Lentiviral-MGMT gene transfer into hematopoietic stem cells

描述(申请人提供)：治疗性干细胞基因转移依赖于通过将新的DNA整合到细胞基因组中而实现的长期基因表达。目前以癌逆转录病毒为特征的临床试验遇到了一些障碍，包括低水平的基因转移，低表达，以及公认的在启动子区域附近的优先插入，这增加了插入突变和白血病的机会。这项提议将把Lentigen公司在大规模生产最新一代慢病毒载体(LV)方面取得的进展与将MGMT基因转移到造血干细胞的临床需求联系起来。这项应用的目标是通过临床前试验优化MGMT慢病毒基因转移的安全性，然后在癌症患者中使用这种载体进行临床试验。MGMT(O6-甲基鸟嘌呤-DNA-甲基转移酶)酶的表达已成为体内选择造血祖细胞的有效方法。MGMT基因产物(也称为AGT，O6-烷基鸟嘌呤-DNA-烷基转移酶)修复被烷化剂破坏的DNA。造血祖细胞(HP)可以用含有MGMT的载体转导，并通过给予DNA损伤药物治疗进行体内选择，即烷化剂替莫唑胺，其效力通过联合给予天然AGT的治疗性抑制剂O6-苄基鸟嘌呤(BG)而增强。利用MGMT的一个特性良好的单点突变P140K，它可以抵抗BG的抑制作用，有可能选择性地保护Hp免受药物组合的影响，为体内多系祖细胞提供了一种独特的浓缩策略。LVS是逆转录病毒载体的一个亚类，与肿瘤逆转录病毒载体相比具有几个优点，包括提高了转导效率，长期基因表达无沉默，以及降低了插入癌的风险。在这项应用的第一阶段，我们将生成具有改进的安全特性的cGMP载体，分析插入位点的频率和偏好，并创建一个公共可用的数据库，以与其他调查人员和监管机构共享这些信息。这将在与人类细胞共享临床基因载体产生的数据方面建立一个新的标准。在这项应用的第二阶段，将启动一项临床试验，旨在证明慢病毒载体的安全使用，有可能改善晚期胶质瘤患者的预后。这项试验将是人类首次对耐药基因介导的体内干细胞选择进行研究。这项试验不仅对胶质瘤患者具有重要意义，而且也是一种有效的手段，证明了一种选择基因修饰干细胞的平台的有效开发，该平台也可以用于纠正许多单基因疾病。 公共卫生相关性：这项提案是第一阶段和第二阶段相结合的应用程序Fast-Track，将在临床前模型和胶质瘤临床试验中评估表达MGMT基因的慢病毒基因载体。本应用的目的是优化MGMT慢病毒基因转移的安全性。在临床研究中证明这种慢病毒载体的安全使用将打开治疗胶质瘤的大门，也将打开治疗其他恶性肿瘤的大门，在这些恶性肿瘤中，血液毒性是主要的副作用，或者恶性干细胞被供者来源的(同种异体)或患者来源的(自体)干细胞取代，可以在体内选择。因此，这项提议代表着通过提供手段将携带基因的细胞数量增加到更大的潜在治疗水平，从而提高干细胞疗法的有效性的第一步。

DESCRIPTION (provided by applicant): Therapeutic stem cell gene transfer relies on long-term gene expression achieved by integration of new DNA into the cellular genome. Current clinical trials featuring oncoretroviruses have encountered a number of roadblocks that include low levels of gene transfer, poor expression, and a recognized preferential insertion near promoter regions that increases the chance for insertional mutagenesis and leukemia. This proposal will link the advances made at Lentigen Corp. in large scale production of the newest generation lentiviral vectors (LV) with the clinical need for transferring the MGMT gene into hematopoietic stem cells. The goal of this application is to optimize the safety of lentiviral gene transfer of MGMT using preclinical assays followed by a clinical trial using this vector in cancer patients. Expression of the MGMT (O6-methylguanine-DNA- methyltransferase) enzyme has been established as an effective method for hematopoietic progenitor (HP) selection in vivo. The MGMT gene product (also known as AGT, O6-alkylguanine-DNA-alkyltransferase) repairs DNA damaged by alkylating agents. Hematopoietic progenitor cells (HP) can be transduced with a vector containing MGMT and selected for in vivo by administration of a DNA damaging drug therapy, i.e. the alkylating agent Temozolomide, whose potency is increase by co-administration of O6-benzylguanine (BG), a therapeutic inhibitor of the naturally occurring AGT. Using a well-characterized singe point mutation of MGMT, P140K, that is resistant to the inhibitory effects of BG, it is possible to selectively protect HP from the drug combination, providing a unique enrichment strategy for multilineage progenitors in vivo. LVs, a subclass of retroviral vectors, offer several advantages over oncoretroviral vectors including increased transduction efficiencies, long-term gene expression without silencing, and decreased risk of insertional oncogenesis. In Phase I of this application we will generate cGMP vector with improved safety characteristics, analyze the insertion site frequency and preference, and create a publically available database to share this information with other investigators and regulatory agencies. This will set a new standard in the field for sharing data generated from the use of a clinical gene vector with human cells. In the Phase II portion of this application, a clinical trial designed to demonstrate safe use of lentiviral vector with the potential to improve outcomes for patients with advanced glioma, will be initiated. This trial will be the first in man study of in vivo stem cell selection mediated by a drug resistance gene. This trial is of importance not only for patients with glioma, but as means to demonstrate the effective development of a platform for selecting gene-modified stem cells that could also be used for the correction of numerous monogenic disorders. PUBLIC HEALTH RELEVANCE: This proposal is a combined phase I and phase II application, Fast-Track, that will evaluate lentiviral gene vectors expressing the MGMT gene in pre-clinical models and in a clinical trial for glioma. The goal of this application is to optimize the safety of lentiviral gene transfer of MGMT. Demonstration of the safe use of this lentiviral vector in clinical studies will open the door to the treatment of not only glioma, but other malignancies in which hematotoxicity is a major side-effect, or in which malignant stem cells are replaced by donor-derived (allogeneic) or patient-derived (autologous) stem cells that can be selected for in vivo. Thus, this proposal represents a first step in improving the efficacy of stem cell therapeutics by providing the means to increase the number of cells carrying the gene to greater and potentially therapeutic levels.