Hematopoietic stem cell (HSC) gene therapy for genetic diseases remains an attractive alternative to allogeneic transplantation. Nonhuman primates have been invaluable in studying HSC gene transfer strategies. In contrast to mouse studies, nonhuman primate studies more closely reflect conditions applicable to humans, e.g., the use of the same HSC enrichment methods and cytokines, the same culture conditions, and similar engraftment issues. We have used nonhuman primates to study different retrovirus vectors, transduction conditions, conditioning regimens and in vivo selection of gene-modified repopulating cells. We have developed transduction and transplantation conditions in the nonhuman primate model resulting in high- level in vivo marking in hematopoietic repopulating cells. Using these optimized conditions and in vivo selection we have achieved marking levels sufficient to cure not only immunodeficiencies, but also genetic diseases which require relatively high marking levels including hemoglobinopathies. These studies, however, required high-dose conditioning with total body irradiation (TBI), which would not be an appropriate conditioning regimen for such genetic diseases. We have also used the nonhuman primate model to perform extensive insertion site analyses and found that lentivirus vectors and foamy virus vectors maybe less prone for insertional mutagenesis than gammaretroviral vectors. Based on these findings, we propose here to develop safer and more clinically applicable HSC gene therapy protocols for genetic diseases, especially for hemoglobinopathies.
In aim 1, we propose to study an inducible promoter system to more safely select gene- corrected cells in vivo. We will also test and compare the use of lentivirus and foamy virus vectors in this setting and analyze integration sites before and after in vivo selection using whole genome-adapted pyrosequencing.
In aim 2, we will determine whether we can expand HSC and select gene-corrected cells before infusion into the recipient.
In aim 3, we will evaluate engraftment in a nonmyeloablative transplant setting using a combination of busulfan and stem cell mobilization..
In aim 4, we will evaluate our optimum conditions with a clinical globin vector to determine the efficacy and safety of our strategies to treat hemoglobinopathies. Protocols developed in the nonhuman primate model should be readily translatable to the clinical setting.

Public Health Relevance

We propose to develop safer and more clinically applicable gene therapy treatment plans for genetic diseases in blood-forming stem cells, especially for defects in red blood cells which carry oxygen.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL098489-02
Application #
8015603
Study Section
Special Emphasis Panel (ZRG1-TAG-M (01))
Program Officer
Qasba, Pankaj
Project Start
2010-01-21
Project End
2013-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
2
Fiscal Year
2011
Total Cost
$704,921
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
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Radtke, Stefan; Haworth, Kevin G; Kiem, Hans-Peter (2016) The frequency of multipotent CD133(+)CD45RA(-)CD34(+) hematopoietic stem cells is not increased in fetal liver compared with adult stem cell sources. Exp Hematol 44:502-7
Younan, P M; Polacino, P; Kowalski, J P et al. (2015) Combinatorial hematopoietic stem cell transplantation and vaccination reduces viral pathogenesis following SHIV89.6P-challenge. Gene Ther 22:1007-12
Gori, Jennifer L; Butler, Jason M; Chan, Yan-Yi et al. (2015) Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. J Clin Invest 125:1243-54
Younan, Patrick M; Peterson, Christopher W; Polacino, Patricia et al. (2015) Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates. Mol Ther 23:943-951
Watts, Korashon L; Beard, Brian C; Wood, Brent L et al. (2014) No evidence of clonal dominance after transplant of HOXB4-expanded cord blood cells in a nonhuman primate model. Exp Hematol 42:497-504
Kiem, Hans-Peter; Arumugam, Paritha I; Burtner, Christopher R et al. (2014) Pigtailed macaques as a model to study long-term safety of lentivirus vector-mediated gene therapy for hemoglobinopathies. Mol Ther Methods Clin Dev 1:14055
Adair, Jennifer E; Johnston, Sandra K; Mrugala, Maciej M et al. (2014) Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients. J Clin Invest 124:4082-92
Beard, Brian C; Adair, Jennifer E; Trobridge, Grant D et al. (2014) High-throughput genomic mapping of vector integration sites in gene therapy studies. Methods Mol Biol 1185:321-44
Wang, Cathy X; Sather, Blythe D; Wang, Xuefeng et al. (2014) Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells. Blood 124:913-23

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