Nonviral vehicles targeting liver cell-subtypes for nuclear delivery via rafts

描述（由申请人提供）：尽管最近取得了进展，但核酸（NA）疗法的发展继续受到有效递送的挑战的阻碍。GeneSegues，Inc. (GSI)已经开发了一种非病毒递送技术，亚50纳米（s50）胶囊，其选择筏/小窝摄取路径用于NA药物的有效细胞内递送。筏/小窝路径是一种内源性的细胞内转运机制，可以有效地将内化的复合物递送到细胞中，避免炎症和药物降解机制，这些机制一直阻碍NA药物的有效递送。利用这种受体介导的，尺寸敏感的路径实现了一种新的胶囊设计，包括一个单层结晶聚合物壳屏蔽NA药物，空间稳定的外壳，并靶向胶囊。在肝脏中，GSI已经证明了肝脏靶向的功能性质粒DNA的细胞亚型递送的初步概念验证。使用靶向脱唾液酸糖蛋白受体（ASGPr）的脱唾液酸类粘蛋白（ASOR）配体的s50胶囊证明了小鼠中的肝细胞特异性递送，而使用靶向HA内吞受体（HARE）的透明质酸（HA）包被的s50胶囊实现了向LSEC的递送。我们现在建议开始临床前开发这些肝靶向的、携带质粒的s50胶囊用于治疗应用。此外，鉴于RNAi有希望的治疗潜力，我们还建议协同评估s50胶囊用于将治疗性RNAi寡核苷酸全身性体内递送至限定的肝脏靶位点的效用。因此，我们提出的具体目标是（1）使用已发表的siRNA序列，确定s50肝细胞靶向胶囊用于全身递送双链和单链RNAi寡核苷酸的实用性和安全性（siApoB-1）封装在s50 ASOR配体胶囊中，以及（2）评估靶向LSEC的s50胶囊用于全身递送质粒DNA的安全性，使用包封在s50 HA-配体胶囊中的表位标记的报告基因。将通过放射性标记评估经由HA包被的以及ASOR包被的s50胶囊递送的NA的生物分布，并且将评估质粒和RNAi货物的早期炎症反应的任何证据。如果成功的话，我们预计这项工作将为与NIH和/或药物开发人员合作提供一个坚实的框架，以推进肝病的封装NA疗法。公共卫生相关性：核酸药物为治疗肝脏疾病提供了很大的希望。然而，将核酸递送到肝脏的疾病细胞中是非常成问题的，因为（1）肝脏是具有许多不同细胞类型的异质器官，并且（2）核酸一旦进入细胞内就可以引发药物降解和毒性反应。我们建议进一步开发能够进入和穿透肝脏内特定细胞类型并避免细胞内防御的核酸递送技术，以有效治疗肝脏疾病。

DESCRIPTION (provided by applicant): Despite recent advances, development of nucleic acid (NA) therapies continues to be impeded by the challenges of effective delivery. GeneSegues, Inc. (GSI) has developed a nonviral delivery technology, sub-50 nanometer (s50) capsules, which co-opt the raft/caveolar uptake path for efficient intracellular delivery of NA drugs. The raft/caveolar path is an endogenous, intracellular transport mechanism that can efficiently deliver internalized complexes into the cell, avoiding the inflammation and drug degradation mechanisms that have consistently thwarted effective delivery of NA drugs. Exploiting this receptor-mediated, size-sensitive path is achieved with a novel capsule design comprised of a single-layer crystallized polymer shell which shields the NA drug, sterically stabilizes the shell, and targets the capsule. In liver, GSI has demonstrated initial proof-of-concept for liver-targeted, cell-subtype delivery of functional plasmid DNA. Hepatocyte-specific delivery in mice was demonstrated with s50 capsules employing asialoorosomucoid (ASOR) ligands targeting the asialoglycoprotein receptor (ASGPr), while delivery to LSEC's was achieved using hyaluronan (HA)-coated s50 capsules targeting the HA receptor for endocytosis (HARE). We now propose to initiate preclinical development of these liver-targeted, plasmid-bearing s50 capsules for therapeutic application. Additionally, in view of the promising therapeutic potential of RNAi, we propose to also synergistically assess the utility of s50 capsules for systemic in vivo delivery of therapeutic RNAi oligonucleotides to defined liver target sites. Therefore, our proposed specific aims are to (1) determine utility and safety of s50 hepatocyte-targeted capsules for systemic delivery of double and single stranded RNAi oligonucleotides, using a published siRNA sequence (siApoB-1) encapsulated in s50 ASOR-ligand capsules and (2) assess safety of LSEC-targeted s50 capsules for systemic delivery of plasmid DNA, using an epitope-tagged reporter gene encapsulated in s50 HA-ligand capsules. Biodistribution will be assessed by radiolabel for NA's delivered via HA-coated as well as ASOR-coated s50 capsules, and any evidence of early inflammatory responses will be assessed for plasmid and RNAi cargos. If successful, we expect this body of work will provide a solid framework for partnering with NIH and/or drug developers to move forward encapsulated NA therapies for liver disease. PUBLIC HEALTH RELEVANCE: Nucleic acid drugs offer much promise for treating diseases of the liver. However, delivering nucleic acids into disease cells of the liver is very problematic because (1) the liver is a heterogeneous organ with many different cell types, and (2) nucleic acids can trigger drug degradation and toxic reactions once inside the cell. We propose to further develop nucleic acid delivery technology capable of accessing and penetrating specific cell types within the liver, and of avoiding intracellular defenses, for effective treatment of liver disease.