Lentiviral-MGMT gene transfer into hematopoietic stem cells

描述（由申请人提供）：治疗性干细胞基因转移依赖于通过将新DNA整合到细胞基因组中实现的长期基因表达。目前以肿瘤逆转录病毒为特征的临床试验遇到了许多障碍，包括低水平的基因转移、表达差以及在启动子区域附近公认的优先插入，这增加了插入突变和白血病的机会。该提案将Lentigen Corp.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中的临床需求联系起来。本申请的目的是使用临床前试验优化MGMT的慢病毒基因转移的安全性，然后在癌症患者中使用该载体进行临床试验。MGMT（O 6-甲基鸟嘌呤-DNA-甲基转移酶）酶的表达已被确立为体内造血祖细胞（HP）选择的有效方法。MGMT基因产物（也称为AGT，O 6-烷基鸟嘌呤-DNA-烷基转移酶）修复被烷化剂损伤的DNA。造血祖细胞（HP）可以用含有MGMT的载体转导，并通过施用DNA损伤药物疗法（即烷基化剂替莫唑胺）进行体内选择，所述烷基化剂替莫唑胺的效力通过共同施用O 6-苄基鸟嘌呤（BG）（天然存在的AGT的治疗性抑制剂）而增加。使用MGMT的充分表征的单点突变P140 K，其对BG的抑制作用具有抗性，可以选择性地保护HP免受药物组合的影响，为体内多谱系祖细胞提供独特的富集策略。LV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，包括增加的转导效率，长期基因表达而不沉默，以及降低插入肿瘤发生的风险。在本申请的I期，我们将生成具有改善的安全性特征的cGMP载体，分析插入位点频率和偏好，并创建药物学可用的数据库，以与其他研究者和监管机构共享此信息。这将在该领域建立一个新的标准，用于共享使用临床基因载体与人类细胞产生的数据。在本申请的II期部分，将启动一项临床试验，旨在证明慢病毒载体的安全使用，并有可能改善晚期胶质瘤患者的结局。这项试验将是第一个在人体内进行的由耐药基因介导的体内干细胞选择研究。这项试验不仅对神经胶质瘤患者很重要，而且作为证明选择基因修饰干细胞的平台的有效开发的手段，该平台也可用于纠正许多单基因疾病。 公共卫生相关性：该提案是一个合并的I期和II期申请，快速跟踪，将评估慢病毒基因载体表达MGMT基因在临床前模型和神经胶质瘤的临床试验。本申请的目的是优化MGMT慢病毒基因转移的安全性。证明这种慢病毒载体在临床研究中的安全使用将打开大门，不仅治疗胶质瘤，但其他恶性肿瘤，其中血液毒性是一个主要的副作用，或其中恶性干细胞被替代供体来源的（同种异体）或患者来源的（自体）干细胞，可以选择在体内。因此，该提议代表了通过提供将携带基因的细胞的数量增加到更大和潜在治疗水平的手段来改善干细胞治疗剂的功效的第一步。

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.