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Nouvelle approche pour modifier le tropisme des vecteurs adenoviraux a l'aide de ligands bispecifiques.

机译：使用双特异性配体修饰腺病毒载体嗜性的新方法。

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摘要

Adenoviruses have been studied as a way to develop new treatments for different diseases. Adenoviral vectors (AdV) are considered interesting tools for this propose, because they can be produced at high titers (1X10 12 particles per millilitre) in laboratory and they have the capacity to infect non-dividing and dividing cells. AdV have been often modified in order to obtain the ability to kill tumour cells or to deliver exogenous genetic sequences essential to treat monogenic disease. However, weak expression of the primary adenovirus receptor, the CAR (Coxsackie and adenovirus receptor) reduces greatly the transduction efficiency of AdV for the tumour cells. Moreover, some normal tissues express low amount of CAR, like the skeletal muscle, reducing the appeal of using AdV as a gene delivery vehicle for this tissue. To address this problematic, many modifications were done on the adenoviral capsid. The goal of these modifications were to generate an AdV able to target specific cellular receptors that were expressed in tumour cells but not in normal cells. Several approaches were done to modify the tropism of AdV, such as incubation with a bispecific ligands, incorporation of peptides within the adenoviral fiber structure or substitution of the viral fiber with a different serotype fiber.;The hypothesis of my project was to determine if an interaction domain fused within a ligand could bind the complementary domain incorporated on a virus and change the tropism of the AdV. The first step was to include a synthetic interaction domain, the K-Coil, within specific region of the adenoviral fiber, as well as inserting two point mutations to abolish the natural tropism. To target the EGF-R, IGF-IR and the CEA6, we fused the complementary interaction domain, the E-Coil, to the respective ligand known as the EGF and the IGF-I or to a single domain antibody (known as AFAI) that bind specifically to CEA6. The specific interaction between the E-Coil and K-Coil was used to associate the ligand with the fiber in order to retarget the AdV toward the selected receptor.;We showed that the different ligands as well as the modified fibers could be produced and that both E-Coil and K-Coil expressing partners could interact together. We optimized the viral production by using an iodixanol purification protocol. More importantly, we clearly demonstrated that the ligand association with the fiber could increase the transduction efficiency between 2 to 21 fold against various tumour cells. The difficulty of adenovirus to infect muscle cells because of the lack of CAR expression brought us to evaluate the potential of our retargeted AdV to increase the transduction for the tissue. We showed that the use of IGF-E5 could increase the transduction efficiency in myoblasts as wells as in myotubes. We finally demonstrated that our retargeting system could increase the transduction efficiency for skeletal muscle by 1,6 fold in new born MDX mice. In conclusion, our results show that the retargeting system is indeed functional. This system could be assessed using vectors that express therapeutic genes.;Key words: Adenovirus, Retargeting, IGF-IR, Tumours, Muscles.
机译：已经研究了腺病毒作为开发针对不同疾病的新疗法的方法。腺病毒载体（AdV）被认为是该提议的有趣工具，因为它们可以在实验室中以高滴度（每毫升1X10 12个颗粒）生产，并且具有感染未分裂和分裂细胞的能力。为了获得杀死肿瘤细胞或递送治疗单基因疾病必不可少的外源遗传序列的能力，经常对AdV进行修饰。然而，主要腺病毒受体，CAR（柯萨奇和腺病毒受体）的弱表达大大降低了AdV对肿瘤细胞的转导效率。此外，某些正常组织表达的CAR数量很少，例如骨骼肌，从而降低了将AdV用作该组织的基因传递载体的吸引力。为了解决这个问题，对腺病毒衣壳进行了许多修饰。这些修饰的目的是产生能够靶向在肿瘤细胞中表达但在正常细胞中不表达的特定细胞受体的AdV。已经采取了几种方法来改变AdV的向性，例如与双特异性配体一起孵育，将肽掺入腺病毒纤维结构中或用另一种血清型纤维替代病毒纤维。;我项目的假设是确定是否融合在配体中的相互作用域可以结合并入病毒的互补域，并改变AdV的向性。第一步是在腺病毒纤维的特定区域内包括一个合成的相互作用域K-Coil，并插入两个点突变以消除自然向性。为了靶向EGF-R，IGF-1R和CEA6，我们将互补相互作用域E-Coil融合到称为EGF和IGF-1的各个配体或单个域抗体（称为AFAI）上与CEA6特异性结合的蛋白。 E-Coil和K-Coil之间的特定相互作用被用来使配体与纤维缔合，从而将AdV重新定向到选定的受体。我们证明了可以产生不同的配体以及改性纤维，并且E-Coil和K-Coil表达合作伙伴都可以一起互动。我们通过使用碘克沙醇纯化方案优化了病毒生产。更重要的是，我们清楚地证明了与纤维的配体缔合可以将针对各种肿瘤细胞的转导效率提高2至21倍。由于缺乏CAR表达，腺病毒难以感染肌细胞，这使我们评估了重新靶向的AdV增加组织转导的潜力。我们表明，使用IGF-E5可以提高成肌细胞以及肌管中的转导效率。最后，我们证明了我们的重定向系统可以使新生MDX小鼠的骨骼肌转导效率提高1.6倍。总之，我们的结果表明，重新定向系统确实可以正常运行。可以使用表达治疗性基因的载体对该系统进行评估。关键词：腺病毒，重定向，IGF-IR，肿瘤，肌肉。

著录项

  • 作者

    Pinard, Maxime.;

  • 作者单位

    Universite de Montreal (Canada).;

  • 授予单位 Universite de Montreal (Canada).;
  • 学科 Biology Virology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 332 p.
  • 总页数 332
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 肿瘤学;
  • 关键词

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