A major 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. 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. We have been very interested in the CXCR4/SDF-1 axis that is very important to enhancing engraftment and to the trafficking of other blood cells, particularly neutrophils and B lymphocytes. Finally, as a major effort of our stem cell program, we have initiated a program of development and study of induced pluripotent 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. We also have been involved with collaborators in the Fauci lab (LIR, NIAID) and with the Gallin lab (LHD, NIAID) in elucidating abnormalities of B lymphocytes in patients with CGD. 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 generation of induced pluripotent stem cells and gene correction of iPSC: A major accomplishment of our iPSC program over the past year and a half has been the development of an efficient method for reprogramming generation of iPSC from the small numbers of CD34 hematopoietic stem cells found in peripheral blood. This method was published this year (Merling RK, et al, Blood 121:e98-107, 2013). In the context of this work we have generated over 25 iPSC lines from patients with the X-linked from and all 4 of the autosomal recessive genetic forms of CGD and from patients with X-linked severe combined immune deficiency (X-SCID). We are in the process of developing methods to genetically correct the CGD defect for all 5 forms of CGD in these iPSC. This work has great potential for developing future treatments for CGD, including the generation of autologous gene corrected neutrophils derived from CGD to help control severe infections in patients with CGD that are not responding to conventional therapy. We have established a collaboration with Dr. David Stroncek in the Department of Transfusion Medicine, CC, NIH to establish GMP conditions for generating and maintaining iPSC. This is an important element of the long term goal of developing new treatments for CGD and X-SCID using autologous iPSC. 2. 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 (LMI, DIR, NIAID, NIH), 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). 3. New treatments for allogeneic transplant related graft versus host disease (GVHD): 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 was published from our laboratory in the past year reporting that the effect is likely due to the agonists induction of production of FOXP3 positive regulatory T lymphocytes (Han KL, et al., J Immunol 190:458-68, 2013). 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. Interactions of neutrophils with bacterial pathogens: In work done by our collaborators in the Leto lab (LHD, NIAID) it was shown that substances produced byt the bacterial pathogen, Pseudomonas aerugenosa can induced neutrophil nets (DNA meshworks derived from neutophils important in host defense against infections) but that these bacterial induced nets require hydrogen peroxide produced by the neutrophil. CGD patient neutrophils cannot produce the nets and this defect may play a role in CGD susceptibility to infection (Rada B, et al., PLoS One 8:e54205, 3013). In other studies with the Gallin laboratory (LHD, NIAID) and the Holland laboratory (LCID, NIAID) we have collaborated in achieving better understanding of the pathogenic potential of Granulobacter bethesdensis, showing how innate immunity to this organism is impaired in CGD patients (Zarember KA, et al, Infect Immun 80:975-81, 2012) and in achieving better understanding of the frequency of infection with this organism as indicated by serum reactivity (Greenberg DE, et al, J Infect Dis 206:943-51, 2012). 5. B cell defects in patients with CGD: Although the oxidative defect in CGD results primarily in defects of innate immunity, it is becoming increasingly evident that the maturation and function of B lymphocytes in CGD patients is also abnormal. ) B-cell activating factor (BAFF) is elevated in Chronic Granulomatous Disease (Matharu K, et al., Clin Immunol 148:258-264, 1913). Sevearl years ago we showed that CGD patients have low levels of memory B cell, but we now should that they none-the-lessmaintain humoral immunologic memory despite these low frequencies of circulating memory B cells, because an alternate type of memory B cells pick up this function. (Moir S, et al, Blood. 120:4850-4858, 2012). 6. 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 (Tewari P, et al, Biol Blood Marrow Transplant 18:1368-77, 2012;Kang and Malech, Methods Enzymol 507:125, 2012). We also assist collaborators with their projects, such as our assistance to the Kwong laboratory (Surgical Branch, NCI) in a project they had to deliver inhibitory siRNAs to cancer cells using nanoparticles (Tobin LA, et al, Biomaterials 34:2980-90, 2013)

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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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