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)疗法的发展仍然受到有效给药挑战的阻碍。GeneSegues，Inc.(GSI)开发了一种非病毒递送技术，亚50纳米(S50)胶囊，它利用RAFT/空泡吸收途径有效地在细胞内递送NA药物。RAFT/Caveole通路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体输送到细胞内，避免了一直阻碍NA药物有效输送的炎症和药物降解机制。利用这种受体介导的、尺寸敏感的途径是通过一种新颖的胶囊设计实现的，该胶囊设计由单层结晶聚合物外壳组成，该外壳屏蔽NA药物，在空间上稳定外壳，并靶向胶囊。在肝脏方面，GSI已经证明了最初的概念验证，即肝脏靶向的、细胞亚型的功能质粒DNA的传递。以去唾液酸糖蛋白受体(ASGPr)为靶点的S50胶囊为靶向去唾液酸糖蛋白受体(ASGPr)配体的S50胶囊，而以透明质酸(HA)为靶向内吞(HARE)的透明质酸(HA)包被的S50胶囊可实现对LSEC的肝细胞特异性递送。我们现在建议启动这些肝靶向、携带质粒的S50胶囊的临床前开发，用于治疗应用。此外，鉴于RNAi具有良好的治疗潜力，我们还建议协同评估s50胶囊在系统体内将治疗性RNAi寡核苷酸递送到确定的肝脏靶点的有效性。因此，我们提出的具体目标是：(1)确定s50肝细胞靶向胶囊用于系统递送双链和单链RNAi寡核苷酸的实用性和安全性，使用已发表的封装在s50 ASOR-配体胶囊中的siRNA序列(siApoB-1)；(2)评估LSEC靶向s50胶囊用于系统递送质粒DNA的安全性，使用封装在s50 HA-配体胶囊中的表位标记报告基因。通过HA包衣和ASOR包衣的S50胶囊，NA的生物分布将由放射性标记进行评估，任何早期炎症反应的证据都将针对质粒和RNAi货物进行评估。如果成功，我们预计这项工作将为与NIH和/或药物开发商合作推进肝脏疾病的胶囊NA疗法提供坚实的框架。与公共卫生相关：核酸药物为治疗肝脏疾病提供了很大的希望。然而，将核酸输送到肝脏的疾病细胞中是非常有问题的，因为(1)肝脏是一个具有许多不同细胞类型的异质器官，(2)核酸一旦进入细胞内就会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.