Inter and intra-cellular trafficking pathways for natural and therapeutic RNAs

描述（由申请人提供）：寡核苷酸从细胞外向细胞内区室的高度低效转运，其中寡核苷酸的功能活性发挥作用 位置是实际实现基于阿替尼的疗法的全部潜力的最严重限制。几类寡核苷酸治疗剂（ONT），包括反义寡核苷酸（阿索）、疏水修饰的siRNA（hsiRNA）、GalNAc缀合的siRNA和LNP配制的siRNA，具有经验证的生物学功效并且在临床中[1，2]。在所有情况下，到达生物功能的预期位置的寡核苷酸的分数令人惊讶地低，大多数分子被捕获在错误的细胞区室中，导致低效率和临床限制毒性。同时，有大量证据表明细胞间天然小RNA运输是细胞内通讯的一种手段。考虑到阿托伐他汀介导的基因沉默的极高功效（每个细胞25-100个Ago 2负载的siRNA分子[3]），利用细胞内小RNA运输的天然途径打开了显著提高ONT功效的可能性。我们假设，利用自然的，进化上保守的机制和途径跨细胞边界的小RNA的运输是从根本上提高ONT的效率。该提案的目标是阐明天然小RNA和ONT运输所涉及的途径和机制，并利用这些知识来提高寡核苷酸疗法的疗效。我们开发了一套独特的实验工具和分析方法，使我们能够系统地绘制和研究内源性小RNA以及ONT的运输途径。我们已经开发了（1）一类新的疏水siRNA（siRNAs），其有效地进入细胞和组织;（2）通过使用装载hsiRNA的外来体作为模型来可视化天然RNA运输的方法，（3）使用一组小分子来解剖生产性和非生产性ONT运输的方法，所述小分子通过改变它们的细胞分布来显著增强siRNAs功效;（4）一组四种主要类型的ONT，其利用不同的摄取机制并使临床相关的靶亨廷顿蛋白（htt）沉默，（5）一组工程化细胞系（运输标志物的GFP融合物）以研究内化途径。使用这些新的实验工具，沿着TESM细胞内成像，高分辨率质谱和功能和生化分析，我们将确定天然和合成寡核苷酸的生产性和非生产性细胞摄取机制。该项目的完成将导致（1）我们对小调控RNA细胞外摄取的天然机制的理解取得重大进展，（2）绘制不同类型的ONT的吸收途径（3）开发依赖于天然吸收机制的新型RNA化学物质的概念框架。这些发现将导致新一代的ONT具有显着改善的临床疗效。

DESCRIPTION (provided by applicant): Highly inefficient transit of oligonucleotides from outside cells to the intracellular compartments where functional activity of oligonucleotides takes place is the most serious limitation to practical realization of a full potential of oligonucleotid-based therapies. Several classes of oligonucleotides therapeutics (ONT), including antisense oligonucleotides (ASO), hydrophobically modified siRNAs (hsiRNA), GalNAc conjugated siRNAs, and LNP formulated siRNAs have validated biological efficacy and are in clinic [1, 2]. In all cases, the fraction of oligonucleotides reaching the intended place of biological function is surprisingly low with the majority of molecules being trapped in wrong cellular compartments, resulting in low efficiency and clinically limiting toxicity. Concurrently, there is a significant nd rapidly expanding body of evidence demonstrating natural small RNA trafficking between cells as a means of intracellular communication. Given the extremely high efficacy of oligonucleotide-mediated gene silencing (25-100 molecules of Ago2 loaded siRNAs per cell [3]), exploiting the natural pathways of intracellular small RNA trafficking opens a possibility to dramatically improve the efficacy of ONTs. We hypothesize that exploiting natural, evolutionarily conserved mechanisms and pathways for trafficking of small RNAs across cellular boundaries is the way to fundamentally improve ONTs efficiency. The goal of this proposal is to elucidate the pathways and mechanisms involved in natural small RNA and ONTs trafficking and leverage this knowledge to increase the efficacy of oligonucleotide therapies. We have developed a unique set of experimental tools and analytical methods which enable us to systematically map and study endogenous small RNA as well as ONTs trafficking pathways. We have developed (1) a novel class of hydrophobic siRNAs (hsiRNAs), which efficiently enter cells and tissues; (2) an approach for visualization of native RNA trafficking through use of hsiRNA loaded exosomes as a model, (3) an approach for dissection of productive and non-productive ONTs trafficking using a panel of small molecules dramatically enhancing hsiRNAs efficacy by altering their cellular distribution; (4) a panel of four major classes of ONTs, which utilize different uptake mechanisms and silence a clinically relevant target huntingtin (htt), (5) a panel of engineered cell lines (GFP-fusions of trafficking markers) to study internalization pathways. Using these novel experimental tools along with TESM intracellular imaging, high resolution mass spectrometry and functional and biochemical analyses, we will identify the productive and non-productive cellular uptake mechanisms for both natural and synthetic oligonucleotides Completion of this project will result in (1) significant progress in our understanding of the natural mechanisms of extracellular uptake of small regulatory RNAs, (2) mapping uptake pathways for different classes of ONTs (3) development of the conceptual framework for generation of novel RNA chemistries relying on native uptake mechanisms. These findings will lead to a new generation of ONTs with dramatically improved clinical efficacy.