Targeted Delivery of c-Myc Inhibitory Polypeptides

描述(申请人提供)：目前实体肿瘤的治疗受到固有的肿瘤对放射或化疗的抵抗力以及全身应用抗肿瘤药物的毒性的限制。我们的长期目标是通过开发一种针对局部肿瘤的靶向治疗方法来克服这些限制，从而提高治疗的特异性和有效性，并降低正常组织的细胞毒性。原癌基因c-Myc是一种转录因子，在细胞的生长和分化过程中起着重要的调节作用，其异常过表达与肿瘤的发生有关。我们已经开发出一种温度响应性多肽，它可以抑制c-Myc转录活性和培养细胞的增殖。我们的假设是，在全身给药后，通过局部加温，基因工程多肽可以靶向肿瘤部位。这将导致药物在肿瘤中积累，从而抑制肿瘤生长。设计的多肽的氨基酸序列是基于类弹性蛋白(ELP)生物聚合物，这些聚合物在生理温度37℃以下可溶于水溶液，但当温度高于41℃时会聚集在一起，细胞穿透肽(CPP)连接到ELP上以促进细胞进入。在CPP-ELP中添加了来自c-Myc螺旋-环-螺旋区域的肽，称为H1，它抑制c-Myc途径。我们的初步体外结果表明，与非热响应控制肽相比，CPP-ELP-H1结构在MCF7细胞中具有非常显著的作用。为了解决这一假说，将针对以下具体目标：(1)建立合适的动物模型，通过将MCF7细胞植入裸鼠大腿肌肉来在体内验证该假说；(2)通过测定CPP-ELP-H1的血药浓度曲线和定量放射自显影技术来测量CPP-ELP-H1在裸鼠模型正常组织和肿瘤组织中的血浆动力学和体内分布。(3)通过反复给予CPP-ELP-H1并结合局部热疗来评价CPP-ELP-H1对局部热疗和非局部热疗治疗裸鼠大腿肿瘤异种移植瘤的疗效。这些研究将为靶向输送特定癌基因抑制物的新技术提供基础。将所提出的治疗性多肽通过局部热疗靶向实体瘤，可提高治疗的特异性和有效性，降低对正常组织的细胞毒性。因此，多肽介导的治疗递送系统的发展将提供一种替代或增强现有治疗方法用于局部肿瘤治疗的替代手段。这项拟议研究的成功完成将提供体内数据，将这种疗法推向人类疗法的翻译阶段。

DESCRIPTION (provided by applicant): Current treatment of solid tumors is limited by inherent tumor resistance to radiation or chemotherapy and toxicity from systemic administration of antineoplastic agents. Our long term goal is to overcome these limitations by developing a targeted therapeutic approach for localized tumors that increases the specificity and efficacy of the therapy and reduces the cytotoxicity in normal tissues. The c-Myc proto-oncogene is a transcription factor which plays a central role in the regulation of cell growth and differentiation, and its aberrant overexpression is associated with carcinogenesis. We have developed a thermally responsive polypeptide which inhibits c-Myc transcriptional activity and proliferation of cells in culture. Our hypothesis is that after systemic administration, genetically engineered polypeptides can be targeted to the tumor site by applying local hyperthermia. This will results in accumulation of the agent in the tumor with subsequent inhibition of tumor growth. The amino acid sequence of the designed polypeptides is based on elastin-like (ELP) biopolymers which are soluble in aqueous solution below physiological temperature 37 degrees C, but aggregate when the temperature is raised above 41 degrees C. A cell-penetrating peptide (CPP) is conjugated to the ELP to facilitate cell entry. To the CPP-ELP is added a peptide from the helix-loop-helix region of c-Myc, called H1, which inhibits the c-Myc pathway. Our preliminary in vitro results demonstrate a very significant effect of the CPP- ELP-H1 construct in MCF7 cells when compared to a non-thermally responsive control peptide. In order to address the hypothesis, the following specific aims will be addressed: (1) Create a suitable animal model to test the hypothesis in vivo through implantation of the MCF7 cells in the thigh muscle of athymic rats, (2) Measure the plasma kinetics and in vivo distribution of CPP-ELP-H1 in normal and neoplastic tissue in an athymic rat model by determining the plasma concentration curve and determination of the tissue concentrations with quantitative autoradiography.and (3) Evaluate therapeutic efficacy of CPP-ELP-H1 in the treatment of neoplastic xenografts in the thigh of athymic rats with and without localized hyperthermia through repeated administration of the agent coupled with local hyperthermia. These studies will provide the basis for a new technology for targeted delivery of specific oncogene inhibitors. Specific targeting of the proposed therapeutic polypeptides to solid tumors by local hyperthermia would increase specificity and efficacy of treatment and reduce the cytotoxicity in normal tissues. Thus, development of the proposed polypeptide-mediated therapeutic delivery system would provide an alternative means to effectively substitute or augment present therapy for treatment of localized tumors. The successful completion of the proposed research will provide In vivo data to move this therapy towards the translational stage of human therapeutics.