The overall goal of this project is developing improved hematopoietic stem cell based therapies to treat primary immune deficiencies. We have a particular interest in chronic granulomatous disease (CGD) and X-linked severe combined immune deficiency (XSCID). Through these studies we aim to increase understanding of the basic biology of trafficking of hematopoietic stem cells and other cells into and out of the bone marrow;to delineate the homeostatic mechanisms that maintain or expand the pool of hematopoietic stem cells;and the mechanisms that induce differentiation of HSC into more mature leukocytes. We also aim to devise improved methods for growing or maintaining HSC in culture without losing reconstitution potential. Also related particularly to allogeneic and matched unrelated donor transplants is to devise new treatments that prevent or treat graft versus host disease. These studies will provide information that will allow therapeutic manipulation of the transplant/engraftment process. There are many clinical settings in which the number of CD34 positive hematopoietic adult human stem cells are limiting (cord blood transplants for example;or settings of gene therapy where the number of gene corrected autologous CD34 positive hematopoietic adult human stem cells are limited). Our studies are currently focussed on, but not limited to the role of HOXB-4 and other upstream and down stream hematopoietic stem cell proliferative signals in maintaining the stem cells population, with a goal of using such information to allow expansion of HSC in culture while maintaining engraftment/reconstitution potential. We have been working with novel systems such as the commercially available, NANEX CD34+ cell expansion system from Arteriocyte Inc, that allows significant expansion of hematopoietic stem cells without losing in vivo engraftment potential. We have been very interested in the CXCR4/SDF-1 axis and regulatory elements of the system such as CD26 a dipeptidyl peptide IV protease as an important receptor/ligand/downregulatory axis that is very important to enhancing engraftment and to the trafficking of other blood cells, particularly neutrophils and B lymphocytes. In that regard we have been collaborating closely with our colleagues David McDermott and Philip Murphy in LMI/NIAID in studying a group of patients with WHIM syndrome caused by C-terminal truncating mutations that lead to hyperactivity of CXCR4. Finally, we have initiated a program of development and study of induced pleuripotent stem cells (iPSC) derived from somatic cells of patients with PIDs with the goal of using iPSC to study the biology of development of HSC and other blood cells from iPSC and to use iPSC as an in vitro models of the immune defects that are manifested in patients with PIDs. We are particularly interested in delineating the the potential of iPS cells derived from patients with PIDs to differentiate into immune leukocytes such as neutrophils, monocytes and lymphocytes. It is also a part of the goal of this project to communicate information about the biology of HSC, transplantation for PID, graft versus host disease, WHIM syndrome, iPSC models of primary immune deficiencies through publication about primary studies and to publish reviews to educate scientific colleagues and the general public about advances in these areas of study. Published results of our accomplishments toward the goals of this project in the past fiscal year include: 1. Studies of the CXCR4 receptor and its importance in trafficking of HSC and other types of blood cells: We have worked closely with our colleagues in the Murphy lab, who have shown that plerixafor (a potent inhibitor of CXCR4 activity also inhibits the abnormally high activity of C-terminal truncated CXCR4 in WHIM syndrome. This has led to a clinical trial conducted by Dr. McDermott on which we are principle collaborators examining the effect of plerixafor to treat patients with WHIM syndrome, finding that this agent increases the number of circulating neutrophils, B-lymphocytes and T-lymphocytes in these patients (McDermott et al, Blood 118:4957, 2011). Dr. McDermott has also identified new mutations causing WHIM syndrome (Liu et al, Blood 120:181, 2012). 