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Engineering RNA editing tools for the generation of functional tRNA-derived small RNAs in the kidney

用于在肾脏中生成功能性 tRNA 衍生小 RNA 的工程 RNA 编辑工具

基本信息

批准号：
10751516
负责人：
JOSEPH VINCENT BONVENTRE
金额：
$ 33.18万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-20 至 2025-09-19
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10751516
关键词：
Acceleration Acute Acute Renal Failure with Renal Papillary Necrosis Alanine-Specific tRNA Animals Area Attenuated Autophagocytosis Binding Proteins Biogenesis Biological Assay Biological Process Biology Cell Death Cells Cellular Stress Chimeric Proteins Chronic Kidney Failure Clustered Regularly Interspaced Short Palindromic Repeats Communities Conserved Sequence Cysteine-Specific tRNA Data Disease Disease Progression Disease model Engineering Future Gene Silencing Generations Genetic Transcription Genome Goals Guanine Guide RNA Histology Human In Vitro Injections Injury to Kidney Kidney Kidney Diseases Messenger RNA Modeling Modification Mus Names Northern Blotting Oligonucleotides Organ Outcome Parents Pathology Phase Play Process RNA RNA Editing RNA-targeting therapy Regulation Reporting Resource Sharing Ribonucleases Ribosomes Role Serotyping Small RNA Stress Structure System Techniques Testing Therapeutic Transcript Transfer RNA Western Blotting adeno-associated viral vector angiogenin base cell type delivery vehicle design epigenetic regulation gain of function in vivo in vivo Model innovation interest kidney cell knock-down mRNA Stability mouse model preservation prevent promoter response stress granule stressor therapeutic RNA tool transcriptome transcriptome sequencing viral gene delivery

项目摘要

1 tRNAs have been recently demonstrated to be processed by ribonucleases into smaller regulatory fragments 2 named tRNA-derived small RNAs (tDRs) with their own distinct function including mRNA stability and silencing, 3 stress granule formation and epigenetic regulation. The function of these tDRs are dependent on their 4 sequence conservation and base modification that in turn determine their structure and protein-binding ability. 5 We have previously detailed the dynamic regulation of tDRs in response to cellular stress and have in the 6 process identified Asp-GTC-3’tDR that plays a critical role in stress-dependent induction of autophagy, is highly 7 expressed in the kidney and appears to be reno-protective in vitro. In several murine in vivo models, cellular 8 levels of Asp-GTC-3’tDR initially increases in the acute phase of renal injury, and subsequently decreases in 9 chronic kidney disease models. Notably in vivo silencing of Asp-GTC-3’tDR in the acute phase of renal injury 10 accelerates cell death and disease progression suggesting a compensatory response. However, tools to 11 manipulate the expression of this and other tDRs in vivo that preserve their cellular modifications presents a 12 significant hurdle in the field. Our preliminary data suggest that the RNA-targeting CRISPR/Cas13, guided by 13 gRNAs, can induce programmable cleavage(s) on tRNAs and generate functional tDRs without disturbing the 14 parent tRNA pool. Notably, we built a functional and smaller Cas13/ANG chimeric protein by replacing two 15 HEPN domains of pspCas13b with Angiogenin (ANG), a small RNase responsible for tDR biogenesis that 16 permits packaging into viral gene delivery vectors. The long-term objective of this proposal is to is to develop a 17 programmable Cas13/ANG platform packaged in AAVs for delivery to kidneys for the biogenesis of 18 endogenous functional tDRs. To achieve our objectives, we plan to: 1) Test the hypothesis that the engineered 19 CAS13/ANG platform, customized with suitable gRNAs, generates endogenous functional tDRs. As a proof-of- 20 concept, we focus on three tDRs with clear readouts: stress granule-inducing Ala-AGC-5’tDRs and Cys-GCA- 21 5’tDRs, and autophagy-inducing Asp-GTC-3’tDRs. These will be tested in HEK cells for their ability to induce 22 robust tDR generation without disturbing the parent tRNA pools; assays checking for stress granule formation 23 and autophagy flux will be used to evaluate the functionality of Cas13/ANG-generated tDRs; and 2) Use AAV- 24 delivered Cas13/ANG machinery to generate functional Asp-GTC-3’tDR in kidneys and determine if this 25 attenuates CKD progression in our mouse CKD model. The functional effects of induction of Asp-GTC-3’tDR 26 will be assessed by northern blotting, histology, immunostaining, western blotting, and RNA-seq to determine 27 effects on autophagy and the progression of CKD. We expect this platform which we aim to make widely 28 available to the scientific community could be used in many different systems to both study the function of 29 tDRs in vivo, and also provide a possible therapeutic avenue for tDR-based therapeutics.

1 tRNAs最近被证明可以被核糖核酸酶加工成更小的调节片段 2命名为tRNA衍生的小RNA（tDRs），具有其自身独特的功能，包括mRNA稳定性和沉默， 3胁迫颗粒的形成与表观遗传调控。这些tDR的功能取决于它们的 4序列保守性和碱基修饰，这反过来又决定了它们的结构和蛋白质结合能力。 5我们之前已经详细描述了tDR响应细胞应激的动态调节，并在 6过程中鉴定的Asp-GTC-3 'tDR在应激依赖的自噬诱导中起关键作用， 7在肾脏中表达，并且在体外似乎具有肾脏保护作用。在几种鼠体内模型中，细胞 8 Asp-GTC-3 'tDR水平最初在肾损伤的急性期升高，随后在急性期降低。 9种慢性肾脏病模型。值得注意的是，在肾损伤的急性期Asp-GTC-3 'tDR的体内沉默 10加速细胞死亡和疾病进展，表明代偿反应。然而，工具 11操纵这种和其他tDR在体内的表达，以保持它们的细胞修饰， 12个重要的障碍在外地。我们的初步数据表明，RNA靶向CRISPR/Cas 13，由 13种gRNA，可以诱导tRNA上的可编程切割并产生功能性tDR，而不干扰转录因子。 14个亲本tRNA库。值得注意的是，我们通过替换两个Cas 13/ANG嵌合蛋白， pspCas 13 b的15个HEPN结构域与血管生成素（ANG），一种负责tDR生物发生的小RNA酶， 16允许包装到病毒基因递送载体中。这项建议的长期目标是制定一项包装在AAV中的17个可编程Cas 13/ANG平台，用于递送至肾脏，用于 18个内源性功能性tDR。为了实现我们的目标，我们计划：1）测试假设，工程 19用合适的gRNA定制的CAS 13/ANG平台产生内源性功能性tDR。作为证据 20概念，我们集中于具有清晰读数的三种tDR：应激颗粒诱导的Ala-AGC-5 'tDR和Cys-GCA-5' tDR。 21 5 'tDR和自噬诱导Asp-GTC-3' tDR。这些将在HEK细胞中测试其诱导在不干扰亲本tRNA库的情况下产生22个稳定的TDR;检测应力颗粒形成 23和自噬通量将用于评估Cas 13/ANG产生的tDR的功能性;和 24递送Cas 13/ANG机器以在肾脏中产生功能性Asp-GTC-3 'tDR，并确定这是否 25在我们的小鼠CKD模型中减弱CKD进展。Asp-GTC-3 'tDR诱导的功能效应将通过北方印迹、组织学、免疫染色、蛋白质印迹和RNA-seq来评估26例受试者，以确定 27对自噬和CKD进展的影响。我们希望这个平台，我们的目标是使广泛科学界现有的28种方法可以用于许多不同的系统， 29个tDR，也为基于tDR的治疗提供了可能的治疗途径。