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。 大约六年前，我们完成了一项临床试验的基因治疗的遗传缺陷的吞噬细胞免疫系统被称为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。 我们自己对来自我们自己先前的CGD基因治疗研究的髓样血细胞的插入分析证明了显著的多克隆性，并且没有证据表明含有MDS 1或其他髓样调节基因中的载体插入的克隆生长。最近德国X-CGD基因治疗研究和我们自己以前的研究在这方面的差异的原因可能与强启动子活性有关，已知与他们的小鼠脾病灶形成病毒载体相关，相对于我们的MFGS载体，其来源于小鼠莫洛尼白血病逆转录病毒。 虽然这项试验中的患者没有治愈，第一名患者实际上死于败血症，但这两名患者确实从治疗中获得了一些临床益处。 这两名患者在移植时都有潜在感染，在克隆生长和转导细胞最终沉默之前的初始移植周期间解决。 因此，我们在2006年发起了一项临床试验，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。 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方法。 该患者最初具有24%阳性细胞的水平，并且在治疗后7个月时，其肝脏肿大消退，其中1.2%的可检测标记持续存在于外周血中。 现在，在治疗后近四年，他的外周血中仍然有0.8%的可检测标记水平。 在德国XCGD试验中没有发现克隆生长或骨髓发育不良的证据。此外，基于每个细胞的氧化酶表达水平继续处于几乎正常的水平，并且患者似乎已经从治疗中受益，现在每年的感染比历史上更少。 他仍然只是部分遵守抗生素预防措施。 第2例患者因胸壁潜在真菌感染接受治疗。 尽管进行了近三年的多种微生物治疗，这种病变仍然存在，因此该患者符合基因治疗方案。 然而，他的过程并不像第一个患者那样成功，因为他似乎对转导的细胞产生了免疫反应，在外周血中最初有5%的标记后，这些细胞迅速清除。 患者随后因感染持续进展而死亡。第三名患者接受真菌性肺部感染治疗，初始标记水平为4%，随后下降至0.03%，治疗后近两年保持稳定。 他的感染已部分消退，但仍有潜在CGD的表现。 他还接受了雷帕霉素治疗，我们将其添加到方案中，以防止可能发生的免疫排斥反应，假设发生在第二例患者中，并且耐受性良好。 我们已经收集了更多患者的细胞，如果这些患者的持续感染没有通过标准护理得到改善，我们将进行基因治疗，但我们正在等待治疗任何患者，直到可以产生更好的滴度载体，无论是逆转录病毒还是慢病毒。 我们目前正在与合作者合作开发基于慢病毒的载体和/或RD 114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 four years post treatment, he continues to have detectable marking levels in the peripheral blood of 0.8%. 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 two years 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 but are waiting to treat any patients until a better titre vector can be produced, either retroviral or lentiviral based. 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.