Fetal Stem Cell Gene Therapy

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DESCRIPTION (adapted from the application) A growing number of hematological diseases can be diagnosed before birth. In some cases, early treatment may benefit the health and survival of the fetus. Either in utero stem cell transplantation (IUT) or fetal gene therapy may treat diseases such as the hemaglobinopathies. This application aims to determine the best method for the introduction of genes into fetal hematopoietic stem cells (HSCs). Fetal HSCs are more proliferative than their adult counterparts and are, therefore, hypothesized to be more susceptible to transduction by retroviral vectors based on murine leukemia virus or human immunodeficiency virus. IUT offers another means of curing a number of hematological diseases by generating a state of hematopoietic chimerism. However, in the absence of any advantage for the donor HSCs, the levels of chimerism that can be achieved by IUT are low. This limits the use of this therapy to very few diseases. Our aim is to extend the use of IUT to the treatment of diseases, such as thalassemia and sickle cell anemia, by engineering HSCs to have a proliferative advantage over normal HSCs. This application will test if introduction of the erythropoietin receptor (EpoR) into HSCs will render these altered cells responsive to erythropoietin (EPO). This will in turn result in the altered HSCs and their progeny having a proliferative advantage over normal progenitors. Truncated forms of EpoR (tEpoR) will also be tested. These tEpoR, having deletions in the negative regulatory region of their cytoplasmic domains, deliver stronger proliferative signals than EpoR. The effects of introducing the EpoR genes on the proliferation and differentiation of HSCs and their progenitor progeny will be determined using various in vitro culture systems. It is hypothesized that ectopic expression of either EpoR or tEpoR will confer the ability of HSCs and early progenitors to proliferate in response to EPO with minimal effect on the differentiation program of these cells. To test if ectopic EpoR or tEpoR expression on HSCs can make these cells more competitive than their normal counterparts, modified HSCs will be tested against control HSCs in a mouse model of human hematopoiesis. The ability of HSCs expressing ectopic EpoR to engraft bone marrow after no or only minimal cytoablation will also be tested. These in vivo experiments will further determine if making HSCs responsive to EPO will have any detrimental effect on the long‑term reconstituting‑ and multilineage‑ potential of HSCs. A positive outcome from the proposed studies would aid in developing treatments for hemoglobinopathies based on generating hematopoietic allochimerism.