Gene Therapy Clinical Trials for Chronic Granulomatous Disease

该项目涉及利用自体血液干细胞靶向基因疗法治疗X连锁慢性肉芽肿疾病的临床治疗试验。CGD患者循环中的中性粒细胞有缺陷，无法产生杀菌过氧化氢。他们反复遭受威胁生命的感染和过早死亡。大约六年前，我们完成了一项针对被称为X连锁慢性肉芽肿病(X-CGD)的吞噬细胞免疫系统遗传缺陷的基因治疗的临床试验。在我们的一些接受基因治疗的患者中，外周血中高达1/400的循环中性粒细胞在基因治疗后表现出功能纠正。这一校正高峰出现在治疗后3-6周，在5例多次输注自体体外基因校正CD34+祖细胞的患者中，有3例可持续一年以上。这些基因治疗研究表明，通过基因治疗可以对患者的CGD缺陷提供低水平的部分和短暂的纠正。2004年，德国的一个治疗X-CGD患者的小组报告了类似的CGD基因治疗试验的结果；然而，他们还包括8 mg/kg剂量的化疗药白消安，以在骨髓中腾出空间，从而改善植入。他们在外周血中达到了20%的初始水平，然而，也有基因校正的髓系细胞生长出来，导致水平上升。然而，这种结果与克隆的寡克隆性和过度表达有关，在这些克隆中，基因治疗载体通过插入突变激活了MDS1和其他与髓细胞发育相关的基因。我们自己对之前CGD基因治疗研究中的髓系血细胞的插入分析表明，存在显著的多克隆性，没有证据表明含有MDS1或其他髓系调节基因载体插入的克隆会生长。德国最近的X-CGD基因治疗研究与我们之前的研究在这方面存在差异的原因可能与他们基于小鼠脾病灶形成病毒的载体相对于我们的MFGS载体具有较强的启动子活性有关，MFGS载体来自小鼠Moloney白血病逆转录病毒。虽然试验中的患者没有治愈，而且第一个患者实际上死于脓毒症，但这两个患者确实从治疗中获得了一些临床好处。两名患者在移植时都有潜在的感染，在移植细胞克隆生长和最终沉默之前，这一感染在移植前的最初移植周期间就消失了。 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，并继续招募患者参加这项试验，方案编号为07-I-0017。根据恒河猴的临床前数据和患者的临床数据，我们在输注转基因细胞之前使用10 mg/kg的白消安。到目前为止，我们已经治疗了三名患者，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术。患者最初有24%的阳性细胞水平，在治疗后7个月他的肝脓肿消失，外周血中持续存在1.2%的可检测标记。现在，在治疗后近三年，他的外周血液中仍然有1%的可检测到的标志性水平。没有证据表明克隆性生长或骨髓发育不良，就像在德国的XCGD试验中看到的那样。此外，每个细胞的氧化酶表达水平继续保持在几乎正常的水平，患者似乎从治疗中受益，现在每年的感染比历史上更少。他仍然只部分遵守他的抗生素预防措施。第二名患者因胸壁潜在真菌感染而接受治疗。尽管进行了近三年的多菌治疗，但这种损害仍然存在，因此患者有资格接受基因治疗方案。然而，他的疗程并不像第一个患者那样成功，因为他似乎对转导细胞产生了免疫反应，在外周血液中最初有5%的标记后，这些细胞迅速清除。由于感染的持续发展，患者随后死亡。第三名患者接受了肺部真菌感染的治疗，最初的评分水平为4%，随后下降到0.03%，治疗后近一年一直保持稳定。他的感染已经部分缓解，但仍有潜在的CGD的表现。他还接受了雷帕霉素的治疗，我们将其添加到方案中，以防止可能的免疫排斥反应，据推测，第二名患者已经发生了排斥反应，这是很好的耐受性。 我们收集了更多患者的细胞，以期在这些患者的持续感染没有随着护理标准的改善而得到改善的情况下进行基因治疗。我们目前正在与合作者合作，开发基于慢病毒的载体和/或RD114假型载体，以增加长期造血干细胞的转导，这将改善整体结果。

This project involves the conduct of therapeutic clinical trials for the treatment of X-linked chronic granulomatous disease 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. 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). 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. Our own insertional analysis of myeloid blood cells from our own previous CGD gene therapy study demonstrated significant polyclonality and no evidence 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 strong promoter activity known to be associated with their murine spleen focus forming virus based vector relative to our MFGS vector which is derived from murine Moloney leukemia retrovirus. Although the patients in this trial were not cured, and the first patient actually expired from sepsis, both patients did have some clinical benefit from the treatment. Both patients 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 initated a clinical trial in 2006 to treat patient with XCGD and an underlying infection and are continuing to enroll patients to this trial, protocol number 07-I-0017. Based on preclinical data in the rhesus as well as clinical data in a patient, we are using busulfan at a dose of 10mg/kg prior to infusion of the genetically modified cells. To date we have 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 blood. Now at almost three years post treatment, he continues to have detectable marking levels in the peripheral blood of 1%. There is no evidence of clonal outgrowth or myelodysplasia as has been seen in the German XCGD trial. Additionally, the level of oxidase expression on a per cell basis continues to be at almost normal levels and the patient appears to have benefited from the treatment with fewer infections per year now than historically. He continues to remain only partially compliant with his antibiotic prophylaxis. The second patient was treated due to an underlying fungal infection of the chest wall. Despite almost three years of ongoing polymicrobial therapy, this lesion persisted, and therefore the patient was eligible for the gene therapy protocol. His course, however, was not as successful as the first patient as he 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 patient subsequently expired due to continued progression of his infection. 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 now out to almost one year post treatment. He has had partial resolution of his infection but continues to have manifestations of his underlying CGD. He was also treated with rapamycin, which we added to the protocol, to prevent possible immune rejection as is hypothesized to have occurred in the second patient and this was well tolerated. We have collected cells on additional patients in anticipation of performing gene therapy if these patients' ongoing infections do not improve with standard of care. We are currently working with collaborators to develop either a lentivirus based vector and or an RD114 pseudotyped vector to increase transduction of long term hematopoietic stem cells, which should improve the overall results.