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 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. The insertion site analysis shows non-random preference for insertions within genes for both MLV and SIV, with SIV insertions distributed evenly over the length of genes and particularly being found in highly gene rich chromosomal regions. MLV instead targets the region around transcriptional start sites. These highly non-random events indicate either a strong non-random preference for integration at these sites, or an in vivo engraftment or survival/proliferative advantage for these clones. 14 independent insertions were localized to the MDS1/EVI1 locus, an area previously implicated in spontaneous leukemias and in retroviral mutagenesis with replication competent viruses. We have found no MDS1/EVI1 insertions using SIV or ASLV vectors. SIV and ASLV vectors have a significantly lower rate of insertion clusters in proto-oncogenes as compared to MLV. These findings have important implications for future gene therapy clinical applications. 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 leukemia, implicating this gene product for the first time as leukemogenic. We have also recently found a profound impact of ex vivo expansion of transduced CD34+ cells on clonal diversity in vivo, with a selection for MDS1/EVI1 insertions after prolonged culture prior to transplantation. In vivo, cytotoxic pressure with busulfan was shown to result in clonal dominance of cells containing vector insertions in specific genes. We have recently utilized """"""""bar coded"""""""" lentiviral vectors as an alternative methodology for performing clonal tracking of HSCs and their progeny in vivo, avoiding the issues with bias and efficiency in attempts to quantify clonal contributions via insertion site tracking.

Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2011
Total Cost
$1,752,245
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
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Zip Code
Koelle, Samson J; Espinoza, Diego A; Wu, Chuanfeng et al. (2017) Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants. Blood 129:1448-1457
Dunbar, Cynthia E (2017) Two Decades of ASGCT: Dreams Become Reality. Mol Ther 25:1057-1058
Dunbar, Cynthia E (2016) Gene and Cell Therapies in Expansion Mode: ASGCT 2016. Mol Ther 24:1333-4
Bat, Taha; Bat, Betul E; El-Moghraby, Ahmed et al. (2016) Thrombopoietic status of patients on haemodialysis. Br J Haematol 172:954-7
Dunbar, Cynthia E (2016) Blood's 70th anniversary: CARs on the Blood highway. Blood 128:1-3
Hong, So Gun; Lin, Yongshun; Dunbar, Cynthia E et al. (2016) The Role of Nonhuman Primate Animal Models in the Clinical Development of Pluripotent Stem Cell Therapies. Mol Ther 24:1165-9
Yu, Kyung-Rok; Natanson, Hannah; Dunbar, Cynthia E (2016) Gene Editing of Human Hematopoietic Stem and Progenitor Cells: Promise and Potential Hurdles. Hum Gene Ther :
Barese, Cecilia N; Felizardo, Tania C; Sellers, Stephanie E et al. (2015) Regulated apoptosis of genetically modified hematopoietic stem and progenitor cells via an inducible caspase-9 suicide gene in rhesus macaques. Stem Cells 33:91-100
Kim, Sanggu; Kim, Namshin; Presson, Angela P et al. (2014) Dynamics of HSPC repopulation in nonhuman primates revealed by a decade-long clonal-tracking study. Cell Stem Cell 14:473-85
Medina-Echeverz, José; Haile, Lydia A; Zhao, Fei et al. (2014) IFN-? regulates survival and function of tumor-induced CD11b+ Gr-1high myeloid derived suppressor cells by modulating the anti-apoptotic molecule Bcl2a1. Eur J Immunol 44:2457-67

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