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，通常在初次诊断后两年内导致死亡。GBM占成人所有原发性脑肿瘤的25%。尽管精确的手术减积、放射和药物治疗有所改善，但GBM患者的预后在几十年内没有显著改变。因此，迫切需要新的治疗方法。最近的研究表明，类似于白血病，神经胶质瘤是一种异质性肿瘤，由分化的肿瘤细胞和称为“脑肿瘤干细胞”（BTSC）的癌性祖细胞组成。已经证明，BTSC占总肿瘤细胞的一小部分，但是是肿瘤块内能够肿瘤更新和肿瘤复发的唯一细胞。这些发现具有重要意义，因为它们表明，应设计新的治疗方法来选择性地靶向BTSC，而不是靶向大块肿瘤块的传统方法。我们已经发现BTSC可能对免疫系统不可见，因为它们缺乏先天性和适应性免疫介导的杀伤所需的细胞表面受体的表达。用干扰素γ（INF-？）治疗BTSC恢复它们的免疫原性和被杀死的易感性。我们正在推进我们的睡美人（SB）转座子质粒为基础的基因转移系统治疗脑肿瘤与INF-？基因治疗与传统的质粒DNA载体相比，SB介导神经胶质瘤细胞中持续的基因表达，因此具有有效的抗肿瘤功效。同时，我们将通过将聚乙烯亚胺（PEI）转染试剂与抗CD 133抗体结合来开发和测试新型BTSC靶向质粒递送载体，因为BTSC表达CD 133受体，但它们的子细胞不表达。PEI/DNA/CD 133单克隆抗体复合物将用于提供一个有效的自杀基因到一个改进的，侵入性生长的小鼠模型GBM单独，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INF-？介导的免疫刺激将通过引起消除残留BTSC的免疫应答而在脑肿瘤免疫治疗中提供突破。该项目将启动这两种药物用于GBM治疗的商业化。在Aim 1中，我们将通过完善PEI/DNA/CD 133 mAb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GBM肿瘤样本进行，以产生用于I期临床试验的靶向药物。在目的2，SB介导的INF-？基因治疗和BTSC靶向自杀基因治疗将在体内进行评估，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，以实现我们为致命性脑肿瘤患者提供安全有效的基因治疗的目标。目前还没有针对胶质母细胞瘤患者的有效治疗方法，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%。大多数患者在确诊后两年内死亡，仅在美国每年就有超过10000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的基因治疗形式，旨在摧毁罕见的“肿瘤干细胞”，这是最终杀死胶质母细胞瘤患者的细胞。

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.