One of the major goals of the studies pursued in our laboratories is to understand the molecular mechanisms which underlie the regulation of proliferation and differentiation of hemopoietic stem cells (HSC). In the course of our studies on regulators of stem cell proliferation, we found that HSC engineered to overexpress Hoxb4 have a 20- to 55-fold repopulation advantage over untransduced cells. Strikingly this capacity of Hoxb4 to selectively enhance HSC regeneration appears to occur very early (i.e., days) after gene transfer. Our goals are to understand in more detail the biological and molecular basis for this """"""""Hoxb4 effect"""""""" on HSC (Aim no. 1 to 3) and to develop inducible gene/protein transfer methods for achieving this """"""""Hoxb4-expanding"""""""" effect on stem cells (Aim no. 4 and 5).
Five specific aims are proposed: 1- To define the early kinetics, duration and magnitude of HoxB4-induced enhancement of HSC expansion in the murine model and to determine the requirement for host conditioning. The capacity for Hoxb4 to induce expansion of human bone marrow cells will also be investigated using bone marrow transplantation into immuno-compromised mice. 2- To identify the domains of Hoxb4 required for its effects on HSC expansion. 3- To identify proteins that specifically interact with subdomains of Hoxb4 by taking advantage of a yeast-two-hybrid screen using Hoxb4 as a bait and a target cDNA library prepared from fetal liver as a hemopoietic enriched population. 4- To develop and test approaches to achieve enhanced HSC expansion based on transient exposure (i.e., defective lentiviral vectors or TAT-protein delivery) to Hoxb4. 5- To develop a dominant, drug -inducible system for Hoxb4 enhanced HSC expansion. With these studies, we hope to improve our understanding of the molecular mechanisms utilized by the Hoxb4 protein in order to expand HSC in a transplantation context in view of eventually developing tools to manipulate the in vivo and in vitro expansion of these cells.
