This project involves the conduct of therapeutic clinical trials for the treatment of X-linked chronic granulomatous disease with autologous blood stem cell targeted gene therapy. Patients with CGD have defective circulating blood neutrophils that fail to produce microbicidal hydrogen peroxide. They suffer from recurrent life threatening infections and premature mortality. About six years ago we completed a clinical trial of gene therapy for the inherited deficiency of the phagocytic cell immune system known as the X-linked form of chronic granulomatous disease (X-CGD). In some of our gene therapy treated patients up to 1 in 400 circulating neutrophils in the peripheral blood demonstrated functional correction following the gene therapy. This peak level of correction occurred at 3 to 6 weeks after therapy and the effect could be sustained for over a year in three of five patients treated with multiple infusions of autologous ex vivo gene corrected CD34+ progenitor cells. These gene therapy studies demonstrated that it is possible to provide a low level partial and transient correction of the CGD defect in patients by gene therapy. In 2004, the results of a similar gene therapy trial for CGD was reported by a group from Germany that treated X-CGD patients;however they also included the chemotherapy agent busulfan at a dose of 8mg/kg to make room in the bone marrow and therefore improve engraftment. They achieved initial levels of 20% in the peripheral blood however, there was also an outgrowth of gene corrected myeloid cells resulting in increasing levels. This outgrowth was however associated with oligoclonality and over-representation of clones in which the gene therapy vector had by insertional mutagenesis activated MDS1 and other genes associated with myeloid cell development. Our own insertional analysis of myeloid blood cells from our own previous CGD gene therapy study demonstrated significant polyclonality and no evidence of outgrowth of clones containing vector insertion in MDS1 or other myeloid regulatory genes. Cause for the differences between the recent German X-CGD gene therapy study and our own previous studies in this regard may relate to the strong promoter activity known to be associated with their murine spleen focus forming virus based vector relative to our MFGS vector which is derived from murine Moloney leukemia retrovirus. Although the patients in this trial were not cured, and the first patient actually expired from sepsis, both patients did have some clinical benefit from the treatment. Both patients had an underlying infection at the time of their transplant, which resolved in the initial peritransplant period prior to the clonal outgrowth and ultimate silencing of the transduced cells. We therefore initated a clinical trial in 2006 to treat patient with XCGD and an underlying infection and are continuing to enroll patients to this trial, protocol number 07-I-0017. Based on preclinical data in the rhesus as well as clinical data in a patient, we are using busulfan at a dose of 10mg/kg prior to infusion of the genetically modified cells. To date we have treated three patients, the first a 28 year old male with multiple liver abscesses, not amenable to surgical or radio frequency ablative approaches. The patient initially had a level of 24% positive cells and at 7 months post treatment had resolution of his liver abscesses, with 1.2% detectable marking persisting in the peripheral blood. Now at almost five years post treatment, he continues to have detectable marking levels in the peripheral blood of 0.8%. There is no evidence of clonal outgrowth or myelodysplasia as has been seen in the German XCGD trial. Additionally, the level of oxidase expression on a per cell basis continues to be at almost normal levels and the patient appears to have benefited from the treatment with fewer infections per year now than historically. He continues too remain only partially compliant with his antibiotic prophylaxis and more than 4 years out has shown no evidence of adverse effects from the gene therapy. The second patient was treated due to an underlying fungal infection of the chest wall. Despite almost three years of ongoing polymicrobial therapy, this lesion persisted, and therefore the patient was eligible for the gene therapy protocol. His course, however, was not as successful as the first patient as he appeared to develop an immune reaction against the transduced cells, with rapid clearance of these cells after initially having 5% marking in the peripheral blood. The patient subsequently expired due to continued progression of his infection. The third patient was treated for a fungal lung infection and had an initial marking level of 4% with a subsequent decline to 0.03% where it has remained stable now out to almost three years post treatment. He is doing well clinically although continues to have the occasional CGD related infection, and also has no signs of adverse events related to the gene therapy. He was also treated with rapamycin, which we added to the protocol, to prevent possible immune rejection as is hypothesized to have occurred in the second patient and this was well tolerated. We have collected cells on additional patients in anticipation of performing gene therapy if these patients'ongoing infections do not improve with standard of care but are waiting to treat any patients with a retroviral vector we produced in collaboration with Ken Cornetta at Indiana University. In 2013 we have also produced a lentiviral vector which we are waiting to use should a patient be eligible for the protocol. Finally we are also collaborating with Don Kohn at UCLA in their lentiviral gene therapy project with vector produced by Genethon, which we hope to be available within the next 6 months. We are continuing to investigate the role of agents such as Sitagliptin, Sr-1 and PTN as well as a novel culturing system using different transduction medias to determine if these may help with maintaining pluripotency of cells during the transduction period as well as improve engraftment.

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