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

• 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
• 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

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 最近已证明 tRNA 可被核糖核酸酶加工成更小的调节片段 2 个名为 tRNA 衍生的小 RNA (tDR) 具有自己独特的功能，包括 mRNA 稳定性和沉默， 3应激颗粒的形成和表观遗传调控。这些 tDR 的功能取决于它们的 4 序列保守和碱基修饰反过来决定了它们的结构和蛋白质结合能力。 5 我们之前详细介绍了 tDR 响应细胞应激的动态调节，并在 6个过程确定了Asp-GTC-3’tDR在应激依赖性自噬诱导中发挥着关键作用，高度 7 在肾脏中表达，并且在体外似乎具有肾脏保护作用。在几种小鼠体内模型中，细胞 8 Asp-GTC-3’tDR 水平在肾损伤急性期最初升高，随后在肾损伤急性期降低 9种慢性肾病模型。尤其是肾损伤急性期 Asp-GTC-3’tDR 的体内沉默 10 加速细胞死亡和疾病进展，表明存在补偿反应。然而，工具 11 在体内操纵该 tDR 和其他 tDR 的表达，以保留其细胞修饰，呈现出 12 领域内的重大障碍。我们的初步数据表明，RNA 靶向 CRISPR/Cas13 在 13 gRNA，可以诱导 tRNA 上的可编程切割并生成功能性 tDR，而不会干扰 14 个亲本 tRNA 池。值得注意的是，我们通过替换两个来构建功能性且更小的 Cas13/ANG 嵌合蛋白 pspCas13b 的 15 个 HEPN 结构域与血管生成素 (ANG)，这是一种负责 tDR 生物合成的小型 RNase， 16允许包装到病毒基因递送载体中。该提案的长期目标是开发一个 17 个可编程 Cas13/ANG 平台封装在 AAV 中，用于递送至肾脏以进行生物合成 18 个内源性功能 tDR。为了实现我们的目标，我们计划： 1) 检验工程假设 19 CAS13/ANG 平台使用合适的 gRNA 进行定制，可生成内源功能 tDR。作为证明—— 20 概念中，我们重点关注三个具有清晰读数的 tDR：应激颗粒诱导的 Ala-AGC-5’tDR 和 Cys-GCA- 21 5'tDR 和自噬诱导 Asp-GTC-3'tDR。这些将在 HEK 细胞中测试其诱导的能力 22 强大的 tDR 生成，不会干扰亲本 tRNA 池；检查应激颗粒形成的测定 23和自噬通量将用于评估Cas13/ANG生成的tDR的功能； 2) 使用 AAV- 24 交付 Cas13/ANG 机器以在肾脏中生成功能性 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 26 将通过 Northern blotting、组织学、免疫染色、western blotting 和 RNA-seq 进行评估，以确定 27 对自噬和 CKD 进展的影响。我们期待这个平台的广泛推广 28 科学界可用的可用于许多不同的系统，以研究 29 体内 tDR，也为基于 tDR 的疗法提供了可能的治疗途径。