Systemic delivery of siRNA by Nanosac for checkpoint blockade immunotherapy of head and neck squamous cell cancer

摘要 头颈部癌症目前通过伴随化疗和放疗来治疗，但局部复发和局部复发的风险仍然很大。 转移性疾病仍然是一个问题。特别是，迫切需要改善对晚期 和转移性头颈部鳞状细胞癌（HNSCC），其具有6至12个月的中位存活期， 即使是单独或与放射或靶向药物联合的积极化疗。检查点阻断 使用抗体的免疫疗法（CBI）仅尝试达到20-30%的应答率。抗 HNSCC的免疫治疗部分归因于免疫抑制的综合性质， 一个检查点的阻断可以被另一个负调控途径抵消， 靶-抗体接合的性质，允许抑制信号的快速恢复。鉴于这些挑战， siRNA被认为是一种有前途的替代抗体，以提高CBI的好处，基于模块化 siRNA，使多靶向治疗，以及RNA干扰的潜力，以提供持久的影响。 然而，在没有可靠的肿瘤递送手段的情况下，siRNA将仍然不能用于CBI和其他靶向治疗。 癌症治疗开发安全的核酸载体，实现良好的生物分布和肿瘤细胞 静脉注射后的摄取将克服siRNA治疗剂临床转化的关键障碍 在癌症中。 本研究的目的是使用Nanosac，一种新型的软， 非阳离子聚多巴胺纳米胶囊，其内部含有siRNA，并采用天然白蛋白的冠， 流通我们通过用siRNA包裹介孔二氧化硅纳米颗粒（MSN），然后用聚合物包裹， ydopamine，并去除牺牲MSN核心。Nanocore递送siRNA并沉默靶基因， 体外毒性，与白蛋白结合，易于外渗和渗透到肿瘤中，抑制肿瘤 作为靶向PD-L1的siRNA的系统性载体生长。我们的中心假设是，虽然阳离子电荷 和/或常规合成基因载体的典型核酸的暴露损害循环时间， Nanosac将通过其柔软性、siRNA的封装性和安全性克服这些挑战。 和非阳离子白蛋白化表面。为了验证这一假设，我们将追求三个具体目标：（一） 优化用于多基因靶向的Nanosac的设计和生产;（ii）为了定义毒性、药代动力学（PK）， （iii）为了利用Nanosac的全身递送， 通过图像引导的放射来靶向肿瘤。这一战略的核心创新是，我们开创了 Nanosac是一种无毒、非阳离子、柔软的纳米胶囊作为核酸载体，它克服了 常规基因载体，其依赖于阳离子电荷用于装载和细胞内递送。因此，我们 不仅有可能克服siRNA系统性递送至实体瘤的持续挑战 而是为其他广泛的基因靶向策略打开了大门。

ABSTRACT Head and neck cancer is currently treated by concomitant chemotherapy and radiation, but local recurrence and metastatic disease remain a concern. In particular, there is a pressing need to improve treatment for advanced and metastatic head and neck squamous cell cancer (HNSCC), which has a median survival of 6 to 12 months, even with aggressive chemotherapy, alone or combined with radiation or targeted agents. Checkpoint blockade immunotherapies (CBI) with antibodies have been tried only to achieve a 20-30% response rate. Resistance to immunotherapy in HNSCC is partly attributable to the comprehensive nature of immunosuppression, where the blockade of one checkpoint can be counterbalanced by another negative regulatory pathway, and the transient nature of target-antibody engagement, allowing rapid recovery of suppressive signals. Given these challenges, siRNAs are considered a promising alternative to antibodies to enhance the benefits of CBI, based on modularity of siRNA, enabling multi-targeted therapy, and the potential for RNA interference to provide long-lasting effects. However, without a means for reliable tumor delivery, siRNA will remain sidelined for CBI and other targeted cancer therapies. Developing a safe nucleic acid carrier that achieves favorable biodistribution and tumor cell uptake upon intravenous injection would overcome the critical barrier to clinical translation of siRNA therapeutics in cancer. The Objective of this study is to enable systemic gene delivery to tumors using Nanosac, a novel soft, non-cationic polydopamine nanocapsule, which holds siRNA inside and adopts a corona of native albumin in circulation. We produce Nanosac by coating mesoporous silica nanoparticles (MSN) with siRNA, then with pol- ydopamine, and removing the sacrificial MSN core. Nanocore delivers siRNA and silences target genes with no toxicity in vitro, binds albumin, and readily extravasates and penetrates into tumors, and suppresses tumor growth as a systemic carrier of siRNA targeting PD-L1. Our Central Hypothesis is that while the cationic charge and/or exposure of nucleic acid typical of conventional synthetic gene carriers compromise circulation time, de- livery to tumors, and safety, Nanosac will overcome these challenges by its softness, encapsulation of siRNA and the non-cationic, albuminylated surface. To test this hypothesis, we will pursue three Specific Aims: (i) To optimize design and production of Nanosac for multigene targeting; (ii) To define toxicity, pharmacokinetics (PK), biodistribution (BD), and pharmacodynamics of Nanosac; (iii) To leverage systemic delivery of Nanosac and tumor targeting by image-guided radiation. The Core Innovation of this strategy is that we have pioneered Nanosac, a non-toxic, non-cationic, soft nanocapsule as a nucleic acid carrier, which overcomes limitations of conventional gene carriers, which rely on cationic charges for loading and intracellular delivery. Thereby, we have the potential not only to overcome the persistent challenge in systemic delivery of siRNA to solid tumors but to open the door to a wide range of other gene-targeting strategies.