CRISPR-enhanced adipocyte browning to improve glucose tolerance in obesity and diabetes

CRISPR 增强脂肪细胞褐变以改善肥胖和糖尿病的葡萄糖耐量

基本信息

批准号：
10335608
负责人：
MICHAEL P CZECH
金额：
$ 64.99万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-17 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10335608
关键词：
Adipocytes Adipose tissue Adrenergic Agonists Agonist Back Biological Blood Glucose CRISPR/Cas technology Cell Therapy Cells Clinical Trials Closure by clamp Clustered Regularly Interspaced Short Palindromic Repeats Complex Consultations Coupled Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Department of Defense Development Diabetes Mellitus Diabetic mouse Endotoxins Energy Metabolism Engineered Gene Engineering Fasting Funding Gene Combinations Gene Targeting Genes Genetic Transcription Goals Health Human Immune Immune response Implant In Vitro Incidence Knock-out Legal patent Light Liver Mediating Metabolic Metabolic Diseases Methods Microscopy Mus NRIP1 gene Non-Insulin-Dependent Diabetes Mellitus Obese Mice Obesity Oxygen Consumption Pathway interactions Patients Phenotype Prevention Protein Subunits Proteins Research Research Personnel Reverse Transcriptase Polymerase Chain Reaction Sampling Signal Transduction Suppressor Genes Technology Testing Therapeutic Thermogenesis Triglycerides Up-Regulation Vascularization Viral Vector Western Blotting Wild Type Mouse Work adipocyte differentiation base comorbidity cost design diet-induced obesity experimental study expression vector genetically modified cells glucose tolerance implantation improved in vivo insertion/deletion mutation mRNA Expression mouse model nerve supply new technology novel pandemic disease progenitor screening small molecule stem cells subcutaneous transcriptome sequencing

项目摘要

The long term goal of this project is to advance a CRISPR-based technology that genetically modifies human white adipocytes to a “brown-like”, thermogenic phenotype with greatly enhanced ability to promote metabolic health when implanted into obese, diabetic mice. Gene targeting is accomplished in this project without viral or expression vectors, but rather by delivering purified, endotoxin-free Cas9 protein and sgRNA complexes to adipocyte progenitors with nearly 100 percent efficiency. The central hypothesis we will test is that simultaneous CRISPR-based disruption of two distinct “browning suppression” pathways will synergize and convert abundant human white adipocytes to large numbers of rare, fully “brown- like”, therapeutic adipocytes. An attractive transcriptional suppressor target is NRIP1/RIP140, which when deleted by CRISPR in adipocytes causes 100-fold increases in expression of UCP1 and other “BAT” genes. We recently found Nrip1KO mouse or human adipocytes improve metabolic health when implanted in obese mice. An attractive suppressor target in a second thermogenic pathway (cAMP signaling) is the inhibitory RIIb subunit (gene Prkar2) of protein kinase A, which when deleted also upregulates UCP1. Remarkably, we found agonist stimulation of cAMP in Nrip1KO adipocytes upregulates UCP1 and beneficial factors in synergistic mode, approaching a full BAT phenotype. Specific Aim 1 will test the central hypothesis in vitro, leveraging lessons we learned during development of this CRISPR method under pilot Department of Defense funding. First, not all sgRNAs that cause indels in the target gene Nrip1 actually delete the protein. Thus, screening 10 to 20 Prkar2 sgRNAs will be performed in mouse and human adipocytes to define the most efficient at RIIb protein deletion and upregulation of UCP1, with least off-target effects. Second, conditions in which multiple sgRNAs function at near 100 percent efficiency will be used, allowing analysis of adipocytes with single Nrip1KO or Prkar2KO vs adipocytes with double Nrip1/prkar2KO. Efficacy of “brown-like” phenotype induction will be analyzed by cAMP determinations, Western blot and RT-PCR for thermogenic and beneficial secreted proteins, RNAseq signatures and glycolytic and oxygen consumption rates. We will thus determine whether deletion of RIIb plus NRIP1 proteins in mouse and human adipocytes is synergistic, converting white adipocytes to a near BAT phenotype. In Specific Aim 2, these CRISPR-engineered mouse and human adipocytes will be implanted in HFD fed wild type mice or HFD fed immune-compromised NSG mice, respectively. These studies will determine whether double Nrip1KO/Prkar2KO adipocytes are most effective in forming brown-like, implanted adipose depots, including extent of innervation and vascularization (light sheet microscopy). The implanted mice will be evaluated for glucose tolerance and energy expenditure (hyperinsulinemic clamp and metabolic cages). This project will thus define whether CRISPR-engineered mouse and human adipocytes doubly depleted of RIIb plus NRIP1 proteins have greater therapeutic potential than single KO or unmodified adipocytes.

