Increasing Stem Cell Numbers While Retaining Stem Cell Function

Abstract:

This Ancillary Project application establishes a new collaboration between an NHLBI Progenitor Cell Biology Consortium Investigator, Dr. Beverly Torok-Storb, PI of the U01 parent grant: 5U01 HL099993 Controlling Hematopoietic Lineage Commitment from ESC to Platelets and a new investigator, Dr. George Georges. In this application, we build on exciting preliminary studies to produce new methods for ex vivo expansion of hematopoietic stem cells (HSC). Dr. Patrick Paddison (UO1 HL099993 investigator) recently showed that treating human HSCs with two small molecule inhibitors targeting G9a histone lysine methyltransferase (UNC0638) and the aryl hydrocarbon receptor (AHR) (SR1) leads to a dramatic ex vivo expansion of primitive CD34+ CD38lo progenitor cells (~120-fold). Our preliminary studies suggest that SR1 and UNC0638 treatment of dog CD34+ cells achieves comparable expansion of HSC and the expanded cells fully reconstitute hematopoiesis in recipient dogs after a myeloablative dose of 9.2 Gray (Gy) total body irradiation (TBI). We now propose to rigorously evaluate SR1/UNC068 treated HSCs in the in vivo dog model of HSC transplantation. We plan to test long term hematopoiesis from the expanded product alone (no back-up source of stem cells) in a large animal model that approximates the proliferative demand over time required for clinical applications. If successful, this protocol can be immediately applied to patients.

Aim 1. Compare engraftment and survival of dogs transplanted with autologous SR1/UNC0638 “2-drug” vs. “no-drug” (no SR1/UNC0638) expanded CD34+ cells after 9.2 Gy TBI.

Aim 2. Determine the long-term persistence of SR1/UNC0638 expanded, lentiviral vector transduced HSC. During ex vivo expansion, CD34 cells will be transduced with non-expressing lentiviral vectors to track progeny

from the expanded HSCs. Autologous expanded transduced HSC will be infused after 9.2 Gy TBI.

Aim 3. Transplant SR1 and UNC0638 expanded HSCs into allogeneic recipient dogs. After 9.2 Gy TBI, MHCmatched expanded HSCs will be infused, and post-grafting immunosuppression will be given to prevent graft rejection. The long term engraftment of expanded cells will be monitored by molecular chimerism studies. The successful completion of these 3 Aims will provide a powerful rationale for the rapid translation to clinical trials of SR1/UNC0638 expanded human HSC for long-term hematopoietic recovery after transplantation.