The overall goal of this project is to comprehensively analyze the safety of viral gene transfer and to explore strategies that reduce the risk of insertional mutagenesis in a clinically highly relevant large animal model. The development of leukemia in two patients enrolled in the French clinical trial of stem cell gene therapy for the treatment of X-linked SCID has led to a reassessment for the field of stem cell gene transfer. Up to this point, oncoretroviral vectors were considered safe, and the focus has therefore been on optimizing efficacy. To that end, we have developed stem cell transducfion conditions that result in long-term gene transfer levels of 20% to 30% in the baboon model. With the report of leukemia in the French trial three years after gene therapy, long-term follow-up data from clinically relevant large animal models with high-level marking has now become a unique and necessary resource to determine the safety of gene therapy utilizing integrating vector systems. Thus, in Specific Aim 1, we propose to monitor baboons with persistent high-level marking and gene expression for the development of monoclonality and leukemia.
In Specific Aim 2, we will use linear amplification mediated (LAM)-PCR analysis to study and comprehensively characterize the proviral integration sites in these animals, and interrogate available human genome databases to determine the position of integration sites and the association to cancer-related genes. We will also develop and evaluate strategies to improve safety for future stem cell gene therapy studies.
In Specific Aims 3 and 4 we propose to reduce the risk of insertional mutagenesis by decreasing the cell dose of gene-modified cells. We will first examine whether intra-marrow infusion will allow for more efficient engraftment of gene-modified stem cells. We will then determine whether more immature and thus smaller numbers ofhematopoietic stem/progenitor cells can be targeted for gene therapy applications.
In Specific Aim 5, we will explore whether nonviral gene transfer strategies can be used to genetically modify stem cells, and we will evaluate their safety compared to viral vector systems. Results obtained in these nonhuman primate studies should be readily translatable and applicable to human gene therapy studies.
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| Gori, Jennifer L; Butler, Jason M; Kunar, Balvir et al. (2017) Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates. Stem Cells Transl Med 6:864-876 |
| Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia (2016) Optimizing autologous cell grafts to improve stem cell gene therapy. Exp Hematol 44:528-39 |
| Li, Li B; Ma, Chao; Awong, Geneve et al. (2016) Silent IL2RG Gene Editing in Human Pluripotent Stem Cells. Mol Ther 24:582-91 |
| Karponi, Garyfalia; Psatha, Nikoletta; Lederer, Carsten Werner et al. (2015) Plerixafor+G-CSF-mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy. Blood 126:616-9 |
| Vierstra, Jeff; Reik, Andreas; Chang, Kai-Hsin et al. (2015) Functional footprinting of regulatory DNA. Nat Methods 12:927-30 |
| Qi, Heyuan; Liu, Mingdong; Emery, David W et al. (2015) Functional validation of a constitutive autonomous silencer element. PLoS One 10:e0124588 |
| Liu, Mingdong; Maurano, Matthew T; Wang, Hao et al. (2015) Genomic discovery of potent chromatin insulators for human gene therapy. Nat Biotechnol 33:198-203 |
| Polak, Paz; Karli?, Rosa; Koren, Amnon et al. (2015) Cell-of-origin chromatin organization shapes the mutational landscape of cancer. Nature 518:360-364 |
| Deyle, David R; Hansen, R Scott; Cornea, Anda M et al. (2014) A genome-wide map of adeno-associated virus-mediated human gene targeting. Nat Struct Mol Biol 21:969-75 |
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