Gene Therapy Clinical Trials for Chronic Granulomatous Disease

该项目涉及用自体造血干细胞靶向基因疗法治疗X连锁慢性肉芽肿病（CGD）的治疗性临床试验。患有CGD的患者具有缺陷的循环血液中性粒细胞，其不能产生杀微生物的过氧化氢。他们经常遭受威胁生命的感染和过早死亡。 1997年，我们完成了一项基因治疗遗传性吞噬细胞免疫系统缺陷的临床试验，这种缺陷被称为X连锁形式的慢性肉芽肿病（X-CGD）。 在我们的一些基因疗法治疗的患者中，外周血中高达1/400的循环中性粒细胞在基因疗法后表现出功能校正。这种峰值水平的校正发生在治疗后3至6周，并且在多次输注自体离体基因校正的CD 34+祖细胞治疗的5名患者中的3名中，效果可以持续超过一年。这些基因治疗研究表明，有可能通过基因治疗对患者的CGD缺陷提供低水平的部分和短暂的纠正。2004年，来自德国的一个治疗X-CGD患者的小组报告了一项类似的CGD基因治疗试验的结果;然而，他们还包括剂量为8 mg/kg的化疗剂白消安，以在骨髓中腾出空间，从而改善植入。 它们在外周血中达到20%的初始水平，然而，也有基因校正的髓样细胞的生长，导致水平增加。然而，这种生长与寡克隆性和克隆的过度表达相关，其中基因治疗载体通过插入诱变激活了MDS 1和其他与骨髓细胞发育相关的基因。尽管该试验中的患者未被治愈，并且第一名患者实际上死于败血症，但两名患者似乎都从治疗中获得了一些临床益处，因为他们在移植时都有潜在的感染，这些感染在克隆生长和最终沉默转导细胞之前的初始移植周期间消退。 因此，我们在2006年启动了一项临床试验，治疗患有XCGD和潜在感染的患者，方案编号为07-I-0017。基于恒河猴的临床前数据以及患者的临床数据，我们在输注转基因细胞之前以10 mg/kg的剂量使用白消安。 我们治疗了三名患者，第一名是28岁的男性，患有多发性肝脓肿，不适合手术或射频消融方法。 该患者最初有24%的阳性细胞水平，治疗后7个月，他的肝脏病变消退，外周血中持续存在1.2%的可检测标记，没有克隆生长的证据，每个细胞基础上的氧化酶表达水平继续处于几乎正常的水平，与他的既往病史相比，感染率降低。 然而，患者出现了进行性肺部病变，尽管尝试了同种异体移植，但仍导致其于2016年死亡。在该试验中治疗的第二名患者似乎产生了针对转导细胞的免疫反应，在外周血中最初具有5%标记后，这些细胞迅速清除。第三名患者接受了真菌性肺部感染治疗，最初的标记水平为4%，随后下降至0.03%，在此保持稳定，直到他因持续感染接受了匹配的无关供体移植。 他现在已经三年了，做得很好。 我们现在已经在一项合作试验中招募患者-方案15-I-0008，使用Genethon生产的慢病毒载体。第一例患者于2015年12月在波士顿接受治疗，并在20-25%的范围内持续标记，表现相对较好。试验中的第二名患者于2016年7月在NIH接受治疗，该患者的评分持续在20-30%范围内超过一年，没有不良事件。 第二名NIH患者（试验中的第四名）在移植后两个多月，他接受治疗的感染有了显著改善。 我们的第三名患者于8月初接受治疗，第四名患者将于9月接受治疗。 试验中的所有患者（迄今为止共治疗了5例）均具有良好的标记，并且没有治疗的不良事件。 我们现在正在努力获得额外的载体，以继续治疗患者，因为我们迄今为止已经看到了非常令人兴奋的结果，因为我们已经在多中心研究中使用了所有可用的插槽。 我们还在研究修正案，以提高这种疗法的易用性和适用性，包括冷冻转导细胞的使用。 我们还根据方案10-I-0016启动了plerixafor/GCSF动员的外周血细胞的累积，以评估plerixafor对这些动员细胞的基因转导的影响（如有）。 最后，我们将继续与华盛顿大学的大卫罗林斯和圣犹大大学的研究人员合作，为Wiskott-Aldrich综合征患者启动临床基因治疗试验。 本研究的结果也有助于提高CGD患者的基因治疗水平。

This project involves the conduct of therapeutic clinical trials for the treatment of X-linked chronic granulomatous disease (CGD) 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. In 1997, 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. Although the patients in this trial were not cured, and the first patient actually expired from sepsis, both patients appeared to have some clinical benefit from the treatment as they each 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 initiated a clinical trial in 2006 to treat patients with XCGD and an underlying infection, protocol number 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 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 bloodthere was no evidence of clonal outgrowth and the level of oxidase expression on a per cell basis continued to be at almost normal levels with a reduction in infection rate compared to his prior history. The patient howevere developed a progressive pulmonary process which despite an attempt at allogeneic transplant, led to his demise in 2016. The second 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 in the peripheral blood. 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 until he underwent a matched unrelated donor transplant due to continued infections. He is now three years out doing well. We have now been enrolling patients in a collaborative trial- Protocol 15-I-0008 using a lentiviral vector produced by Genethon. The first patient was treated in Boston in December 2015, and has done relatively well with persistent marking in the 20-25% range. The second patient on the trial was treated at NIH in July 2016, and this patient continues to have marking in the 20-30% range more than a year out with no adverse events. The second NIH patient (fourth on the trial) is now over two months post transplant with significant improvement in his infection for which he was treated. Our third patient was treated early August and the fourth will be treated in September. All patients on the trial (total of 5 treated to date) have good marking and no adverse events from the therapy. We are now working to obtain additional vector to continue treating patients given the very exciting results we have seen to date as we have used all of our available slots on the multicenter study. We are also working on amendments to improve the ease and applicability of this therapy including the use of freezing the transduced cells. We have also initiated accrual of plerixafor/GCSF mobilized peripheral blood cells on Protocol 10-I-0016, to assess the impact, if any, of plerixafor on gene transduction of these mobilized cells. Finally, we are continuing our collaboration with David Rawlings at the University of Washington and investigators at St Judes to initiate a clinical gene therapy trial for patients with Wiskott-Aldrich Syndrome. Results from this study should be helpful for improving our gene therapy for CGD patients as well.