EXPERIMENTAL THERAPEUTICS EXPLOITING FOLATE RECEPTORS

我们的长期目标是靶向表达叶酸受体（FR）的宫颈癌细胞进行基因治疗，以进一步上调FR表达。 这将以3种方式帮助肿瘤生长控制：首先，由于体内FR过表达和细胞增殖之间的反向关系，肿瘤倍增将显著延迟4.5至9天;第二，转导的细胞将更容易接受包裹在叶酸栓系的脂质体（F）中的顺铂的摄取，所述脂质体通过FR进入细胞，从而预测较低的IC 50和较低的全身毒性;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它无毒浓度的叠氮胸苷（或更昔洛韦）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FR mRNA的5 '-UTR中的18个碱基的顺式元件和46-kDa-反式因子在细胞中FR的翻译中的相互作用的（潜在的临床）意义。 因此，我们将测试的假设，这种顺式元件和反式因子的相互作用是一个关键的决定因素，为FR的合成在培养的HeLa-IU 1细胞通过引入抗血清纯化的反式因子和反义寡核苷酸顺式元件到细胞中通过F和确定是否淬灭FR（在条件下，FR表达是必不可少的细胞的活力）将触发叶酸缺乏的细胞死亡。 我们还将测试的假设，即转导的正义46 kDa-反式因子cDNA（或更高的亲和力突变体）可以上调FR在HeLa-IU 1细胞，减少细胞增殖，并增加TK诱导的细胞毒性更昔洛韦的敏感性。 这可能会发现新的线索，以调节FR表达的手段，而不是通过直接转导FR基因。在这些研究中，我们还将启动一项计划，系统地测试一系列的假设，以确认F的优越性，提供顺铂或逆转录病毒构建体的宫颈癌细胞，并验证更昔洛韦增强细胞毒性的转导的Hela-IU 1细胞过度表达FR在体外和体内荷瘤小鼠。 这将使我们能够最终检验以下假设：通过用含有F的逆转录病毒构建体进行基因治疗，扩增HeLa-IU 1细胞衍生的皮下侧腹肿瘤上的FR，所述逆转录病毒构建体携带HeLa特异性启动子驱动的正义FR cDNA，这将（a）减少肿瘤倍增;（B）使细胞更容易接受随后的F +顺铂摄取，从而导致更大的细胞毒性。 由于TK活性的平行增加，（c）更昔洛韦的加入将导致与F +顺铂对肿瘤细胞的协同细胞毒性作用，从而降低其IC 50和对正常组织的毒性。 这种方法是创新的，因为它固有的“翻译”性质，我们正试图将实验室研究产生的令人兴奋的结果与临床前环境联系起来，作为将这种方法用于晚期宫颈癌女性的前奏。

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.