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), X-linked severe combined immune deficiency (SCID-X-1 or XSCID), and Wiskott-Aldrich syndrome. 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 that are derived from the human immunodeficiency virus (HIV). Important aspects of this study of lentivectors relating to preferred genome insert sites also derive from known information about HIV. 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. This work also involves development of precise gene targeting to achieve integration of a corrective gene into a specific safe-harbor site in the genome, or the precise repair of a disease mutation. In the past fiscal year year we have accomplished the following: 1. Together with our collaborators (Dr. B Sorrentino at St. Jude) we used a high titer lentivirus vector encoding the common gamma chain of the IL2 receptor that we developed together to treat in a gene therapy trial for XSCID three young adult patients and three children. Results from this trial are encouraging in that there has been correction of both cellular (T cell and NK cell) immunity and humoral immunity (B cell antibody production). The first two patients were able to stop their IgG supplementation and responded to immunization by developing protective titers to many standard immunogens. A first report of these studies has been published this year in Science Translational Medicine in April and is included in the bibliography for this report (De Ravin SS, et al, Sci Transl Med. 2016 Apr 20;8(335):335ra57). 2. We have developed a novel method of gene editing using zinc finger nuclease mRNA targeting the AAVS1 safe harbor genomic site from Sangamo BioSciences delivered to human CD34+ hematopoietic stem cells by electroporation using the MaxCyte instrument together with delivery of a donor plasmid in an AAV6 vector to achieve unprecedentedly high levels of insertion of either a fluorescence marker (Venus; >50% gene targeting) or a therapeutic gene for X-linked chronic granulomatous disease (gp91phox; >15% gene targeting). A first report of these studies has been published this year in Nature BioTechnology in April and is included in the bibliography for this report, and as the major scientific advance associated with this Annual Report (De Ravin SS, et al. Nat Biotechnol. 2016 Apr;34(4):424-9). 3. This year we opened here at the NIH a new clinical trial of lentivector gene therapy for X-chronic granulomatous disease using a novel vector developed by Adrian Thrasher. and which we assisted in assessing (see Santilli G, Almarza E, Brendel C, Choi U, Beilin C, Blundell MP, Haria S, Parsley KL, Kinnon C, Malech HL, Bueren JA, Grez M, Thrasher AJ. Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells. Mol Ther 19:122-32, 2011.) We have enrolled our first patient here at the NIH, achieving early gene correction at a level of 28% of circulating neutrophils in this young adult patient becoming oxidase positive. A patient also has been treated with this vector at the Boston Childrens Hospital site. 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) (Merling et al. Mol Ther 2015, 23:147-57). 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 CYBB or NCF1 gene to achieve gene repair for correction of X-linked CGD or the p47phox deficient autosomal recessive form of CGD. 6. With our collaborators, we have developed improved methods of assessing the insert sites of gene therapy vectors in the genome. 7. We have published reviews and an editorial about gene therapy either from our group or as part of a collaborative effort with other investigators in development of gene therapy for PIDs.
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