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：
10649531
负责人：
MICHAEL P CZECH
金额：
$ 64.54万
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/10649531
关键词：
Adipocytes Adipose tissue Adrenergic Agonists Agonist Back Biological Products 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 Hyperinsulinism Immune Immune response Implant In Vitro Incidence Knock-out Learning 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 Deficiency Protein Secretion 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 chimeric antigen receptor T cells 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 synergism 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，该技术通常将人类白脂肪细胞修改为“棕色”的热表型，具有强大的增强的能力，可促进代谢健康，植入肥胖，糖尿病小鼠。基因靶向是在没有病毒或表达载体的情况下完成的，而是通过将纯化的无内毒素Cas9蛋白和SGRNA复合物传递到脂肪细胞祖细胞中，效率几乎为100％。我们将检验的中心假设是，基于CRISPR的两种独特的“褐变抑制”途径的简单破坏将协同并转化丰富的人类白色脂肪细胞，从而将大量稀有的，完全“棕色”的治疗性脂肪细胞结合在一起。一个有吸引力的转录抑制靶标是NRIP1/RIP140，当它被脂肪细胞中的CRISPR删除时，UCP1和其他“ BAT”基因的表达会增加100倍。我们最近发现，当植入肥胖小鼠时，NRIP1KO小鼠或人脂肪细胞改善了代谢健康。在第二个热途径（CAMP信号）中，有吸引力的抑制靶是蛋白激酶A的抑制性RIIB亚基（Gene Prkar2），当被删除时，它也会上调UCP1。值得注意的是，我们发现在NRIP1KO脂肪细胞中对cAMP的激动剂刺激会在协同模式下上调UCP1和有益因素，从而接近完整的BAT表型。具体目标1将在体外测试中心假设，并利用我们在国防部资金开发的CRISPR方法中学到的课程。首先，并非所有引起靶基因NRIP1中插入的SGRNA实际上都删除了蛋白质。那将在小鼠和人脂肪细胞中进行10至20个PRKAR2 SGRNA，以定义RIIB蛋白缺失和UCP1上调最有效的效率，而脱靶效应最少。其次，将使用将多个SGRNA功能在接近100％效率上函数的条件，从而可以分析具有单个NRIP1KO或PRKAR2KO与具有双NRIP1/PRKAR2KO的脂肪细胞的脂肪细胞。通过CAMP确定，Western印迹和RT-PCR，将分析“类似棕色”表型的诱导功效，用于热生和有益的分泌蛋白，RNASEQ特征以及糖酵解和氧气消耗率。因此，我们将确定在小鼠和人脂肪细胞中RIIB加上NRIP1蛋白的缺失是否是协同的，将白色脂肪细胞转化为近BAT表型。在特定的目标2中，这些CRISPR工程的小鼠和人脂肪细胞将分别植入HFD喂养的野生型小鼠或HFD喂养的免疫受损NSG小鼠。这些研究将确定双重NRIP1KO/PRKAR2KO脂肪细胞是否在形成类似棕色的，植入的脂肪沉积物方面最有效，包括神经支配程度和血管化程度（光片显微镜）。将评估植入的小鼠的葡萄糖耐受性和能量消耗（高胰岛素夹和代谢笼）。因此，该项目将定义与单个KO或未修饰的脂肪细胞相比，RIIB Plus NRIP1蛋白的CRISPR工程小鼠和人脂肪细胞的治疗潜力更大。