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

该项目的长期目标是推进基于CRISPR的技术，该技术将人类白色脂肪细胞遗传修饰为“棕色样”产热表型，当植入肥胖糖尿病小鼠时，其促进代谢健康的能力大大增强。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、无内毒素的Cas9蛋白和sgRNA复合物以接近100%的效率递送到脂肪祖细胞。我们将检验的中心假设是，两种不同的“布朗宁抑制”途径的同时基于CRISPR的破坏将协同作用并将丰富的人白色脂肪细胞转化为大量罕见的完全“棕色样”治疗性脂肪细胞。一个有吸引力的转录抑制靶点是NRIP 1/RIP 140，当在脂肪细胞中被CRISPR删除时，它会导致UCP 1和其他“BAT”基因的表达增加100倍。我们最近发现Nrip 1 KO小鼠或人类脂肪细胞在植入肥胖小鼠时可改善代谢健康。在第二产热途径（cAMP信号传导）中的有吸引力的抑制剂靶标是蛋白激酶A的抑制性RIIb亚基（基因Prkar 2），其在缺失时也上调UCP 1。值得注意的是，我们发现Nrip 1 KO脂肪细胞中cAMP的激动剂刺激以协同模式上调UCP 1和有益因子，接近完整的BAT表型。具体目标1将在体外测试中心假设，利用我们在国防部试点资金下开发这种CRISPR方法期间学到的经验教训。首先，并非所有在靶基因Nrip 1中引起indel的sgRNA实际上都会删除蛋白质。因此，将在小鼠和人脂肪细胞中筛选10至20种Prkar 2 sgRNA，以确定在RIIb蛋白缺失和UCP 1上调方面最有效的，具有最少的脱靶效应。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Nrip 1 KO或Prkar 2 KO的脂肪细胞与具有双Nrip 1/prkar 2 KO的脂肪细胞。将通过cAMP测定、蛋白质印迹和RT-PCR分析产热和有益分泌蛋白、RNAseq签名以及糖酵解和耗氧速率来分析“棕色样”表型诱导的功效。因此，我们将确定小鼠和人脂肪细胞中RIIb加NRIP 1蛋白的缺失是否是协同的，将白色脂肪细胞转化为接近BAT的表型。在具体目标2中，这些CRISPR工程化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喂养的免疫受损的NSG小鼠中。这些研究将确定双Nrip 1 KO/Prkar 2KO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（光片显微镜）。将评价植入小鼠的葡萄糖耐量和能量消耗（高胰岛素钳夹和代谢笼）。因此，该项目将确定CRISPR工程化的小鼠和人类脂肪细胞是否双重耗尽RIIb加NRIP 1蛋白，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.