Ex vivo therapy of PAH utilizing hiPSC derived ECs in a SuNx PAH Rat model and development of an organ specific BMPR2 KO animal model in rat and pig

肺动脉高压(PAH)是一种进行性血管收缩疾病。由于缺乏治疗选择，5年死亡率高达50%。在第一个资助期的基础上，本项目的一个主要目标是最终建立新的、创新的治疗理念，其基础是在体外肺灌流系统(EVLP)中用(功能化的)hPSC衍生的内皮细胞替代功能失调的肺内皮细胞。这种细胞体外治疗将在第一个资助期建立的继发性/常氧性PAH大鼠模型中进行评估。由于目前的动物模型不能代表BMPR2基因功能突变的影响，BMPR2基因的功能突变可以在70%的家族性PAH患者中检测到，这一具有临床意义的BMPR2基因敲除(KO)PAH大鼠模型将被开发和建立，用于评估细胞治疗的概念。因此，通过直接靶向EVLP中的肺内皮细胞，将研究大鼠和猪的BMPR2KO-PAH模型，以产生临床相关数据。在EVLP中，创新的基因编辑方法将与非整合病毒载体相结合，实现BMPR2基因在肺内皮细胞中的靶向性KO。除了BMPR2-KO模型中PAH表型的详细特征外，与成熟的sugen/Normoxic模型相比，体外细胞治疗的效果将在两个动物模型中进行评估。对两种动物模型的治疗效果进行详细比较，除其他外，可以得出关于多环芳烃发生的潜在机制的结论。此外，新建立的体外基因编辑将被用来在猪肺内皮层诱导BMPR2 KO，以产生PAH大型动物模型，以便进一步翻译开发已建立的细胞治疗概念。

Pulmonary arterial hypertension (PAH) is a progressivevasoconstrictive disease. Due to missing curative therapy options, ithas a 5-year mortality up to 50%. Based on the first funding periodone major goal of the current project is to finally establish the new,innovative therapy concept, which bases on the replacement of thedysfunctional pulmonary endothelial cells by (functionalized) hiPSCderived endothelial cells in an ex vivo lung perfusion system (EVLP).This cellular ex vivo therapy will be evaluated on the one hand in thesugen/normoxic PAH rat model, established in the first funding period.As current animal models do not represent the impact of loss offunction mutations in the BMPR2 gene, which can be detected in 70%of the patients with familial PAH, this clinically relevant BMPR2 knockout (KO) PAH rat model will be developed and established to be usedfor evaluation of the cell therapy concept. Therefore, by directtargeting of the pulmonary endothelial cells in the EVLP the BMPR2KO –PAH model in rats and in pigs will be investigated for thegeneration of clinically relevant data. In the EVLP innovative geneediting methods will be combined with non-integrating viral vectors toachieve a targeted KO of the BMPR2 gene in the pulmonaryendothelium. Besides the detailed characterization of the PAHphenotype in the BMPR2-KO model compared to the well-establishedsugen/normoxic model the effect of the ex vivo cell therapy will beevaluated in both animal models. Detailed comparison of treatmenteffects in both animal models will allow to draw inter alia conclusionswith respect to the underlying mechanisms for PAH development. Inaddition, the newly established ex vivo gene editing will be utilized toinduce a BMPR2 KO in the pulmonary endothelial layer of pig lungs togenerate a PAH large animal model to allow for further translationaldevelopment of the established cell therapy concept.