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

该项目的长期目标是推进一项基于crispr的技术，将人类白色脂肪细胞基因改造为“棕色样”的产热表型，并将其植入肥胖的糖尿病小鼠体内，大大增强促进代谢健康的能力。在这个项目中，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、不含内毒素的Cas9蛋白和sgRNA复合物以接近100%的效率递送到脂肪细胞祖细胞中。我们将验证的中心假设是，同时基于crispr的两种不同的“褐变抑制”途径的破坏将协同作用并将丰富的人类白色脂肪细胞转化为大量罕见的、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，当在脂肪细胞中被CRISPR删除时，会导致UCP1和其他BAT基因的表达增加100倍。我们最近发现，将Nrip1KO小鼠或人类脂肪细胞植入肥胖小鼠体内，可改善代谢健康。在第二个产热途径（cAMP信号传导）中，一个有吸引力的抑制靶点是蛋白激酶a的抑制性RIIb亚基（Prkar2基因），当它被删除时，也会上调UCP1。值得注意的是，我们发现Nrip1KO脂肪细胞中cAMP的激动剂刺激以协同模式上调UCP1和有益因子，接近完全BAT表型。Specific Aim 1将在体外测试中心假设，利用我们在国防部试点资助下开发这种CRISPR方法期间学到的经验教训。首先，并不是所有导致靶基因Nrip1缺失的sgrna都删除了该蛋白。因此，我们将在小鼠和人脂肪细胞中筛选10 - 20个Prkar2 sgRNAs，以确定RIIb蛋白缺失和UCP1上调的效率最高，脱靶效应最小的Prkar2 sgRNAs。其次，将使用多个sgrna以接近100%的效率发挥作用的条件，允许分析具有单个Nrip1KO或Prkar2KO的脂肪细胞与具有双Nrip1/ Prkar2KO的脂肪细胞。“棕色样”表型诱导的效果将通过cAMP测定、Western blot和RT-PCR分析产热蛋白和有益分泌蛋白、RNAseq特征、糖酵解和耗氧率来分析。因此，我们将确定小鼠和人类脂肪细胞中RIIb + NRIP1蛋白的缺失是否具有协同作用，将白色脂肪细胞转化为接近BAT的表型。在Specific Aim 2中，这些crispr工程小鼠和人类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Nrip1KO/Prkar2KO脂肪细胞是否在形成棕色样植入脂肪库中最有效，包括神经支配和血管化的程度（薄层显微镜）。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crispr工程小鼠和人类脂肪细胞是否比单一KO或未修饰的脂肪细胞具有更大的治疗潜力。

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.