2. We have shown for the first time that a highly specific agonists (activators) of the Adenosine A2A receptor can prevent graft versus host disease in a single mismatch model of GVHD and a paper is in review reporting this finding. Since these highly specific Adenosine A2A small molecule agonists are also in commercial Phase I and II clinical trials to treat the inflammation of asthma and chronic obstructive pulmonary disease there is also real potential of this class of agents in the future being applied to the GVHD prevention in the clinic. 4. A few years ago we, together with our collaborator, Dr. Linzhao Cheng at Johns Hopkins University initiated a project to develop iPS cells from patients with primary immune deficiencies, focusing initially on patients with X-linked chronic granulomatous disease (X-CGD) (a PID caused by a defect of microbicidal hydrogen peroxide production by blood neutrophils). We have successfully established X-CGD iPS cells from bone marrow mesenchymal cells;differentiated X-CGD iPS cells into mature neutrophils to demonstrate the defect in oxidase activity;and used gene transfer methods to correct the deficiency in the X-CGD iPSCs such that when differentiated to neutrophils the oxidase activity is restored. This will establish a model that will allow exploration of the potential of applying methods of gene repair to the X-CGD iPS cells to correct the CGD phenotype in neutrophils that arise from the gene corrected iPS cells (Zou J et al, Blood 117:5561, 2011). This work has great potential for developing future treatments for X-CGD and other primary immune deficiencies. We are working on potentially using iPS cells from CGD patients, gene corrected as a source of autologous corrected neutrophils that could be used to treat CGD patients with severe infections. 3. We were also involved in collaborative studies of the utility of granulocyte transfusions to support CGD patients with severe infections. While there appears to be clinical benefit from granulocyte transfusions with good survival and function of donor neutrophils, the utility of this approach is limited by development of anti-HLA responses (Heim KF et al, Transfusion 251:1154, 2011). 4. Another related goal of this project is to communicate information to the community about advances in hematopoietic stem cells in transplantation to treat primary immune deficiencies, about the function and trafficking of neutrophils, and about differentiation of neutrophils and about defects in neutrophil function and primary neutropenia disorders (Rosenzweig SD and Malech HL, Neutrophil Functional Disorders, in Encyclopedia of Life Sciences;Zarember KA and Malech HL, The roles of neutrophils in innate immunity, In Regulation of Innate Immune Function;Kang EM et al, J Allergy Clin Immunol 127:1319, 2011;Kang and Malech, Methods Enzymol 507:125, 2012).

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De Ravin, Suk See; Li, Linhong; Wu, Xiaolin et al. (2017) CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci Transl Med 9:
Punwani, Divya; Kawahara, Misako; Yu, Jason et al. (2017) Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency. Hum Gene Ther 28:112-124
Hong, So Gun; Yada, Ravi Chandra; Choi, Kyujoo et al. (2017) Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers. Mol Ther 25:44-53
Jones, Karlie R; Choi, Uimook; Gao, Ji-Liang et al. (2017) A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development. Sci Rep 7:44816
Parta, Mark; Kelly, Corin; Kwatemaa, Nana et al. (2017) Allogeneic Reduced-Intensity Hematopoietic Stem Cell Transplantation for Chronic Granulomatous Disease: a Single-Center Prospective Trial. J Clin Immunol :
Marciano, Beatriz E; Allen, Elisabeth S; Conry-Cantilena, Cathy et al. (2017) Granulocyte transfusions in patients with chronic granulomatous disease and refractory infections: The NIH experience. J Allergy Clin Immunol 140:622-625
Sweeney, Colin L; Choi, Uimook; Liu, Chengyu et al. (2017) CRISPR-Mediated Knockout of Cybb in NSG Mice Establishes a Model of Chronic Granulomatous Disease for Human Stem-Cell Gene Therapy Transplants. Hum Gene Ther 28:565-575
Sweeney, Colin L; Zou, Jizhong; Choi, Uimook et al. (2017) Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction. Mol Ther 25:321-330
Pandya, Hetal; Shen, Michael J; Ichikawa, David M et al. (2017) Differentiation of human and murine induced pluripotent stem cells to microglia-like cells. Nat Neurosci 20:753-759
Kuhns, Douglas B; Fink, Danielle L; Choi, Uimook et al. (2016) Cytoskeletal abnormalities and neutrophil dysfunction in WDR1 deficiency. Blood 128:2135-2143

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