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. About three 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. Six years ago we completed a phase I study of gene therapy for the p47phox deficient autosomal recessive form of CGD (AR-CGD). In that study we demonstrated that a single cycle of gene therapy targeting cytokine mobilized and purified stem cells could result in production of small numbers (.004% to .05%) functionally corrected neutrophils in the peripheral blood that persisted for 2 to 6 months. Following completion of this first trial, we incorporated a number of technical improvements in the gene transfer technology into a modification of the gene therapy clinical trial and focused on gene therapy for the X-linked, gp91phox deficient form of CGD. An amended gene therapy protocol for CGD began to enroll patients in April 1998 incorporating these enhancements and enrollment and treatments were completed in March 2000 and results were reported. In some of the 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 demonstrate that it is possible to provide a low level partial and transient correction of the CGD defect in patients by gene therapy. While the level of correction is not a cure and may not be at a level that provides clinical benefit, it represents a very important demonstration of the principle that gene therapy can correct the biochemical defect of CGD in the patient. It may be that even low level and transient correction might provide clinical benefit in the setting of severe recurrent infections. The current study has achieved its scientific goal of demonstrating feasibility. We have continued to follow these patients for several years and have noted the disappearance of marked cells by about a year after the last treatment. No adverse effects of the gene therapy have been observed more than three to five years after the gene therapy. Recently it has become possible to enumerate and determine the genome location through sequencing of retrovirus insertion sites using a novel method called linear amplification modified polymerase chain reaction (LAM-PCR). We also are in the process of delineated insertion site in archived blood samples from the CGD clinical trial. We are in the planning stage of a study to determine if non-ablative marrow conditioning might enhance the level and durability of the effect of gene therapy for CGD. One of the important questions is whether in a clinical setting it is possible to use non-ablative marrow cytoreduction to more safely achieve the engraftment of stem cells. While this question has an impact on achieving success in gene therapy, advances in the use of non-ablative conditioning for allogeneic transplantation have allowed us to explore the potential of this approach to achieve curative allogeneic transplantation for CGD. An ongoing clinical trial was initiated and recently completed in which patient with CGD undergo non-ablative marrow conditioning with immune suppression achieved with a combination of cyclophosphamide, fludarabine and anti-thymocyte globulin. The patients then received a transplant with purified CD34+ peripheral blood stem cells mobilized from a fully 6/6 HLA matched sibling of the CGD patient. The graft is depleted of most lymphocytes and donor lymphocytes are infused at later time points after transplant to help to establish the donor graft. 5 adults and 5 children were transplanted. 4 of 5 children achieved stable long term engraftment that appears to provide a significant level of protection from infection, and all 5 children are alive and well. While 4 of 5 adults achieved long term engraftment, there were three deaths, one from complications of graft versus host disease, one from pneumococcal pneumonia at 1 year post transplant, and one from complications from a second fully ablative salvage transplant procedure. Of the two adult CGD transplant patients who are fully engrafted one remains infection-free for 3 years without any complications and appears cured of his CGD. The other fully engrafted adult transplant patient is also infection-free and appears to be cured of his CGD, but continues to have mild and well controlled graft versus host disease. We conclude that non-ablative matched related allogeneic transplant is a reasonable option in pediatric patients with CGD and a high risk of mortality from recurrent infection. Currently adults appear to have a higher risk of complications from graft versus host disease and delayed recovery of lymphocyte immunity. Because of that we have begun to study the potential of extracorporeal photophoresis to treat chronic graft versus host disease. If graft versus host disease risks can be reduced it would then reduce one of the risks of allogeneic transplantation for inherited immune deficiencies. A follow up trial of non-ablative allogeneic transplantation in children with high risk CGD has begun using modified procedures to enhance engraftment and reduce the risks of graft versus host disease. A similar transplant trial has been developed as a salvage therapy for CGD patients with incurable infection. An important goal within our program is to also develop gene therapy for X-linked severe combined immune deficiency. However, within the past year there has been a report of two out of 9 infants with XSCID cured with gene therapy in France who at 2 to 3 years after the gene therapy developed lympocytic leukemia as a result in insertion al mutagenesis in the LMO-2 oncogene. Regulatory bodies such as the Food and Drug Administration and the Recombinant DNA Advisory Committee of the Office of Biotechnology Activities have determined that gene therapy for XSCID should be restricted to patients who do not have other therapeutic alternative. We have studied a series of patients with XSCID without a matched sibling who have failed to achieve significant benefit from the standard therapy of a haploidentical marrow transplant from a parent. These patients have recurrent infections and extreme failure to grow normally expressing significant pulmonary impairment. We have been developing the tools to conduct a clinical trial of ex vivo gene therapy for this group of XSCID patients who are doing poorly and lack other therapeutic alternatives.
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