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，该技术将人的白色脂肪细胞基因改造成一种“棕色”的生热表型，将其植入肥胖、糖尿病小鼠体内，极大地增强了促进代谢健康的能力。在这个项目中，基因打靶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、无内毒素的Cas9蛋白和sgRNA复合体以近100%的效率传递给脂肪细胞前体细胞。我们将测试的中心假设是，基于CRISPR的两条不同的“褐化抑制”途径的同时中断将协同作用，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”的治疗性脂肪细胞。一个有吸引力的转录抑制靶点是NRIP1/RIP140，当它被CRISPR在脂肪细胞中删除时，会导致UCP1和其他“BAT”基因的表达增加100倍。我们最近发现，Nrig1KO小鼠或人类脂肪细胞移植到肥胖小鼠体内后，可以改善代谢健康。在第二个产热途径(cAMP信号)中，一个有吸引力的抑制靶点是蛋白激酶A的抑制性RIIb亚单位(基因Prkar2)，当缺失时，它也上调UCP1。值得注意的是，我们发现Nlip1KO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，接近完整的BAT表型。特定目标1将在体外测试中心假设，利用我们在国防部资助的试点项目下开发CRISPR方法期间学到的经验教训。首先，并不是所有导致目标基因Nrig1中的indels的sgRNAs实际上都会删除这种蛋白质。因此，将在小鼠和人类脂肪细胞中筛选10到20个Prkar2 sgRNAs，以确定在RIIb蛋白缺失和UCP1上调方面最有效的，而非靶标影响最小。第二，将使用多个sgRNAs以接近100%的效率发挥作用的条件，允许分析具有单一Nlip1KO或Prkar2KO的脂肪细胞与具有双Nrig1/Prkar2KO的脂肪细胞。“棕色样”表型诱导的有效性将通过cAMP测定、Western印迹和RT-PCR对产热和有益的分泌蛋白、RNAseq特征以及糖酵解和耗氧率进行分析。因此，我们将确定小鼠和人类脂肪细胞中RIIb和NRIP1蛋白的缺失是否具有协同作用，将白色脂肪细胞转变为接近蝙蝠的表型。在特定目标2中，这些CRISPR工程小鼠和人类脂肪细胞将分别植入喂养HFD的野生型小鼠或HFD喂养的免疫受损的NSG小鼠。这些研究将确定双Nrig1KO/Prkar2KO脂肪细胞在形成棕色样植入性脂肪库方面是否最有效，包括神经支配和血管形成的程度(光镜)。植入的小鼠将接受葡萄糖耐量和能量消耗(高胰岛素钳夹和代谢笼)的评估。因此，该项目将确定双缺乏RIIb和NRIP1蛋白的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.