Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells, and using genetic marking techniques to answer important questions about in vivo hematopoiesis. In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and using genetic marking techniques to understand stem cell behavior in vivo. We have continued to further enhance gene transfer efficiency into rhesus engrafting cells, resulting in early levels of marked cells as high as 50-80%, with stable levels of 5-35% in all lineages, a range with clinical utility. These levels can be achieved with traditional amphotropic MLV vectors, as well as with SIV-based and modified HIV-based lentiviral vectors. We have developed avian sarcoma leukocytosis virus (ASLV) vectors and site-specific non-viral vectors based on phage for hematopoietic target cell applications, due to more favorable insertion site profiles. ASLV can transduce rhesus long-term repopulating cells, as first demonstrated in our in vivo autologous transplantation model. We have discovered that transduction under hypoxic conditions can improve engraftment and long-term modification of hematopoietic stem cells. We have continued to utilized the LAM-PCR technology, most recently utilizing a high throughput modification, and a non-biased restriction-enzyme free procedure to improve the technology for insertion site retrieval and tracking. We retrieve and analyze clonal contributions to peripheral blood populations following transplantation of CD34+ transduced progenitor cells. Given the occurence of leukemia in now seven patients receiving gene therapy for severe immunodeficiencies with retrovirally-transduced hematopoietic stem cells, we have performed large scale sequencing of retroviral insertion sites in rhesus macaques transplanted with cells transduced either with MLV or SIV vectors, and we continue to follow animals transplanted up to 15 years ago with transduced CD34+ cells, a unique resource for predicting the long-term safety and utility of retroviral gene transfer. We continue to explore the mechanism of clonal expansion and leukemogenesis in primitive transduced hematopoietic cells, now using overexpression vectors to study the impact of BCL2A1 and MDS1/EVI1 on immortalization or transformation. BCL2A1 over-expressed in murine HSCs results in clonal primarily B cell leukemias, implicating this gene product for the first time as leukemogenic. Attrition of telomeres can serve as a mitotic clock for stem and progenitor cells and their progeny. We examined telomere lengths in hematopoietic cells of macaques transplanted 1-15 years previously with lentivirally-transduced CD34+ cells, comparing transduced and untransduced myeloid and lymphoid cells, and found no significant differences in telomere length, suggesting no impact of vector integration on proliferative/self-renewal behavior. We also compared gene expression profiles in transduced and untrasnduced marrow CD34+ cells from these animals, and found no genes consistently up or down regulated and no differences in expression of proto-oncogenes. We have successfully developed two suicide gene strategies allowing ablation of vector-containing hematopoietic cells in vivo, following transplantation of transduced cells. The first utilizes an optimized and highly sensitive herpes tk mutant transgene, which is activated by ganciclovir. We have shown complete ablation of all detectable retrovirus vector containing cells with a non-toxic 21 day treatment course of ganciclovir in non-human primates transplanted 4-6 months previously, with stable vector marking levels pre ganciclovir. The second utilizes an engineered inducible caspase 9 suicide gene which can be activated by the small molecule dimerizer AP1903. Stably engrafted animals had greater than 90% of their vector-containing cells ablated with short treatment courses of AP1903, and we continue to optimize this system, showing that higher dosing of the dimerizer does not improve suicide-gene mediated ablation, but instead that expression level of the iCasp transgene impacts on ablation, and low-expressing cells persist. We have also documented upregulation of the anti-apoptotic gene product Bcl2 in cells persisting in vivo and in vitro following dimerizer exposure.

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National Heart, Lung, and Blood Institute
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Wu, Chuanfeng; Li, Brian; Lu, Rong et al. (2014) Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells. Cell Stem Cell 14:486-99
Sellers, Stephanie E; Dumitriu, Bogdan; Morgan, Mary J et al. (2014) No impact of lentiviral transduction on hematopoietic stem/progenitor cell telomere length or gene expression in the rhesus macaque model. Mol Ther 22:52-8
Malide, Daniela; Metais, Jean-Yves; Dunbar, Cynthia E (2014) In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy. J Vis Exp :
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Wu, Chuanfeng; Jares, Alexander; Winkler, Thomas et al. (2013) High efficiency restriction enzyme-free linear amplification-mediated polymerase chain reaction approach for tracking lentiviral integration sites does not abrogate retrieval bias. Hum Gene Ther 24:38-47
Hong, So Gun; Dunbar, Cynthia E; Winkler, Thomas (2013) Assessing the risks of genotoxicity in the therapeutic development of induced pluripotent stem cells. Mol Ther 21:272-81
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