CAREER: Developing albumin-binding responsive polypeptides for nucleic acid delivery

基于核酸的药物已被确定为各种疾病的有效治疗方法，包括癌症、病毒感染和遗传疾病。在2019冠状病毒病大流行期间，信使RNA （mRNA）疫苗的使用突出了这一点。由于核酸固有的物理化学性质，包括负电荷、高亲水性和对酶降解的敏感性，通常需要带正电荷的递送载体才能成功发挥其功能。然而，目前大多数的载体存在毒性不理想和递送效率低的问题。这限制了核酸疗法在临床中的广泛应用。本CAREER项目提出了开发生物友好、安全、有效的核酸载体的创新方法。这些以多肽为基础的载体毒性较小，可以有效地将核酸运送到患病部位。多肽的结构将被系统地研究以优化其功效。蛋白质结合分子也将用于进一步降低载体的毒性，同时提高递送效率。该项目为核酸传递和许多其他生物医学应用在疾病治疗中的生物材料的发展提供了重要的见解。结合研究成果，首席研究员提出了一系列教育项目，以帮助K-12学生理解化学和科学。该项目旨在培养生物材料领域的研究生和本科生，尤其是女性和少数族裔，并最终为下一代STEM劳动力的发展做出贡献。本职业项目旨在开发安全有效的核酸治疗用高分子载体，包括小干扰RNA （siRNA）和信使RNA （mRNA）。siRNA和mRNA是一类具有深远治疗潜力的“信息药物”，已被研究用于治疗各种疾病，包括癌症、病毒感染、遗传和代谢紊乱。然而，由于核酸不稳定，除了具有免疫原性外，还不能有效地穿过细胞膜，因此核酸疗法的广泛临床应用受到了挑战。为了应对核酸疗法在临床应用中的挑战，该项目致力于开发具有最小毒性的白蛋白结合反应性多肽。因此，这些多肽可以携带、保护、传递和释放核酸到靶组织和细胞中，重点是传递过程。这将通过两种创新方法来实现，以减少毒性和提高核酸治疗的有效性：1)开发氧化还原反应多肽，其中悬垂的阳离子胺通过二硫键连接到多肽主干；2)将白蛋白结合分子Evans Blue与多肽结合（EBylation），搭便车携带内源性白蛋白，屏蔽和伪装正电荷，从而降低细胞毒性和免疫毒性。本项目旨在系统地研究反应胺部分与核酸传递效率之间的结构-性质关系。此外，我们还将研究乙酰化对核酸结合、细胞摄取和转染的影响。可逆蛋白结合的概念也将被引入自组装材料的伪装，以增强生物相容性和减少不良的相互作用。这项提案将有助于培养一群多样化的本科生和研究生，以应对社会挑战和复杂的科学问题。它将与K-12学生的外展活动和努力相辅相成，最终为下一代STEM劳动力的发展做出贡献。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

PART 1: NON-TECHNICAL SUMMARYNucleic acid-based drugs have been identified as an effective treatment for various diseases including cancer, viral infections, and genetic disorders. This was highlighted by the use of messenger RNA (mRNA) vaccines during the Covid-19 pandemic. Due to the intrinsic physicochemical properties of nucleic acids, including negative charges, high hydrophilicity, and susceptibility to enzymatic degradation positively charged delivery carriers are typically required to successfully exert their functions. However, most of the current carriers suffer from unsatisfactory toxicities and low delivery efficiencies. This limits the widespread use of nucleic acid therapeutics in a clinical setting. This CAREER project proposes innovative approaches to developing biologically friendly, safe, and effective nucleic acid delivery carriers. These polypeptide-based carriers are less toxic and can effectively deliver nucleic acids to diseased sites. The structure of the polypeptide will be systematically investigated for optimized efficacy. Protein-binding molecules will also be used to further reduce the carrier’s toxicity along with an increase in delivery efficiency. This project provides critical insights into the development of biomaterials for nucleic acid delivery and many other biomedical applications in disease treatment. Integrated with the research effort, the principal investigator proposes a series of educational projects to help K-12 students understand chemistry and science. This program aims to train graduate and undergraduate students, particularly women and underrepresented minorities, in the field of biomaterials, and ultimately contribute to the development of the next-generation STEM workforce.PART 2: TECHNICAL SUMMARYThis CAREER project aims to develop safe and effective polymeric delivery carriers for nucleic acid therapeutics, including small interfering RNA (siRNA) and messenger RNA (mRNA). siRNA and mRNA are a class of “information drugs” with far-reaching therapeutic potential and have been investigated for the treatment of various diseases, including cancers, viral infections, and genetic and metabolic disorders. However, broad clinical adoption of nucleic acid therapeutics has been challenged by the fact that nucleic acids are unstable and cannot efficiently cross cell membranes besides being immunogenic. To tackle the challenges of nucleic acid therapeutics for clinical adoption, this project is dedicated to the development of albumin-binding responsive polypeptides that have minimal toxicity. Therefore, these polypeptides can carry, protect, deliver, and release nucleic acid into target tissues and cells, with an emphasis on the delivery process. This will be accomplished through two innovative approaches to reduce toxicity and improve the effectiveness of nucleic acid therapeutics: 1) development of redox-responsive polypeptides, wherein pendant cationic amines are connected via disulfide bonds to polypeptide backbones; 2) conjugation of the albumin-binding molecule Evans Blue to polypeptides (EBylation) to hitchhike endogenous albumin for shielding and camouflaging the positive charge for minimal cytotoxicity and immunotoxicity. This project seeks to systematically investigate the structure-property relationship between responsive amine moieties and nucleic acid delivery efficiency. Additionally, the effect of EBylation on nucleic acid binding, cellular uptake, and transfection will be studied. The concept of reversible protein binding will also be introduced to camouflage the self-assembled materials for enhancement of biocompatibility and reduction of undesirable interactions. This proposal will help prepare a diverse group of undergraduate and graduate students to tackle social challenges and complex scientific problems. It will be complemented with outreach activities and efforts for students in K-12, ultimately contributing to the development of the next-generation STEM workforce.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.