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个tRNAs最近被证明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RNAs(TDR)，具有其独特的功能，包括mRNA稳定和沉默， 3应激颗粒形成与表观遗传调控。这些TDR功能取决于它们的 4序列保守和碱基修饰，进而决定其结构和蛋白质结合能力。 5我们之前已经详细介绍了TDRS在细胞应激反应中的动态调节，并在 6过程确定的Asp-GTC-3‘TDR在应激依赖的自噬诱导中起关键作用，是高度 7在肾脏中表达，在体外似乎具有肾脏保护作用。在几个小鼠体内模型中，细胞 8肾损伤急性期Asp-GTC-3‘TdR水平最初升高，随后下降。 慢性肾脏病模型9只。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，提示一种代偿反应。然而，工具可用于 11在体内操纵该TDR和其他TDR的表达，保持其细胞修改呈现一种 12该领域的重大障碍。我们的初步数据表明，以RNA为靶点的CRISPR/Cas13在 13个gRNA，可诱导tRNA上的程序性切割(S)，并在不干扰TDR的情况下产生功能性TDR 14个亲本tRNA池。值得注意的是，我们通过替换两个 PspCas13b的15个HEPN结构域与血管生成素(Ang)结合，Ang是一种负责TDR生物发生的小核糖核酸酶 16允许包装成病毒基因递送载体。这项提议的长期目标是开发一种 17个可编程的Cas13/Ang平台，包装在AAVs中，用于运送到肾脏进行生物发生 18个内源性功能性TDRs。为了实现我们的目标，我们计划：1)测试工程设计的 19个CAS13/Ang平台，定制合适的gRNAs，产生内源性功能TDR。作为证明- 20概念，我们重点研究了三种具有清晰读数的TDRs：应激颗粒诱导的ALA-AGC-5‘TDRS和Cys-GCA-TDRS- 21 5‘TDRS和自噬诱导的Asp-GTC-3’TDRS。这些细胞将在HEK细胞中进行测试，以了解它们诱导 22在不干扰亲本tRNA池的情况下产生稳健的TDR；检查应激颗粒形成的分析 23和自噬通量将用于评估Cas13/Ang产生的TDRs的功能；和2)使用AAV- 24台提供了Cas13/Ang的机器，以在肾脏中产生功能性的Asp-GTC-3‘TDR并确定这是否 在我们的小鼠CKD模型中，25可以减缓CKD的进展。天冬氨酸-GTC-3‘-TdR诱导的功能效应 26将通过Northern印迹、组织学、免疫染色、Western blotting和rna-seq来确定。 27对自噬和慢性肾脏病进展的影响。我们期待着我们的目标是广泛地制作这个平台 科学界可以在许多不同的系统中使用28种方法来研究 29 TDR在体内的表达，也为基于TDR的治疗提供了一条可能的治疗途径。