Restoration of endogenous transcription factor activity by nuclease-deficient Cas9VPR prevents cardiac failure upon stress II

Cardiac remodeling is linked to maladaptive transcriptional networks, but identifying and targeting key factors with potential to reverse these processes is still a challenge. We employed network-based analysis of single cell heart transcriptomes to compute the transcription factor activities triggering heart failure. We found that the regulation of KLF15 activity is a major driver of cardiomyocyte failure. Through the use of CRISPR/nuclease-deficient (d)Cas9-based transcriptional regulation, we demonstrated that restoration of endogenous Klf15 expression and activity in cardiomyocytes was sufficient to mitigate pathological transcriptional reprogramming and preserved cardiac function in mice and human myocardial models. We also showed that KLF15 is a downstream targetable factor in the broad TGFß-mediated stress response in human cardiomyocytes, controlling transcriptional embryonalization. Lastly, we engineered a therapeutically applicable mini-dCas9VPR variant suitable for adeno-associated viral delivery, eliciting endogenous KLF15 transcriptional activation in human cardiomyocytes. This case study presents a therapeutic strategy for restoring the stress-induced downregulation of essential genes to prevent heart failure with broader implications for other disease contexts. Overall design: Engineered heart muscle (EHM) were generated by combining CRISPRa hiPSC-cardiomyocytes (3.85x10^5) transduced with either non-targeted control gRNAs (NT) or KLF15 gRNAs and with human foreskin fibroblasts (1.65x10^5) in a bovine collagen matrix. These EHM were cast into a 48-well plate (myriamed GmbH) and cultured initially in IMDM (Gibco) supplemented with 4% B27 minus insulin, 100 ng/mL IGF-1, 5 ng/mL VEGF165, 10 ng/mL FGF-2, 100 U/mL penicillin, 100 µg/mL streptomycin, and 5 ng/mL TGF-ß1 for 3 days. From day 4 onwards, TGF-ß1 was removed. Starting from day 14, the medium was switched to alpha-MEM (Gibco) with 4% B27 minus insulin, 1% MEM non-essential amino acids, 100 ng/mL IGF-1, 5 ng/mL VEGF165, 10 ng/mL FGF-2, 100 U/mL penicillin, and 100 µg/mL streptomycin. On day 28, EHM were either cultured on flexible, silicone poles (control, healthy = Flex) or subjected to mechanical stress by culturing them on rigid metal poles (stressed = Fix) for an additional 7 days. Total RNA was extracted for RNA sequencing to analyze gene expression changes under the different experimental conditions (NT vs. KLF15 gRNA and Flex vs. Fix).

心脏重构与适应不良的转录网络密切相关，但识别并靶向具备逆转此类病理过程潜力的关键因子仍是一项挑战。本研究通过对心脏单细胞转录组开展网络分析，计算触发心力衰竭的转录因子活性。研究发现，Krüppel样因子15（Krüppel-like factor 15, KLF15）的活性调控是心肌细胞衰竭的核心驱动因素。借助基于CRISPR/核酸酶缺陷型（nuclease-deficient, d）Cas9的转录调控技术，我们证实，在心肌细胞中恢复内源性Klf15的表达与活性，足以逆转病理性转录重编程，并在小鼠及人类心肌模型中维持心脏功能。我们还发现，KLF15是人类心肌细胞中转化生长因子β（TGF-β）介导的广泛应激反应通路的下游可靶向因子，可调控转录层面的胚胎样转化。最后，我们构建了一款可用于治疗的微型dCas9VPR变体，适配腺相关病毒（adeno-associated virus, AAV）递送载体，可在人类心肌细胞中诱导内源性KLF15的转录激活。本案例研究提出了一种治疗策略，通过恢复应激诱导下调的核心基因表达以预防心力衰竭，该策略对其他疾病场景亦具有广泛借鉴意义。

整体实验设计：将转染非靶向对照向导RNA（non-targeted control gRNAs, NT）或KLF15向导RNA（KLF15 gRNAs）的CRISPR激活型人类诱导多能干细胞衍生心肌细胞（CRISPRa hiPSC-cardiomyocytes, 3.85×10^5），与人包皮成纤维细胞（1.65×10^5）结合于牛胶原基质中，构建工程化心肌组织（engineered heart muscle, EHM）。将此类EHM接种于48孔板（myriamed GmbH），初始培养采用伊斯科夫改良达勒姆培养基（IMDM, Gibco），添加4%无胰岛素B27添加剂、100 ng/mL胰岛素样生长因子1（IGF-1）、5 ng/mL血管内皮生长因子165（VEGF165）、10 ng/mL成纤维细胞生长因子2（FGF-2）、100 U/mL青霉素、100 μg/mL链霉素及5 ng/mL转化生长因子β1（TGF-β1），培养3天。自第4天起移除培养体系中的TGF-β1。自第14天起，将培养基更换为α-最低必需培养基（α-MEM, Gibco），添加4%无胰岛素B27添加剂、1% MEM非必需氨基酸、100 ng/mL IGF-1、5 ng/mL VEGF165、10 ng/mL FGF-2、100 U/mL青霉素及100 μg/mL链霉素。第28天时，将EHM分别接种于柔性硅胶支架（对照组，健康状态组=Flex），或通过接种于刚性金属支架（应激组=Fix）施加机械应激，继续培养7天。提取总RNA进行RNA测序，以分析不同实验条件下（NT组vs. KLF15 gRNA组、Flex组vs. Fix组）的基因表达变化。