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. 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 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 two and one half years to treat in a gene therapy trial for XSCID two young adult patients and three children. Results from this trial are encouraging, and analysis of the patterns of lentivector insert in blood cells from these two patients farthest out shows excellent polyclonality with no persistant dominant clones. The 5 patient in this trial are 30, 24, 5, 1.5, and 1 month post-treatment. All patients are stable with no safety problems encountered to date. Marking in the 1st three patients in myeloid cells is between 8% and 12%, and marking in lymphocytes in the first three patients is increasing as expected. The first two patients have acquired the ability to produce immunoglobulins with normal responses to immunizaton, and freedom from requirement for periodic supplemental immunoglobulin intravenous infusions for the first time in their lives. This is an unprecedented result for gene therapy in patients with XSCID. The results of the first two subjects to date farthest out from treatment are quite extraordinary and life-changing for these patients. Subject one required life-long intravenous gamma globulin, had chronic protein losing enteropathy resulting in an IVIG requirement of weekly infusions to maintain IgG levels just barely in the sub-normal acceptable levels, recurrent viral infections, chronic norovirus, and had a BMI less than 16 (very malnourished), with absent NK cells and abnormally functioning T cells. Subject one now has been totally off IVIG for 21 months with a blood IgG in the normal range, has responded to influenza and tetanus immunization with normal titers, no longer has protein losing enteropathy nor the norovirus, has had no significant infections, has a BMI well above 18 in the normal range, has acquired NK cells and has normal T cell functional assays. Subject two has had a similar response. As part of this study we have shown for the first time that treatment with lentivirus vector can result in patients acquiring false positive DNA pcr test for HIV. While this carries no medical risk to patients, it is an important informed consent issue for such clinical trials (See: De Ravin, et al. Mol Ther 2014, 22:244-5) 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. Work toward the next phase of development of gene therapy for X-CGD will involve a multicenter study using a lentivector 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.) 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 30% of the neutrophils that arise from gene corrected stem cells. This lentivector developed by our collaborators in London and Frankfurt uses a hybrid promoter from Fes plus Cathepsin G genes that provides myeloid specificity to expression. 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 fall 2015. 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. 6. 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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