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

描述(申请人提供)：低效率地将寡核苷酸从细胞外转移到细胞内的隔间，在那里寡核苷酸具有功能活性 地方是实际实现基于寡核苷酸的治疗的全部潜力的最严重的限制。包括反义寡核苷酸(ASO)、疏水修饰的siRNAs(HsiRNA)、GalNAc结合的siRNAs和LNP形成的siRNAs等几类寡核苷酸疗法(ONT)已经证实了生物学效应，并正在临床应用[1，2]。在所有情况下，达到预期生物功能位置的寡核苷酸比例令人惊讶地低，大多数分子被困在错误的细胞隔间，导致效率低，临床毒性有限。同时，大量迅速扩大的证据表明，细胞间的天然小RNA运输是细胞内交流的一种方式。鉴于寡核苷酸介导的基因沉默的极高效率(每个细胞25-100个携带Ago2的siRNAs[3])，利用细胞内小RNA运输的自然途径为显著提高ONTs的效率打开了可能性。我们假设，利用自然的、进化上保守的机制和途径跨越细胞边界运输小RNA是从根本上提高ONTs效率的方法。这项建议的目的是阐明天然小RNA和ONTs贩运所涉及的途径和机制，并利用这一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小RNA以及ONTs的贩运途径。我们开发了(1)一类新型的疏水性siRNAs(HsiRNAs)，它可以有效地进入细胞和组织；(2)一种通过使用hsiRNA负载的外切体作为模型来可视化天然RNA运输的方法；(3)一种利用一组小分子来解剖生产性和非生产性ONTs运输的方法，该方法通过改变hsiRNAs的细胞分布来显著提高hsiRNAs的效率；(4)一组由四种主要类型的ONTs组成的小组，它们利用不同的摄取机制并沉默一个临床相关的靶标记物(HTT)，(5)一组工程细胞系(GFP-运输标记的融合)来研究内化途径。使用这些新的实验工具以及TESM细胞内成像、高分辨率质谱仪以及功能和生化分析，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。完成该项目将导致(1)我们在理解细胞外摄取小调节RNA的自然机制方面取得重大进展，(2)绘制不同类别ONTs的摄取途径图(3)建立依赖于天然摄取机制的新型RNA化学生成的概念框架。这些发现将导致新一代ONTs的临床疗效显著提高。

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.