC, amphotropic virus particles are used. We have compared the frequency of transduction of these two different viruses on highly enriched mouse PHSC and found that the ratio of amphotropic virus to ecotropic virus in repopulated mice is approximately 0.1, similar to the differences described above. This result appears to be a function of the level of expression of the mRNAs for the two retrovirus receptors. The level of ecotropic receptor is much greater than the level of amphotropic receptor in both mouse and human PHSC. Currently we are attempting to manipulate amphotropic mRNA levels to increase gene transfer efficiency using these vectors. We intend to test these approaches on human PHSC using the transgenic SCIDS mouse we have developed. These mice express the human IL-3, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), and SCF genes, and we have shown that they can support human hematopoiesis for up to 24 weeks in vivo, a significant improvement on the previous models. We are evaluating strategies for gene transfer into human PHSC using this mouse model. One example of this is a comparison of the efficiency of transduction of amphotropic vectors and vectors pseudo typed with the VSV-G envelope. It is anticipated that the use of this mouse model will reduce the dependence on the Rhesus monkey model for testing large animal gene transfer strategies. We recently showed that the retroviral transfer of the GATA-1 transcription factor caused an increase in erythroid progenitor cells in repopulated mice. To further study the effects of the GATA family of transcription factors on hematopoiesis, we have generated transgenic mice with either the GATA-1 gene, or the GATA-2 gene under the control of a tetracycline responsive promoter. These mice will be valuable in the enumeration of the effects target cells for both transcription factors, and their role in hematopoiesis.
