Hematopoietic stem cell (HSC) gene therapy (GT) holds promise for curing diseases for which there is currently inadequate or highly toxic treatment. There remains an unmet need to develop effective HSC-GT that achieves a greater level of engraftment with reduced conditioning regimen toxicity and without risk of immunogenicity. We propose to use the dog model of HSC-GT to study these key problems that can translate into significant advances for treatment of hematological and other diseases.
The aims of this proposal are designed to improve the efficacy and safety of autologous HSC-GT with (1) increased engraftment by infusion of greater numbers of ex vivo expanded gene modified (GM)-HSC, (2) reduced toxicity by eliminating or reducing chemo/radiotherapy conditioning prior to GM-HSC infusion, and (3) inducing immune tolerance to the therapeutic protein / neoantigen produced by GM-HSC and their progeny. The studies are designed to achieve results that will be translated into improving human HSC-GT.
Aim 1. Increase the cell dose of autologous GM-HSC with a 2-week ex vivo expansion of CD34+ cells. During expansion, transduce the CD34+ cells with a non-immunogenic molecular """"""""barcode"""""""" lentiviral vector. Use high throughput sequencing to track engraftment of GM-HSC progeny after transplantation long term. Compare 14-day expanded GM-HSC to 3-day cultured GM-HSC in dog competitive repopulation assays.
Aim 2. Reduce the cytotoxic conditioning regimen needed for long term engraftment of GM-HSC. To increase the competitive repopulating advantage of ex vivo expanded GM-HSC, mobilize endogenous HSC with the CXCR4 antagonist plerixafor (AMD3100) just prior to low-dose total body irradiation (TBI) followed by infusion of GM-HSC. Next, assess if KIT (CD117)-specific tyrosine kinase inhibitor (TKI) can increase engraftment of GM-HSC. We will test the hypothesis that the GM-HSCs have a competitive advantage over the plerixafor mobilized, or TKI-treated endogenous HSC. If successful, we would combine plerixafor + TKI to assess if this non-cytotoxic regimen could achieve GM-HSC engraftment without TBI. Plerixafor + TKI followed by GM-HSC infusion may be repeated multiple times to further increase engraftment of GM-HSC.
Aim 3. Establish immune tolerance to GM-HSC expressed neoantigens with CTLA4-Ig costimulatory blockade and pharmacologic immunosuppression (cyclosporine and mycophenolate mofetil). Finally, we will test the optimal GM-HSC transplant regimen to correct the erythroid disease in pyruvate kinase (PK) deficiency dogs with the R-type PK gene to achieve a functional cure of anemia. Upon completion of these three aims, we will have defined highly translatable approaches to increase engraftment of GM-HSC, reduce conditioning regimen toxicity and induce immune tolerance to GM-HSC

Public Health Relevance

This project is directly relevant to improving the health of patients with diseases that can be treated with hematopoietic stem cell gene therapy. Diseases include genetic diseases, inborn errors of metabolism, immune deficiencies, and also cancer. This project focuses on: (1) increasing the number of gene modified hematopoietic stem cells infused in order to improve the outcome of the gene therapy, (2) eliminating the chemotherapy/radiation conditioning regimens that have been traditionally used for gene therapy treatment, and (3) preventing immune responses to the gene therapy cells.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
High Priority, Short Term Project Award (R56)
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Special Emphasis Panel (ZRG1)
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Welniak, Lisbeth A
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Fred Hutchinson Cancer Research Center
United States
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Georges, George E; Storb, Rainer (2016) Hematopoietic stem cell transplantation for acquired aplastic anemia. Curr Opin Hematol 23:495-500