Engineering human-derived programmable RNA effectors to retune gene expression

遗传缺失（单倍不足）或重复事件通常与多种多样的 这些疾病包括：癌症、心血管和代谢疾病以及神经病。因为许多基因 对过度表达和表达不足都很敏感，基因产物在任何一个方向上的不平衡都会导致 疾病，严格的监管控制是治疗的必要性。然而，RNA靶向技术提供了一种 在转录物水平上适当和可逆地调节基因表达的机制，导致可调的， 细胞特异性修复基因剂量的致病变化。我们最近开发的CRISPR-Cas- 受启发的RNA靶向系统（CIRTS），将小的人源蛋白和引导RNA整合到一个容易的 在转录水平上进行可调基因表达操作的编程和包装技术。 值得注意的是，CIRTS比功能相关但细菌衍生的CIRTS具有主要的临床优势。 CRISPR/Cas系统：最大限度地降低使用人类蛋白质部分产生的免疫原性风险。这 该提案寻求使用蛋白质工程和基于细胞的筛选来创建高效，优化的CIRTS- 的技术-无论是基因激活和失活-并评估他们的普遍性， 疾病相关的目标，并在体内测试该技术的潜力。初步筛选将侧重于 基因PMP 22，一个典型的基因，过度和不足的激活导致不同的，但相关的，遗传的， 紊乱这为优化CIRTS降解剂（Aim 1）和活化剂（Aim）提供了一个肥沃的试验平台 2）。在目标1中，人类蛋白质组将被挖掘以识别和表征可以被识别的功能域。 整合到CIRTS中以可编程地降解PMP 22 RNA。靶RNA着陆位点如何与 每个成功设计的功能性结果，以及对另一个目标SNCA的一般性，将是 同时追求。该提案的目标2侧重于开发CIRTS激活剂， 蛋白质组的新功能域和使用新的报告系统，以有效地筛选和表征 最后，评估另外两个靶基因SCN 1a（大脑）和JAG 1（肝脏）的先导设计。目标3将 评估系统的选择性和特异性约束，以及CIRTS技术在 一系列的生物学背景。在这里，领先的CIRTS架构将以最先进的 基于Cas 13的系统，比较体内免疫原性并测试基于CIRTS的调节性免疫应答的功效。 系统在“真实的世界”的背景下，即小鼠模型的PMP 22为基础的基因剂量失调。关键是，数据 这三个目标相互补充、相互影响，形成了设计-建造-试验三方面的优化 周期该项目的完成将产生一个多模态，可编程的CIRTS工具包的操纵 在RNA水平上的基因表达。除了提供该系统的原理验证外， 在一个典型的临床应用的潜力，这项工作也将产生一个独特的套件RNA靶向效应 其在各种生物医学研究和合成生物学领域具有广泛的用途。

Project Summary Genetic deletion (haploinsufficiency) or duplication events are often associated with a variety of diseases, including: cancers, cardiovascular and metabolic disease, and neuropathies. Because many genes are sensitive to both over- and under-expression, with imbalances in gene product in either direction leading to disease, tight regulatory control is a therapeutic necessity. RNA-targeting technologies, however, provide a mechanism to appropriately and reversibly modulate gene expression at the transcript level, resulting in tunable, cell-specific remediation of disease-causing shifts in gene dosage. Our recently developed CRISPR- Cas- inspired RNA targeting system (CIRTS), integrates small, human-derived proteins and guide RNA into an easily programmed and packaged technology for tunable gene expression manipulation at the transcript level. Notably, CIRTS imparts a major clinical advantage over that of functionally-related, but bacteria-derived CRISPR/Cas systems: minimized immunogenic risk stemming from the use of human protein parts. This proposal seeks to use protein engineering and cell-based screens to create highly efficient, optimized CIRTS- based technologies – both for gene activation and deactivation – and assess their generality across a panel of disease-relevant targets, and to test the potential of the technology in vivo. Initial screening will focus on the gene PMP22, an exemplar gene for which both over- and under-activation result in distinct, but related, genetic disorders. This provides a fertile testbed for optimization of both CIRTS degraders (Aim 1) and activators (Aim 2). In Aim 1, the human proteome will be mined to identify and characterize functional domains that can be integrated into CIRTS to programmably degrade PMP22 RNA. How target RNA landing sites correlate with the functional outcomes of each successful design, as well as generality against another target, SNCA, will be simultaneously pursued. Aim 2 of the proposal focuses on developing CIRTS activators, mining the human proteome for novel functional domains and using novel reporter systems to efficiently screen and characterize them, and finally, assessing lead designs in two other target genes, SCN1a (brain) and JAG1 (liver). Aim 3 will assess selectivity and specificity constraints of the system, and the generality of the CIRTS technology across a range of biological contexts. Here, lead CIRTS architectures will be benchmarked against state-of-the-art Cas13-based systems, comparing immunogenicity in vivo and testing the efficacy of CIRTS-based regulatory systems in “real world” contexts, namely mouse models of PMP22-based gene dosing disorders. Critically, data from all three aims feed off of and inform one another, resulting in a tripartite design-build-test optimization cycle. Completion of this project will generate a multi-modal, programmable CIRTS toolkit for the manipulation of gene expression at the RNA level. In addition to providing proof-of-principle validation of this system’s potential in one exemplar clinical application, this work will also produce a unique suite RNA-targeting effectors with broad utility across a wide variety of biomedical research and synthetic biology fields.