Brain Tumor Stem Cell-targeted Gene Therapy

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是一种致命的脑肿瘤，通常在最初诊断后两年内导致死亡。GBM占成人原发性脑肿瘤的25%。尽管精确的手术切除、放疗和药物治疗有了进步，但几十年来GBM患者的预后并没有显著改变。因此，迫切需要新的治疗方法。最近的研究表明，与白血病类似，胶质瘤是由分化的肿瘤细胞和称为“脑肿瘤干细胞”（BTSCs）的癌前细胞组成的异质性肿瘤。研究表明，BTSCs仅占肿瘤细胞总数的一小部分，但却是肿瘤团块中唯一具有肿瘤更新和肿瘤复发能力的细胞。这些发现具有重要意义，因为它们表明应该设计新的治疗方法来选择性地靶向BTSC，而不是传统的靶向大块肿瘤的方法。我们发现BTSCs可能对免疫系统是不可见的，因为它们缺乏先天和适应性免疫介导杀伤所需的细胞表面受体的表达。用干扰素γ (INF-?)治疗BTSCs可恢复其免疫原性和被杀死的易感性。我们正在推进睡美人（SB）转座子质粒基因转移系统，用于INF-？基因治疗。与传统的质粒DNA载体相比，SB在胶质瘤细胞中介导持续的基因表达，因此具有强大的抗肿瘤功效。同时，我们将开发和测试一种新的btsc靶向质粒传递载体，通过偶联聚乙烯亚胺（PEI）转染试剂与抗CD133抗体，因为btsc表达CD133受体，但它们的子细胞不表达。PEI/DNA/CD133mAb复合物将被用于将一个有效的自杀基因传递到单独的、侵袭性生长的GBM小鼠模型，或与sb介导的INF-？基因治疗。我们认为btsc靶向细胞死亡与持续的INF-？介导的免疫刺激将通过唤起消除残留BTSCs的免疫反应，为脑肿瘤免疫治疗提供突破。该项目将启动这两种药物用于GBM治疗的商业化。在Aim1中，我们将通过完善PEI/DNA/CD133mAb复合物来开发和优化btsc靶向基因递送。这些实验将在体外使用人类GBM肿瘤样本进行，以生产用于I期临床试验的靶向药物。在Aim 2中，sb介导的INF-？基因疗法和btsc靶向自杀基因疗法将在体内进行评估，为大型动物研究和最终的IND应用做准备。这些研究的完成对于加速SB和btsc靶向基因治疗载体的商业化至关重要，以实现我们为致命脑肿瘤患者提供安全有效的基因治疗的目标。胶质母细胞瘤是一种致命的脑肿瘤，占成人原发性脑肿瘤的25%，目前尚无有效的治疗方法。大多数患者在确诊后两年内死亡，仅在美国每年就有超过1万人死于胶质母细胞瘤。在这个项目中，我们将开发并开始商业化一种新型的基因疗法，旨在破坏罕见的“肿瘤干细胞”，这种细胞最终会杀死胶质母细胞瘤患者。

DESCRIPTION (provided by applicant): Glioblastoma Multiforme (GBM) is a lethal brain tumor that typically causes death within two years after initial diagnosis. GBM accounts for 25% of all primary brain tumors in adults. Despite improvements in precise surgical de-bulking, radiation, and drug therapy, the prognosis for GBM patients has not significantly changed in decades. Novel therapies are therefore desperately needed. Recent studies have revealed that similar to leukemia, a glioma is a heterogeneous tumor comprised of differentiated tumor cells and cancerous progenitor cells known as "brain tumor stem cells" (BTSCs). It has been demonstrated that BTSCs account for a minor fraction of total tumor cells, but are the only cells within the tumor mass that are capable of tumor renewal and tumor recurrence. These findings have significance because they suggest that novel therapy should be designed to selectively target BTSC, rather than conventional approaches that target the bulk tumor mass. We have discovered the BTSCs may be invisible to the immune system because they lack expression of cell surface receptors needed for both innate and adaptive immune-mediated killing. Treatment of BTSCs with interferon gamma (INF-?) restores their immunogenicity and susceptibility to being killed. We are moving forward with our Sleeping Beauty (SB) Transposon plasmid-based gene transfer system for treatment of brain tumors with INF-? gene therapy. In contrast to conventional plasmid DNA vectors, SB mediates sustained gene expression in glioma cells and therefore has potent anti- tumor efficacy. In parallel, we will develop and test a novel BTSC-targeted plasmid delivery vector by conjugating polyethylenimine (PEI) transfection reagent with anti-CD133 antibody because BTSCs express the CD133 receptor but their daughter cells do not. PEI/DNA/CD133mAb complexes will be used to deliver a potent suicide gene into an improved, invasively growing mouse model of GBM alone, or in combination with SB-mediated INF-? gene therapy. We contend that the combination of BTSC-targeted cell death with sustained INF-? mediated immune stimulation will provide a breakthrough in brain tumor immunotherapy by evoking an immune response that eliminates residual BTSCs. This project will initiate the commercialization of these two drugs for GBM therapy. In Aim1, we will develop and optimize BTSC-targeted gene delivery by perfecting PEI/DNA/CD133mAb complexes. These experiments will be done in vitro using human GBM tumor samples to produce a targeted drug for a phase I clinical trial. In Aim 2, the efficacy of SB-mediated INF-? gene therapy and BTSC-targeted suicide gene therapy will be evaluated in vivo in preparation for large animal studies and eventual IND application. Completion of these studies is crucial to accelerate commercialization of SB and BTSC-targeted gene therapy vectors, in order to meet our goal of providing safe and effective gene therapy for treatment of patients with fatal brain tumors. There is currently no effective therapy for patients with glioblastoma, a lethal brain tumor that accounts for 25% of primary brain tumors in adults. Most patients die within two years after diagnosis and over 10, 000 people die each year from glioblastoma in the United States alone. In this project we will develop and begin to commercialize a novel form of gene therapy that is intended to destroy rare "tumor stem cells", which are the cells that ultimately kill glioblastoma patients.