Development and conduct of allogeneic stem cell transplant and autologous stem cell gene therapy for inherited immune deficiencies

该项目的第一部分涉及原发性免疫缺陷患者同种异体移植的调理方案的发展。2007年，我们启动了一项使用busulfan、Campath和低剂量TBI的临床方案，治疗了44例CGD患者，其中39例接受了非亲属供体（MUD）移植。研究结果发表在《临床免疫学杂志》上。(Parta等。江森自控)。该队列包括两名P40型CGD患者，在这一独特的亚群中表现出难治性结肠炎的完全逆转。在该研究的后续研究中，我们在之前的研究结果的基础上建立了一个新的方案模型，使用更高的细胞剂量和移植后环磷酰胺来改善移植，同时减轻较大移植物引起的移植物抗宿主病（GvHD）的风险。到目前为止，我们已经招募了20名患者，其中3人死于进行性肺病。对所有移植患者的回顾性评估表明，移植前C反应蛋白（CRP）升高是一个常见的危险因素，因此修改了方案以排除CRP升高的患者。我们随后又移植了5例患者，结果良好。一名患者由于细胞剂量低而未接受移植后环磷酰胺治疗，尽管最初植入，但在第85天失去了移植物。他仍然健在。第二例患者尽管在移植后使用环磷酰胺，但仍出现移植物丢失。其余的患者都很好，没有患者发展成严重的移植物抗宿主病。

The first part of this project involves the development of conditioning regimens for allogeneic transplantation of patients with primary immunodeficiencies. In 2007 we initiated a clinical protocol using busulfan, Campath and low dose TBI and treated 44 patients with CGD, 39 of whom received an unrelated donor (MUD) graft. The results were published in the Journal of Clinical Immunology. (Parta et al. JCI). Included in this cohort were two patients with the P40 form of CGD demonstrating complete reversal of refractory colitis in this unique subset. In follow up to that study we have opened a new protocol modeling on our previous results using a higher cell dose and post-transplant cyclophosphamide to improve engraftment but mitigate the increased risk of Graft versus Host Disease (GvHD) from the larger graft. We have enrolled 20 patients so far with 3 deaths due to progressive pulmonary disease. A retrospective evaluation of all transplanted patients suggested that an elevated C reactive protein (CRP) prior to the transplant itself was the one common risk factor and the protocol was thus modified to exclude patients who have an elevated CRP. We subsequently transplanted 5 more patients with good outcomes. One patient did not receive the post-transplant cyclophosphamide due to a low cell dose and despite initial engraftment, lost the graft at day 85. He remains alive and well. A 2nd patient experienced graft loss despite the use of post-transplant cyclophosphamide. The remainder of the patients have all done well with no patients developing severe GvHD. To expand eligibility, in 2014 we opened a protocol using haploidentical donors. The 1st patient had an ongoing infection refractory to all standard therapy involving the heart and is now 5 years out with complete resolution of his infection. (J Clin Immunol. 2015 Oct;35(7):675-80). We enrolled a total of 7 patients on this protocol but saw severe GvHD in the last 3 patients with 2 of the patients succumbing to its' complications but the 3rd patient recovering and now doing well. This protocol is now closed and a new protocol is now open to accrual (19-I-0080). This new protocol uses both early and late Campath along with busulfan, TBI, and post-transplant cyclophosphamide. One patient has been treated to date and has done very well with full engraftment, and no evidence of GvHD. Accrual and treatment has been slowed due to Covid-19 but we have a 2nd patient planned for August. A protocol for X-linked and JAK-3 SCID (20-I-0080) has also been opened for accrual, but no patients have yet been enrolled. We are also working towards developing antibody based conditioning regimens for CGD and SCID patients in order to further reduce regimen toxicity. As a member of the Primary Immune Deficiency Treatment Consortium (J Allergy Clin Immunol. 2014 Feb;133(2):335-47) we developed a collaborative protocol (6903) to review the results of transplants done for CGD in North America. We enrolled over 100 transplanted patients and published the results on a subgroup of patients with inflammatory bowel disease (Marsh et al, JCI 2019). We are now in the midst of analyzing the data from the overall study. We have also been involved in a microbiome analysis, (a substudy done in collaboration with Emilia Falcone) with a manuscript now in preparation. We are also finalizing a new CGD related protocol (6908) which will specifically evaluate the autoinflammatory aspects of CGD patients pre and post-transplant. In the laboratory, our post bacs Caroline Kreitzer and Nicole Fama, have been working on developing murine models of engraftment syndrome and graft failure as these both occur with significant frequency in our CGD patients and improved understanding of the pathophysiologies should lead to better prevention and treatment. Utilizing our established murine models of GvHD, we are modifying the conditioning regimens to induce graft rejection and/or engraftment syndrome with various cytokines to mimic the inflammatory milieu seen in patients as well as manipulating the graft cell composition to assess any donor graft effects. Although progress has been made in these efforts, there have been delays due to Covid-19. The second part of this project involves the use of genetically modified autologous cells for the treatment of patients with XCGD and other immunodeficiencies. We initiated a clinical trial in 2006 to treat XCGD patients and an underlying infection, protocol 07-I-0017. Based on preclinical data in the rhesus as well as clinical data in a patient, we used busulfan at a dose of 10mg/kg prior to infusion of the genetically modified cells. We treated three patients, the results of which were published in Blood. Of the three patients the first had persistent levels of detectable oxidase positive cells more than 7 years post gene therapy; however he developed a progressive pulmonary process which despite an attempt at allogeneic transplant, led to his demise in 2016. The 2nd patient treated on this trial appeared to develop an immune reaction against the transduced cells, with rapid clearance of these cells after initially having 5% marking. 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 remained stable until he underwent a MUD transplant due to continued infections. He is now more than 5 years out doing well. In 2015 we developed a collaborative study, Protocol 15-I-0008, using a lentiviral vector for XCGD. The 2nd patient on the trial was treated at NIH in 2016, and continues to have marking in the 20-30% range more than 3 years post treatment. The 2nd NIH patient is now over 2 years post transplant with resolution of his underlying pulmonary fungal infection. Our 3rd patient was treated in early August of 2017 and unfortunately developed autoimmune thrombocytopenia, unrelated to the gene therapy, and died of a cerebral hemorrhage. Our most recent patient was treated in 2019 and is doing well with more than 70% oxidase cells at his last evaluation. A total of 9 patients have been treated at the various sites. Unfortunately 3 patients, including the last patient treated in 2020, have had loss of their marking. The results, including patients from a London trial that used the same type of conditioning and vector have been published. (Kohn et al. Nature Medicine 2020.) An addendum protocol to treat patients with the same vector but using a modified conditioning regimen is now being developed to reduce the risk of graft loss/rejection. We are also developing a collaborative study to treat patients with the P47 autosomal recessive form of CGD using a lentiviral vector and hope to start enrolling patients by late 2020 (delayed due to the Covid-19). Future plans will include incorporating transduction enhancers to improve the efficiency of transduction and reduce the amount of vector needed to treat individual patients. We are also planning to involve the use of antibody-based conditioning similar to our efforts in allogeneic transplantation. Uimook Choi and Nicole Fama are also working on developing vectors for the P67 and P22 forms of CGD. Finally, Caroline Kreitzer, in collaboration with the laboratory of Mihailis Lionakis, has been developing a CARD 9 lentivector with initial data showing efficacy in in vitro studies. In vivo mouse studies will be initiated this fall after work optimizing the vector to achieve higher protein expression.