该项目的长期目标是推进基于 CRISPR 的技术，将人类白色脂肪细胞基因改造为“棕色”的产热表型，当植入肥胖、糖尿病小鼠体内时，其促进代谢健康的能力大大增强。该项目无需病毒或表达载体，而是通过将纯化的、无内毒素的 Cas9 蛋白和 sgRNA 复合物以近 100% 的效率传递给脂肪细胞祖细胞来完成。我们将测试的中心假设。基于 CRISPR 的同时破坏两种不同的“褐变抑制”途径将协同作用并将丰富的人类白色脂肪细胞转化为大量稀有的、完全“棕色样”的治疗性脂肪细胞，一个有吸引力的转录抑制靶点是 NRIP1/RIP140。当通过 CRISPR 在脂肪细胞中删除时，UCP1 和其他“BAT”基因的表达增加 100 倍，我们最近发现 Nrip1KO 小鼠或人类脂肪细胞得到改善。当植入肥胖小鼠体内时，第二个产热途径（cAMP 信号传导）中一个有吸引力的抑制目标是蛋白激酶 A 的抑制性 RIIb 亚基（基因 Prkar2），该亚基在被删除后也会上调 UCP1。值得注意的是，我们发现了 cAMP 的激动剂刺激。 Nrip1KO 脂肪细胞以协同模式上调 UCP1 和有益因子，接近完整的 BAT 表型具体目标 1 将在体外检验中心假设。利用我们在国防部资助下开发这种 CRISPR 方法的经验教训，首先，并非所有导致目标基因 Nrip1 中插入缺失的 sgRNA 都会真正删除该蛋白质，因此，将在小鼠和人类中进行 10 至 20 个 Prkar2 sgRNA 的筛选。脂肪细胞确定 RIIb 蛋白删除和 UCP1 上调最有效，脱靶效应最小。其次，多个 sgRNA 在附近发挥作用的条件。将使用 100% 效率，允许对具有单个 Nrip1KO 或 Prkar2KO 的脂肪细胞与具有双 Nrip1/prkar2KO 的脂肪细胞进行分析。将通过 cAMP 测定、Western blot 和 RT-PCR 来分析生热和有益的“棕色样”表型诱导功效。因此，我们将确定小鼠和人类中是否删除 RIIb 加 NRIP1 蛋白。脂肪细胞具有协同作用，可将白色脂肪细胞转化为接近 BAT 的表型，这些研究将确定将这些 CRISPR 工程改造的小鼠和人类脂肪细胞分别植入 HFD 喂养的野生型小鼠或 HFD 喂养的免疫受损的 NSG 小鼠中。双倍 Nrip1KO/Prkar2KO 脂肪细胞是否最有效地形成棕色样植入脂肪库，包括神经支配和血管化的程度（浅表）将评估植入小鼠的葡萄糖耐量和能量消耗（高胰岛素钳和代谢笼），因此该项目将确定双倍去除 RIIb 和 NRIP1 蛋白的 CRISPR 工程小鼠和人类脂肪细胞是否比单一 KO 具有更大的治疗潜力。或未修饰的脂肪细胞。