Methods for Treating Genetic Disorders

Hematopoiesis

The study of hematopoiesis, particularly at the level of stem cell biology, has been a long-standing interest in our laboratory. Our original entrée into the field, over a decade ago, involved attempts to use retroviral-mediated gene transfer to develop methods for the efficient transduction of murine hematopoietic stem cells. It became clear at that time that a greater understanding of the fundamental biological properties of hematopoietic stem cells was necessary, particularly with regard to their potential for self-renewal and differentiation in vivo and in vitro and their ability to engraft bone marrow transplant (BMT) recipients. Our current efforts are still focused primarily in these two areas.

A major program in the laboratory is the purification and functional characterization of hematopoietic stem cells, particularly the factors influencing the balance between the self-renewal and differentiation of stem cells, and the use of that information to design more-effective stem cell transduction and BMT protocols. Our efforts in the area of stem cell purification are based on our fortuitous recent finding that all of the stem cells present in normal murine bone marrow can be isolated using a simple one-step protocol. This procedure involves the staining of bone marrow cells with the DNA-binding vital dye Hoechst 33342 and the FACS (fluorescence-activated cell-sorting) isolation of cells that are simultaneously weakly staining at two different wavelengths (red and blue).

The fraction of cells isolated by this method, termed the side population, represents ~0.1 percent of murine bone marrow cells and possesses cell surface markers consistent with other less efficient purification methods (e.g., Sca+, c-kit+, linlow). We have used this purification method to demonstrate the existence of a small population of naturally cycling cells (~1-3 percent) and the ability to isolate the population physically. Through competitive repopulation studies, we have shown that the naturally replicating cells are able to engraft lethally irradiated recipients efficiently.

Studies with murine stem cells purified by these methods focus on (1) determining whether the replicating cells we are able to isolate remain in continuous cycle or are only transiently in replication; (2) analyzing the responses of both replicating and quiescent fractions of stem cells to specific combinations of hematopoietic growth factors, with a particular interest in whether the replicating cells respond differently; (3) examining the relative ability of replicating and quiescent cells to engraft BMT recipients under conditions of competitive repopulation involving specific populations of cells, as some of our previous work suggests that different populations may compete for engraftment. From these studies, we hope to test the hypothesis that the naturally replicating fraction of cells may be a critical cell population for BMT.

We are also attempting to apply the Hoechst purification to human hematopoietic stem cells. To date, determination of the surface phenotype of human hematopoietic stem cells has relied exclusively on in vitro assays that do not directly assay cells that are capable of the engraftment of BMT recipients. Consequently, the identification of the optimal populations of human cells for transplantation purposes (and/or gene transfer) has been problematic. We are attempting to determine whether the Hoechst purification protocol offers a novel means of determining the validity of the available in vitro assays and perhaps even provides a means of defining the true cell surface phenotype of human stem cells capable of reconstitution.

Finally, we continue to apply the knowledge obtained in the study of the biology of stem cells to the practical development of gene therapies for diseases involving hematopoietic cells. Our major focus in this area involves the analysis of the efficiency of transfer and expression of human b-globin genes in hematopoietic cells in vivo. Such studies are aimed at the development of effective therapies for b-thalassemia and sickle cell anemia.

Manipulation of the Immune Response

The basic premises of this research program are that the ability to modify genetically either tumor cells or antigen-presenting cells may make it possible to manipulate immune responses in ways that have not previously been possible and that such manipulations might lead to more effective immunotherapies for cancer and infectious disease and perhaps even to novel immunosuppressive therapies.

Our efforts are focused on the notion that the localized expression of cytokines, growth factors, and other gene products at the site of an infection or other immunological insult plays a major role in immune responses and that the presence, absence, or relative amounts of different gene products likely dictate the ultimate outcome of the response. Our current efforts are aimed at the development of immunotherapies for both cancer and human immunodeficiency virus (HIV) infection. We also recently initiated a pilot effort to evaluate the possibility of preventing particular antigen-specific immune responses.

Vector Development

Our long-standing interest in the development of mammalian transducing vectors continues. One effort is focused on the development of fourth-generation retrovirus-packaging cell lines, in which nonretroviral transcriptional signals are used to provide expression of the retroviral gene products necessary for packaging. We recently succeeded in generating stable cell lines that express the retroviral gag-pol gene product in a constitutive fashion and the vesicular stomatitis virus G protein in an inducible fashion. The G protein, which is toxic to cells when stably expressed, confers wide host range and makes it possible to concentrate retroviral pseudotypes. In other studies, we are attempting to generate murine leukemia virus-based viruses able to integrate in resting cells. These studies make use of recent information regarding the role of nuclear localization sequences and phosphorylation in the ability of HIV to integrate in resting cells.