Gene Therapy and Allogeneic Transplantation For Immune D

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Abstract

This project involves the conduct of therapeutic clinical trials for the treatment of inherited immune deficiencies using hematopoietic stem cell therapies including allogeneic transplantation and autologous blood stem cell targeted gene therapy. This project also includes associated studies of the diagnostic procedures (including genetic diagnosis), and treatment modalities that are alternatives to transplantation and gene therapy for those patients with the inherited immune deficiencies that are the target diseases for our transplant and gene therapy program. During the past year we have completed the first phase of a clinical trial of gene therapy for X-linked severe combined immune deficiency. Retroviral gene therapy can restore immunity to infants with X-linked severe combined immunodeficiency (XSCID) caused by mutations in the IL2RG gene encoding the common gamma chain (gc) of receptors for interleukins (IL)-2, -4, -7, -9, -15 and -21. We investigated the safety and efficacy of gene therapy as salvage treatment for older XSCID children with inadequate immune reconstitution despite prior bone marrow transplant from a parent. Subjects received retrovirus transduced autologous peripherally mobilized CD34+ hematopoietic cells. Multi-lineage retroviral marking and improvements in health occurred in all 3 children, and T cell function significantly improved in the youngest subject (age 10 years). Further follow-up of clinical, immunologic and molecular parameters in our patients will establish the long-term safety and efficacy of this approach to gene therapy for pre-adolescents with XSCID who have failed to achieve or maintain immune reconstitution after BMT. 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). 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. 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. This past year the results of a similar gene therapy trial for CGD was reported by a group from Germany that X-CGD patients conditioned with the chemotherapy agent busulfan to make room in the bone marrow before the gene therapy had outgrowth of gene corrected myeloid cells resulting in high level correction. However, this outgrowth was 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 eveidence 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 busulfan conditioning in the German study or it may relate to the strong promoter activity known to be associated with their murine spleen focus forming virus based vector relative to our MFGS which is derived from murine Moloney leukemia retrovirus. Other clinical research accomplishments this year include increasing recognition that autoimmune problems can affect patients with a variety of primary immune deficiencies (PID). Many types of PID are more aptly characterized as diseases of immune dysregulation rather than just as immune deficiency with recurrent infections. We published a paper describing two patients in which chronic granulomatous disease appears to have triggered the development of sarcoidosis, an autoimmune disease not generally seen in CGD. This was the first report of sarcoidosis in CGD, but Crohn's disease, discoid lupus erythematosis and rheumatoid arthritis have been noted in CGD patients. We proposed in this paper an important new paradigm in understanding CGD, suggesting that there is immune dysregulation associated with CGD that may trigger autoimmune diseases in a subset of patients where the specific autoimmune disease triggered likely related to an individual patient's genetic predisposition to a particular autoimmune disease. This has important therapeutic management implications in that specific therapies proven to be effective for the specific autoimmune disease triggered by CGD must be used in such patients rather than just the general clinical management modalities designed to prevent infections or control the general inflammation common to most CGD patients. In the area of allogeneic transplantation for CGD, we previously reported the successful use of non-ablative conditioning to achieve successful long term engraftment and cure of CGD patients using HLA-matched sibling donors as the source of the hematopoietic stem cell graft. One of the problems with this approach was the high rate (30%) of graft failure or very low engraftment. This year we performed a follow up transplant on an X-CGD child previously transplanted by us who had achieved high level donor T cell engraftment but less than 1% long term myeloid engraftment. We demonstrated successful permanent conversion to almost 100% donor chimerism in the lymphoid and myeloid lineages using conditioning with only busulfan at 10 mg/kg. This strongly supports the use of this approach to rescue low engraftment rather than using a fully myelo- and lympho-ablative conditioning regimen for such salvage therapy. Finally, in other studies we are conducting a clinical trial to examine the potential of extracorporeal photophoresis to treat chronic graft versus host disease and to understand how this modality works immunologically. If graft versus host disease risks can be reduced it would then reduce one of the risks of allogeneic transplantation for inherited primary immune deficiencies. In order to reduce graft versus host disease we have begun a clinical trial to study the effect of extracorporeal photopheresis (ECP) on cGVHD. We found novel immunologic changes in ratios of central and effector memory T-cells that correct following three months of ECP. Chronic graft versus host disease (cGvHD) remains a problematic complication of allogeneic hematopoietic stem cell transplantation. Laboratory parameters correlated with cGvHD have not been fully defined, though changes in CD4/CD8 ratios occur and a decrease in CD4+ central memory T-cells has been noted. Extracorporeal photopheresis (ECP) is an effective therapy for steroid-refractory cGvHD. We have noted changes in lymphocyte subsets following ECP. CD4+ and CD8+ T-cell central and effector memory populations were enumerated by flow cytometry in a cohort of 37 patients post-allogeneic transplantation with symptomatic cGvHD. 7 of the patients with symptomatic cGvHD were treated with ECP over 6 months and prospectively assessed for changes in lymphocyte subsets. There was a highly significant correlation of an increase in CD8+ central memory cells and a concomitant decrease in CD4+ central memory cells. These data indicate a high correlation between disturbances in the balance of central and effector memory populations and cGvHD suggesting utility in following responses to therpy.