This project involves laboratory studies and studies in animal models of the tools and methods that need to be developed to correct or repair the genetic defects causing the gp91phox deficient X-linked form of chronic granulomatous disease (X-CGD), as well as the p47phox, p67phox, p22phox and p40phox deficient autosomal recessive forms of CGD (AR-CGDs), and X-linked severe combined immune deficiency (SCID-X-1 or XSCID). This work involves studies of a variety of lentivirus vectors and the critical functional sub-elements that go into the design of safe and effective lentivirus vectors. The work also involves studying vectors in a variety of cell types and in particular optimizing gene transfer into human CD34+ hematopoietic stem cells (HSC). This project also involves the engineering of induced pluripotent stem cells from adult somatic cells of patients with CGD or XSCID for the purpose of achieving gene correction of the functional immune defect in the iPSC, including the differentiation in culture to the mature blood or immune cells affected by the primary immune deficiency under study. In the past fiscal year year we have accomplished the follow: 1. Together with our collaborators (Dr. B Sorrentino at St. Jude) we have developed a high titer lentivirus vector encoding the common gamma chain of the IL2 receptor. This vector has has already been used in the past year to treat in a gene therapy trial for XSCID two adult patients. Early results from this trial are encouraging, and analysis of the patterns of lentivector insert in blood cells from these two patients shows excellent polyclonality with no dominant clones. The first patient in this trial is now 9 months post-treatment and the second patient is 3 months post treatment, and both patients are well with no safety problems encountered to date. Marking in both patients in myeloid cells is between 10% and 17%, and marking in lymphocytes is increasing as expected. The companion clinical trial of gene transfer at St. Jude will study treatment of infants newly diagnosed with XSCID, and they are open and recruiting. 2. A few years ago we completed a retrovirus vector clinical trial of gene therapy for X-CGD patients with severe ongoing infection not responsive to conventional therapy using a murine retrovirus vector and busulfan conditioning. All three patients demonstrated early marking with appearance in the circulation of 24%, 5% and 4% neutrophils that were oxidase normal. However, marking persisted in only two of the patients such that after the first year to the third year marking was 1% and 0.03% , respectively. In the two patients with long term marking their infections cleared but one eventually succumbed to his pre-gene therapy severe infection. Laboratory assessment of gene insertions sites showed no clonal dominance. We conclude that even when not curative or permanent, gene therapy can provide clinical benefit in the treatment of persistent severe infection in CGD. (Kang EM et al, Blood 115:783, 2010). We continue to monitor marking in the two surviving patients with marking persisting in one of the patients at 0.5% of neutrophils almost six years after treatment. This supports the concept that non-ablative busulfan conditioning will result in persistence of gene marked cells. 3. Preclinical work toward the next phase of development of gene therapy for X-CGD has involved the development and study of a new lentivirus vector with features very similar to the CL20 lentivector that we have developed for the clinical trial XSCID noted in section 1. above except that this vector includes the codon optimized cDNA encoding the gp91phox gene product of the CYBB gene. Using our NSG mouse model that can engraft human hematopoietic stem cells we have shown that this vector can achieve full functional oxidase correcion up to 50% of the neutrophils that arise from gene corrected stem cells. We are currently in the proceess of production of a clinical lot of this vector in collaboration with the Indiana University Vector Production Facility (Dr. Kenneth Cornetta). We have also used the NSG mouse system to test an alternate lentivector developed by our collaborators in London and Frankfurt that uses a hybrid promoter from Fes plus Cathepsin G genes that provides myeloid specificity to expression. This vector also has excellent properties and our European collaborators plan to bring that vector to the clinic (Santilli G et al, Mol Ther 19:122, 2011) in their program. Recently we were invited to participate in a joint European and US consortium of gene therapy using the vector described in the Santilli paper. Dr. Donald Kohn at UCLA, Los Angeles, CA is the lead for the US portion of the consortium. We have agreed to participate in this consortium clinical trial which likely will open in the US by spring 2014. 