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 vector 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 pleuripotent 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 (dogs are collaboration with Dr. P Felsburg at the Univ of Pennsylvania). Most important is that this construct does not activate LMO2 when inserted into the first intron of this gene (Zhou et al, Blood 116:900, 2010). This critical pre-clinical work allowed the development of clinical protocols of gene therapy for XSCID using this vector that have been approved by the IRBs 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 clinical trial of gene transfer at St. Jude will study treatment of infants newly diagnosed with XSCID. 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 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). With our other collaborators we are exploring other envelops and lentivector systems with the ultimate aim of bringing the best vector to the clinic (Sakuma T et al, Hum Gen Ther 21:1665, 2010). 5. 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 demonstrate 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). 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;and Kang et al, J Allergy Clin Immunol 127:1319, 2011).
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