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), the p47phox deficient autosomal recessive form of CGD (AR-CGD), 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. These function sub-elements include assessment of gene promoters or hybrid promoter constructs, assessment of insulator elements that may protect nearby genes from activation by vector inserts in the genome, assessment of selectable elements that could increase level of gene marking, development of novel pseudotyping envelopes. 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, competed and/or published in final form the following results toward the general goals of the project: 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 for a planned gene therapy trial for XSCID. The CL20 backbone-based lentivector has the following safety elements: self-inactivating lesions in the 3LTR, internal promoter that is the elongation factor 1 alpha short version (EF1a-s), 400 bp version of the Chicken H4 globin insulator, codon-optimized therapeutic transgene cDNA. This vector appears to perform well at transducting human hematopoietic stem cells and correcting the immune defect in both XSCID mice and XSCID dogs. Most important is that this construct does not activate LMO2 when inserted into the first intron of this gene. During the past year clinical lots of vector have been produced and are available for treating patients. Also during the past year, a clinical protocols of gene therapy for XSCID using this vector that have been approved by the IRBs, IBCs, reviewed by the RAC, and the IND process completed at the FDA for protocols at the NIH and at St. Jude Childrens Research Hospital. The clinical trial in our program at the NIH will study treatment of older children with XSCID who had received lymphocyte depleted haploidentical transplants from a parent, but whose immunity had not been adequately restored or was waning. The first patient in this trial is scheduled for treatment in October 2012. The clinical trial of gene transfer at St. Jude will study treatment of infants newly diagnosed with XSCID, and they are open and recruiting. 2. We completed the laboratory assessment of a completed 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. 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 X-CGD. (Kang EM et al, Blood 115:783, 2010). 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 process 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. 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 now moved forward in the laboratory with Zinc Finger Nucleases to demonstrate that this general approach of targeting a corrective minigene to the AAVS1 safe harbor site can be applied to 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. We have also 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. 6. We have published a number of chapters and reviews about gene therapy, 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 (Segal et al, Biol Blood Marrow Transplant 17(S-1): S123, 2011;Kang et al, J Allergy Clin Immunol 127:1319, 2011);Kand and Malech, Methods Enzymol 507:125, 2012;Corrigan-Curay, J et al, Mol Ther 20:1084.

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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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