5. Beginning a few years ago, together with our collaborator (Dr. L Cheng at Johns Hopkins Sch of Medicine) we have developed iPSC from the somatic cells of a patient with X-CGD, demonstrated that neutrophils differentiated from patient iPSC do not have oxidase activity but those from normal iPSC do, recapitulating the disorder. We also demonstrated that gene transfer can correct the oxidase defect in the X-CGD iPSC in that neutrophils differentiated from the gene corrected X-CGD iPSC have restored oxidase activity (Zou J et al, Blood 117:5561, 2011). We have developed a novel highly efficient method for reprogramming iPSC lines derived from the CD34+ hematopoietic stem cells present in only 10-20ml of peripheral blood and applied this method to generate iPSC lines from many of our patients with CGD, XSCID and some other inherited immune deficiencies (Merling RK, et al, Blood 121:e98-107, 2013). Using these iPSC we have demonstrated AAVS1 safe harbor site minigene targeing correction of iPSC lines derived from patients with each of the four autosomal recessive forms of CGD (p47phox, p40phox, p22phox and p67phox deficient CGD). We have also developed ZNFs and TALENs that target the CYBB gene to achieve insertion of a minigene designed to correct X-linked CGD, and to target the NCF1 gene to achieve gene repair for correction of the p47phox deficient autosomal recessive form of CGD. 6. We have published a number of chapters and reviews about gene therapy either from our group or as part of a collaborative effort with other investigators, thus communicating to the scientific community and to the general public information about progress in the field of gene therapy in general and for gene therapy of CGD and XSCID in particular (Kang and Malech, Methods Enzymol 507:125, 2012;Corrigan-Curay, J et al, Mol Ther 20:1084, 2012). 7. We have also participated efforts to better understand the problems affecting our patients with CGD in natural history studies that are important for future planning of gene therapy protocols for these patients (Alimchandani M, et al, Am J Surg Pathol, in press 2013;Alvarez-Downing MM, et al, Dis Colon Rectum 56:609-14, 2013;Leiding JW, et al, Clin Infect Dis 54:694-700, 2012;Leiding JW, et al, J Clin Immunol 33:725-30, 2013).

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De Ravin, Suk See; Wu, Xiaolin; Moir, Susan et al. (2016) Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med 8:335ra57
Sweeney, Colin L; Teng, Ruifeng; Wang, Hongmei et al. (2016) Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis. Stem Cells 34:1513-26
De Ravin, Suk See; Reik, Andreas; Liu, Pei-Qi et al. (2016) Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol 34:424-9
Felsburg, Peter J; De Ravin, Suk See; Malech, Harry L et al. (2015) Gene therapy studies in a canine model of X-linked severe combined immunodeficiency. Hum Gene Ther Clin Dev 26:50-6
McDermott, David H; Gao, Ji-Liang; Liu, Qian et al. (2015) Chromothriptic cure of WHIM syndrome. Cell 160:686-99
Marciano, Beatriz E; Spalding, Christine; Fitzgerald, Alan et al. (2015) Common severe infections in chronic granulomatous disease. Clin Infect Dis 60:1176-83
Panch, Sandhya R; Yau, Yu Ying; Kang, Elizabeth M et al. (2015) Mobilization characteristics and strategies to improve hematopoietic progenitor cell mobilization and collection in patients with chronic granulomatous disease and severe combined immunodeficiency. Transfusion 55:265-74
Hahn, Katherine J; Ho, Nancy; Yockey, Lynne et al. (2015) Treatment With Anakinra, a Recombinant IL-1 Receptor Antagonist, Unlikely to Induce Lasting Remission in Patients With CGD Colitis. Am J Gastroenterol 110:938-9
Malech, Harry L; Ochs, Hans D (2015) An emerging era of clinical benefit from gene therapy. JAMA 313:1522-3
Pripuzova, Natalia S; Getie-Kebtie, Melkamu; Grunseich, Christopher et al. (2015) Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs) using global quantitative proteome analysis. Stem Cell Res 14:323-38

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