Therapeutic efficacy of antisense oligonucleotides targeting PHLPP in preclinical models of diabetes

1型糖尿病(T1D)和2型糖尿病(T2D)在病理生理上高度复杂，最终都是由胰岛β细胞衰竭引起的，这是糖尿病的标志和根本原因。潜在的分子机制仍然没有完全确定，以便为治愈提供贝塔细胞特异性治疗。因此，调节高功能β细胞的存活和/或增强β细胞的再生能力是治疗糖尿病的一种有吸引力的方法。丝氨酸-苏氨酸磷酸酶(STPs)，如PHLPP1/2(PH域富含亮氨酸重复蛋白磷酸酶-1/2)，调节细胞存活，是药物开发的潜在靶点。了解PHLPPs在β细胞死亡调控中的作用和机制具有重要的生物学意义和临床意义。我们已经证实，在代谢应激的人和啮齿动物糖尿病β细胞中，PHLPPs水平高度升高。在体外、体内和T2D患者胰岛中，PHLPPs的基因抑制显著改善了糖尿病实验模型中β细胞的存活和功能，使PHLPPs成为糖尿病胰岛β细胞功能再生治疗的有希望的靶点。为了以β细胞特异性的治疗方式抑制PHLPP，本项目将利用配体诱导的胰升糖素样肽-1受体(GLP1R)内化的新策略，通过将PHLPP反义寡核苷酸(PHLPP-ASO)连接到GLP1R多肽激动剂上，有效并特异性地将PHLPP反义寡核苷酸(PHLPP-ASO)输送到胰岛β细胞。我们将测试GLP1-PHLPP-ASOS在体内和体外恢复β细胞存活和功能以及使血糖正常化的能力。此外，我们还将通过测序在整个(整个胰岛)和单个细胞(β细胞/内分泌细胞)水平上识别PHLPP缺失的β细胞中直接或间接转录基因调控的调控网络。这将有助于我们进一步研究潜在的功能代谢、增殖和生存途径，重点放在β细胞生存信号上，如mTOR、HIPPO、AKT和OXPHOS。这项建议的目的是将我们最初非常有希望的结果推进到转译研究中；建立以前未知的PHLPP1/2信号通路，作为治疗药物干预的新靶点，以恢复糖尿病患者的胰岛β细胞功能和正常血糖。

Both type 1 diabetes (T1D) and type 2 diabetes (T2D), highly complex in their pathophysiology, finally result from pancreatic beta cell failure; hallmark and fundamental cause of diabetes. Underlying molecular mechanisms are still not fully characterized in order to provide beta cell specific therapies for a cure. The modulation of the survival of highly functional beta cells and/or enhancement of beta cell regenerative capacity therefore represent an attractive therapeutic approach for diabetes. Serine-threonine phosphatases (STPs) such as PHLPP1/2 (PH domain leucine-rich repeat protein phosphatase-1/2) regulate cell survival and serve as potential targets for drug development. Our understanding of the function and mechanism of PHLPPs in beta cell death regulation has important biological and clinical significance. We have identified that PHLPPs levels were highly elevated in metabolically stressed human and rodent diabetic β-cells. Genetic inhibition of PHLPPs markedly improved beta cell survival and function in experimental models of diabetes in vitro, in vivo and in islets from patients with T2D, presenting PHLPPs as promising targets for functional regenerative therapy of pancreatic beta cells in diabetes. With the aim of therapeutically inhibiting PHLPPs in a beta cell specific manner, this project will exploit a novel strategy of ligand-induced internalization of the glucagon-like peptide-1 receptor (GLP1R) to deliver PHLPP antisense oligonucleotides (PHLPP-ASOs) potently and specifically to pancreatic beta cells by conjugating them to a GLP1R peptide agonist. We will test GLP1-PHLPP-ASOs for their ability to restore beta cell survival and function and to normalize glycemia in vitro und in vivo. Also, we will identify the regulatory networks of direct or indirect PHLPP-driven transcriptional gene regulation in PHLPP-depleted β-cells at the bulk (whole islets) and single cell level (beta cells/endocrine cells) by using RNA-sequencing. This will help us to further characterize potential functional metabolic, proliferative and survival pathways with major focus on beta cell survival signaling such as mTOR, Hippo, AKT and OXPHOS. The purpose of this proposal is to move forward our initial highly promising results into translational research; to establish the previously uncharacterized PHLPP1/2 signaling pathway as a novel target for curative pharmacological intervention to restore functional pancreatic beta cells and normoglycemia in diabetes.