Small circular mRNA vaccines

小环状RNA疫苗 摘要： 疫苗每年拯救了无数人的生命。基于病原体、DNA、蛋白质或 多肽与较差的药代动力学、有限的生物稳定性、预先存在的抗病毒载体免疫、 免疫原性弱，或对基因组整合或毒力逆转的安全担忧。新兴的信使核糖核酸 疫苗有可能克服上述药代动力学和安全性问题，使用mrna和 先进的药物输送系统。然而，尽管有效传递，目前的信使核糖核酸疫苗依赖于长信使核糖核酸。 这仍然与1)有限的生物稳定性有关，尽管经过了广泛的修饰，以及由此导致的有限的货架期 即使使用冷链，抗原转译效率也很低，2)酶法生产复杂。至 针对这些局限性，我们建议开发抗原编码的小环状RNA(CircRNA)，它是 高度生物稳定性和现有纳米载体高效传递，作为一种新的平台的信使核糖核酸疫苗。小的 CircRNA是由翻译多肽抗原的最小RNA元件组成的。我们的初步数据显示 小CircRNA疫苗的几个显著特征：1)与长mRNA不同，小CircRNA利用 自动RNA合成器，实现快速、高效、精确的化学合成和多功能 修饰；2)即使没有任何生物稳定的修饰，无末端的CircRNA也能阻止核酸外切酶 在冷冻、冰箱和环境温度下降解并延长其在体内的半衰期和保质期； 结果表明，CircRNA具有有效的抗原翻译和免疫调节作用；3)小分子CircRNA疫苗 利用目前的药物输送系统有效地输送到所需的组织和细胞，以及小的CircRNA 相对于体积较大的信使核糖核酸，大小可以增加其在纳米载体中的载量；4)CircRNA疫苗是自我 由于固有的免疫刺激而得到佐剂；5)在细胞中，CircRNA产生真正的长肽抗原， 诱导强大的免疫力；6)CircRNA疫苗的模块化使其易于广泛应用。AS 结果，低剂量的CircRNA纳米颗粒在成年小鼠和老年小鼠中都能引起强烈的免疫反应。 小鼠，保护小鼠免受靶细胞攻击，并介导强大的肿瘤免疫治疗。在此应用程序中， 目的1利用CircRNA优化生物稳定的小环状RNA疫苗的抗原翻译和呈递 工程学；目标2将研究纳米颗粒将CircRNA输送到免疫组织和细胞，以及稳定性， CircRNA疫苗在交付到目标细胞时的完整性和抗原翻译动力学；Aim 3将破译 纳米颗粒递送的小环状RNA疫苗的免疫调节机制；目标4将使用人类 以人乳头瘤病毒(HPV)相关肿瘤为试验床，评价复方丹参的预防和治疗效果 同基因和转基因小鼠肿瘤模型中的纳米颗粒小环状RNA疫苗。PI(早期阶段 调查员)组建了一个具有互补专业知识的团队来进行这项研究。如果成功，这项研究 为将CircRNA发展成为广泛应用的新型RNA疫苗平台奠定了基础 预防和治疗疾病，如癌症和传染病。 第1页

Small circular mRNA vaccines Abstract: Vaccines save numerous lives each year. Conventional vaccines based on pathogens, DNA, proteins, or peptides are associated with poor pharmacokinetics, limited biostability, preexisting anti-viral-vector immunity, weak immunogenicity, or safety concerns over genomic integration or virulent reversion. The emerging mRNA vaccines hold the potential to overcome the above issues with pharmacokinetics and safety using mRNA and advanced drug delivery systems. However, despite efficient delivery, current mRNA vaccines rely on long mRNA that is still associated with 1) limited biostability, though extensively modified, and the resulting limited shelf-life even using cold chains and poor antigen translation efficiency, and 2) complicated enzymatic production. To address these limitations, we propose developing antigen-encoding small circular mRNA (circRNA), which are highly biostable and efficiently delivered by existing nanocarriers, as a novel platform of mRNA vaccines. Small circRNA is comprised of minimal RNA elements to translate peptide antigens. Our preliminary data showed several notable features of small circRNA vaccines: 1) in contrast to long mRNA, small circRNA leverages automated RNA synthesizers for fast, efficient, and precise chemical synthesis and versatile functional modifications; 2) even without any biostabilizing modifications, terminus-free circRNA prevents exonuclease degradation and prolongs its in vivo half-life and shelf-life under freezing, fridge, and ambient temperatures; as a result, circRNA showed efficient antigen translation and immunomodulation; 3) small circRNA vaccines leverage current drug delivery systems for efficient delivery to desired tissues and cells, and the small circRNA sizes can increase its loading capacity in nanocarriers, relative to bulky mRNA; 4) circRNA vaccine is self- adjuvanted due to intrinsic immunostimulation; 5) in cells, circRNA produces long genuine peptide antigens that elicit potent immunity; and 6) the modularity of circRNA vaccine allows its easy adaption for wide application. As a result, low-dose circRNA nanoparticles elicited potent immune responses in both young adult mice and aged mice, protected mice from target cell challenge, and mediated robust tumor immunotherapy. In this application, Aim 1 will optimize the antigen translation and presentation of biostable small circRNA vaccines by circRNA engineering; Aim 2 will study nanoparticle delivery of circRNA into immune tissues and cells, and the stability, integrity, and antigen translation kinetics of circRNA vaccines upon delivery to target cells; Aim 3 will decipher the immunomodulation mechanism by nanoparticle-delivered small circRNA vaccines; and Aim 4 will use human papillomavirus (HPV)-associated cancer as a test bed to assess the prophylaxis and therapeutic efficacy of nanoparticulate small circRNA vaccine in syngeneic and transgenic mouse tumor models. The PI (Early Stage Investigator) has assembled a team with complementary expertise to conduct this study. If successful, this study will lay the foundation to develop circRNA as a novel platform of mRNA vaccines for wide application in the prophylaxis and treatment of diseases, such as cancer and infectious diseases. Page 1