This project involves the conduct of therapeutic clinical trials for the treatment of inherited immune deficiencies using hematopoietic stem cell transplantation. 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. In 2004 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. We have now opened a clinical trial to treat patients with immunodeficiencies using either a matched related, matched unrelated, or cord blood product and a tolerance inducing conditioning regimen consisting of Campath 1-H and busulfan with sirolimus for graft versus host disease (GvHD) prophylaxis. For patients receiving an unrelated product, total body irradiation is also added to the regimen. To date we have transplanted a total of 44 patients on this protocol, 39 of whom received an unrelated donor graft. In patients not receiving a stem cell boost we have we have seen very limited GvHD with only one patient having Grade 2 skin GvHD and three patients with Grade 1 GvHD of the gut. We have used cord blood in 3 cases. One was for a patient with P67 deficient CGD and another with the X-linked form, both of whom failed to engraft. With the first patient, the thinking was that the Campath impacted negatively with the ability of the more naive T cells in a cord blood graft preventing engraftment, and the protocol was modified to use ATG in the case of a cord blood product. However with the second graft failure in a CGD patient, we have stopped using cord blood products for these patients. This data is also correlated by results from Duke University where even with a myeloablative regimen, the engraftment rate using cord blood products in CGD patients is between 40 to 50%. The third patient to receive a cord blood graft was transplanted for X-linked Severe Combined Immunodeficiency. He was conditioned with ATG instead of Campath and is currently 7 years out with full engraftment, no evidence of graft versus host disease, and normal immune function. We now have had a total of nine deaths on the protocol with none in the last year. Two of the deaths were unrelated to the transplant (refusal to continue dialysis, and an accidental drug overdose). One patient developed a transfusion related acute lung injury after a platelet transfusion and three patients died from infection after developing GvHD (Klebsiella infection, adenovirus, and aspergillus brain abscess). One patient had pulmonary hemorrhage and CMV and a final patient developed HHV6 after prolonged pancytopenia after a late graft failure and stem cell boost. Despite these outcomes, our overall mortality rate has been low relative to bone marrow transplantation in general using unrelated donors. Further we have transplanted a number of patients with ongoing infections including fungal osteomyelitis of the spine and/or meninges and in some cases have used granulocyte infusions during the period of transplant-induced neutropenia with no adverse effects. Overall our results for the CGD patients in particular are especially promising with an overall survival of 38 out of 47 and an overall long term engraftment rate of 35 out of 38 evaluable. We are also the only centre to have transplanted patients with the P40 form of CGD demonstrating complete reversal of refractory colitis in this unique subset. (Manuscript in preparation.) Given the graft rejection rates we have now opened a modified protocol using a higher cell dose and post transplant cyclophosphamide to mitigate the increased risk of GvHD from the higher cell dose. At this point, we have enrolled 3 patients on this protocol with one death due to progressive underlying pulmonary disease. The other two patients are doing well, engrafted, without any evidence of GvHD to date. At the end of 2014, we also initiated a clinical trial using haploidentical donors and transplanted a patient with no matched donors available. This patient had an ongoing infection refractory to all standard therapy involving the heart and we therefore proceeded with a parental graft and using post transplant cyclophosphamide. We obtained good engraftment with only limited graft versus host disease. This patient is now almost 2 years out with complete resolution of his infection. We have now enrolled an additional 4 patients, including a 26 yo female with an invasive aspergillus infection of the sternum. We have seen mild GvHD and mild hemorrhagic cystitis as a result of the high dose post transplant cyclophosphamide, but all patients have done well to date. In order to determine the outcomes of transplant for CGD in general, we have become a part of the Primary Immune Deficiency Treatment Consortium and have initiated a collaborative protocol to review the results of transplant done for CGD in North America. The protocol has now been approved and is being implemented in various centers. We have started to enroll patients for both the retrospective and prospective transplant studies. We have also been obtaining stool samples pre and post transplant for microbiome analysis, which is a substudy being done in collaboration with Emilia Falcone (NIAID) and Julie Segre (NHGRI). In related laboratory pre-clinical studies, we have been investigating the use of an adenosine A2a receptor agonist to prevent or treat graft versus host disease (GVHD). Prior studies have shown that agonists specific to this receptor improve outcomes in ischemia models of tissue damage. We have seen benefit in attenuating the onset and severity of GVHD in our F1-parental transplant model and have published this data in the Journal of Leukocyte Biology. Further studies have shown a role for T regulatory cells (Tregs) as part of the mechanism of the drugs effects. We have now established a CRADA with Lewis and Clark Pharmaceuticals (formally Adenosine Therapeutics/PgxHealth/Forest Labs) to study other formulations of the A2a receptor agonists and have seen similar effects on T regs, both in vitro and invivo, by these agonists. This data was published in the Journal of Immunology and we recently published a review paper regarding the use adenosine agonists in GVHD. Dr Sharma a postdoctoral fellow was the first author. Moreover, we have started to study these drugs in a colitis model given their benefit in GvHD of the gut. We are now in the midst of submitting a paper describing a new method to assess these agonists in a more rapid and efficient manner prior to beginning in vivo murine studies. We have also started a collaboration with Gabriel Dvesklar of UHSUS to study Pregnancy Specific Glycoprotein -1 and 9 (PSG-1 or 9) as immunomodulators and a possible therapy for GvHD. Preliminary in vitro and in vivo studies have been promising with reduced GvHD seen in either in vitro studies or our mouse models treated with the proteins. We have published one paper describing our results with PSG-9 in collaboration with Dr Dvesklar and Dr Sharma has presented some of this data at the annual American Society of Hematology meeting. The data from our results with PSG-1 are being readied for publication with only a few more analyses pending.
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