miRNA-Nanotechnology as a novel regenerative therapy for lymphangioleiomyomatosis

摘要 淋巴管平滑肌瘤病（LAM）是一种与硬化症相关的疾病。两者都是由于 TSC 1或TSC 2基因中的遗传性或散发性突变，其功能为阴性 mTOR通路的调节剂。不受控制的mTORC 1活性导致肿瘤 肺中异常平滑肌细胞（LAM细胞）的增殖， 呼吸，复发性气胸，以及肺组织结构和功能的损失，主要是 在女人身上。第一个也是唯一一个FDA批准的LAM治疗方法是免疫抑制剂 西罗莫司，2015年由辉瑞公司上市。它是目前的标准治疗，通过抑制 mTORC 1。西罗莫司有几个临床缺点，包括相当数量的非- 反应者，由于其免疫抑制特性和妊娠导致的严重不良事件 C类，限制其在育龄妇女中使用。因此，存在高度未满足的医疗需求 为LAM和TS开发替代和更安全的治疗方案。我们已经确定了 miRNA 302 b模拟物作为LAM/TS的潜在新疗法。使用小鼠肺损伤 我们的合作者Hao Shen能够证明，使用miRNA 302 b的非靶向治疗 中性脂肪乳剂模拟物改善肺功能、宿主恢复和肺泡上皮细胞 再生小鼠我们还证实了在人的肝细胞中存在一种“停滞”的AT 2-AT 1转分化状态。 LAM，表明受损的AT 2适应性可能导致肺泡结构的丧失。我们的目标 该基金的目的是研究靶向肺上皮的miRNA 302 b模拟脂质 通过增强AT 2细胞再生，可以提高纳米颗粒（LNP）治疗LAM的功效。要求1 由miRNA-302 b模拟脂质纳米颗粒的体外表征组成， 研究了几种体内靶向策略。我们的PI博士杰克布伦纳将设计和 在他的生物工程实验室里制造纳米粒子。我们将进行3D类器官实验 在存在LNP[miR 302 b]的情况下，用人和小鼠AT 2细胞，随后进行体内研究 比较不同的靶向策略，以使用转基因生物获得所需的组织和细胞类型， LAM模型，由我们的另一位PI教授Vera Krymskaya开发。使用最有效的瞄准 策略，目标2将是使用相同转基因鼠模型的体内概念验证研究 我们的治疗是否能阻止怀孕前和怀孕后的空气 扩大并导致肺恢复。术后观察4周和8周 LNP[miR 302 b]施用。终点将包括肺功能、BALF分析、qPCR和 组织学肺切片针对疾病相关标志物和细胞类型进行染色。所有合作者 他们是各自领域的专家，因此有能力成功完成这个项目， 汇集了许多独特和创新的方面来治疗这种毁灭性的罕见疾病。

ABSTRACT Lymphangioleiomyomatosis (LAM) is a Tuberous sclerosis-related disorder. Both occur due to an inherited or sporadic mutation in either the TSC1 or TSC2 gene, which function as negative regulators of the mTOR pathway. Uncontrolled mTORC1 activity leads to the neoplastic proliferation of abnormal smooth muscle cells (LAM cells) in the lungs, progressive shortness of breath, recurrent pneumothoraxes, and loss of pulmonary tissue structure and function primarily in women. The first and only FDA-approved treatment for LAM is the immunosuppressant sirolimus, marketed since 2015 by Pfizer. It is the current standard-of-care and acts by inhibiting mTORC1. Sirolimus has several clinical disadvantages, including a considerable number of non- responders, severe adverse events due to its immunosuppressive properties and pregnancy category C, limiting its use in women of childbearing age. Thus, there is a high unmet medical need to develop alternative and safer treatment options for LAM and TS. We have identified treatment with miRNA302b mimics as a potential novel therapy for LAM/TS. Using a murine lung injury model, our collaborator Hao Shen was able to show that non-targeted treatment with miRNA302b mimics as neutral lipid emulsion improved lung function, host recovery, and alveolar epithelial regeneration mice. We also demonstrated a “stalled” AT2-AT1 transdifferentiation state in human LAM, suggesting that impaired AT2 fitness may contribute to loss of alveolar structure. Our goal for this grant is the investigation of pulmonary-epithelium targeted miRNA302b mimic lipid nanoparticles (LNP) efficacy for the treatment of LAM by enhancing AT2 cell regeneration. Aim 1 is composed of in vitro characterization of the miRNA-302b mimic lipid nanoparticle and investigation of several targeting strategies in vivo. Our PI Dr. Jake Brenner will design and construct the nanoparticles in his bioengineering lab. We will perform 3D organoid experiments with human and mouse AT2 cells in the presence of the LNP[miR302b] followed by an in vivo study comparing different targeting strategies to reach the desired tissue and cell type using a transgenic model of LAM, developed by our other PI Prof. Vera Krymskaya. Using the most effective targeting strategy, Aim 2 will be an in vivo proof-of-concept study using that same transgenic murine model pre and post-pregnancy to answer the question whether our treatment will prevent airspace enlargement and lead to lung recovery. We will observe mice for 4 and 8 weeks after intratracheal LNP[miR302b] administration. Endpoints will include lung function, BALF analysis, qPCR, and histological lung sections stained for the disease related markers and cell types. All collaborators are experts in their respective field and thus well equipped to successfully complete this project, bringing together a number of unique and innovative aspects to treat this devastating rare disease.