EXPERIMENTAL THERAPEUTICS EXPLOITING FOLATE RECEPTORS

利用叶酸受体的实验治疗

• 批准号: 6129304
• 负责人: Asok Antony
• 金额: $ 20.13万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-16 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6129304
• 关键词: HeLa cells; Retroviridae; antisense nucleic acid; apoptosis; athymic mouse; cervix neoplasms; cis platinum compound; complementary DNA; drug interactions; folate; ganciclovir; gene therapy; genetic promoter element; human papillomavirus; immunoglobulin G; liposomes; neoplasm /cancer therapy; nonhuman therapy evaluation; oligonucleotides; receptor expression; transcription factor; vitamin receptor; zidovudine

Our long-term goal is to target folate receptor (FR)-expressing cervical carcinoma cells for gene therapy to further up-regulate FR expression. This will assist tumor growth Control in 3 ways: first, because of an inverse relationship between FR overexpression and cell proliferation in vivo, tumor doubling will be dramatically delayed from 4.5 to 9 days; second, transduced cells will be more receptive to the uptake of cisplatin that is encapsulated in folate-tethered liposomes (F ) which enter cells via FR, thereby predicting a lower IC50 with less systemic toxicity; and third, the increase in thymidine kinase (TK) activity which accompanies FR overexpression will render these cells hypersensitive to otherwise nontoxic concentrations of azidothymidine (or ganciclovir). While pursuing this goal, we propose to continue our basic studies to elucidate the (potential clinical) significance of the interaction of the 18-base cis-element in the 5'-UTR of FR mRNA and 46-kDa-trans-factor in the translation of FR in cells. So we will test the hypothesis that this cis-element and trans-factor interaction is a critical determinant for the synthesis of FR in cultured HeLa-IU1 cells by introducing antisera to the purified trans-factor and antisense oligonucleotides to the cis-element into cells via F and determining if quenching FR (under conditions when FR expression is essential for the viability of the cell) will trigger a folate-deficient cell death. We will also test the hypothesis that transduction of the sense 46-kDa-trans-factor cDNA (or higher-affinity mutant) can up-regulate FR in HeLa-IU1 cells, decrease cell proliferation, and increase susceptibility to TK-induced cytotoxicity by ganciclovir. This could identify new leads to modulate FR expression by means other than through direct transduction of FR genes. During these studies, we will also initiate a program to systematically test a series of hypotheses to confirm the superiority of F to deliver either cisplatin or retroviral constructs to cervical carcinoma cells, and to verify that ganciclovir augments the cytotoxicity of transduced Hela-IU1 cells that overexpress FR in vitro and in vivo in tumor-bearing mice. This will place us in a position to eventually test the hypothesis that amplification of FR on HeLa-IU1 cell-derived subcutaneous flank tumors by gene therapy with F containing retroviral constructs bearing HeLa-specific promoter-driven sense FR cDNA will (a) reduce tumor doubling; (b) render cells more receptive to subsequent uptake of F +cisplatin resulting in greater cytotoxicity. And because of the parallel increase in TK activity, (c) the addition of ganciclovir will lead to synergistic cytotoxic effects to tumor cells with F +cisplatin thereby reducing its IC50 and toxicity to normal tissues. This approach is innovative because of its inherent 'translational' nature wherein we are attempting to bridge exciting results that have arisen from bench research to the preclinical setting as a prelude to using this approach for women with advanced cervical cancer.

我们的长期目标是靶向叶酸受体（FR）表达宫颈癌细胞进行基因治疗，以进一步上调FR表达。 这将以三种方式有助于肿瘤生长控制：首先，由于FR过表达与体内细胞增殖之间存在反比关系，因此肿瘤的加倍将从4.5天大幅度延迟到9天；其次，转导的细胞将更容易接受顺铂的吸收，将其封装在叶酸螺旋体脂质体（F）中，这些脂质体（F）通过FR进入细胞，从而预测较低的IC50具有较低的系统性毒性。第三，伴随FR过表达的胸苷激酶（TK）活性的增加将使这些细胞对其他偶氮苷（或Ganciclovir）的无毒浓度过敏。 在追求这一目标的同时，我们建议继续我们的基础研究，以阐明在FR mRNA的5'-UTR和46 kDa-trans因子中的（潜在的临床）意义（潜在的临床）意义在细胞翻译中。 因此，我们将检验以下假设：这种顺式元素和跨因素的相互作用是通过将抗血清引入纯化的反式因素和反义寡核苷酸的抗元素和反义寡核苷酸来通过f和确定fr表达的细胞，这是在纯化的元素中通过静脉元素引入元素，即是否在FR表达中，是否是通过FR表达在FR的情况下（是否是通过dive），这是否是通过FR表达的范围，这是在较大的情况下（是否是通过diver），这是在较重要的情况下（是否是通过dive），这是在培养的Hela-IU1细胞中合成FR的至关重要的决定因素（是否是通过FRENINGINITING）。 死亡。 我们还将测试以下假设：46-kDa-trans因子cDNA（或高亲和力突变体）的转导可以上调HELA-IU1细胞中的FR，减少细胞增殖，并增加Ganciclovir对TK诱导的细胞毒性的敏感性。 这可以通过直接转导FR基因来确定新的引线来调节FR表达。 During these studies, we will also initiate a program to systematically test a series of hypotheses to confirm the superiority of F to deliver either cisplatin or retroviral constructs to cervical carcinoma cells, and to verify that ganciclovir augments the cytotoxicity of transduced Hela-IU1 cells that overexpress FR in vitro and in vivo in tumor-bearing mice. 这将使我们能够最终检验以下假设：通过基因治疗含有含有HELA特异性启动子驱动的感觉FR cDNA的F cDNA的F cDNA在HELA-IU1细胞衍生的皮下侧面肿瘤上放大FR在衍生的皮下侧面肿瘤（a）减少肿瘤倍增； （b）使细胞更容易接受，随后摄取F +顺铂导致细胞毒性更大。 （C）（c）添加Ganciclovir将导致对具有F +顺铂的肿瘤细胞的协同细胞毒性作用，从而降低其IC50和对正常组织的毒性。 这种方法具有创新性，因为它具有固有的“翻译”性质，在这种性质中，我们试图弥合从台式研究到临床前环境产生的令人兴奋的结果，以此作为对晚期宫颈癌女性使用这种方法的前奏。