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

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 preserve 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: Double transgenic Myh6-dCas9VPR/Klf15 knockout (KO) mice were generated by crossing Myh6-dCas9VPR mice with Klf15 KO mice. Wild-type C57BL/6N mice were used as controls. Mice (both male and female, 4-6 months old) were injected via the tail vein with either rAAV2/9-TRISPR-Klf15 or rAAV2/9-TRISPR-NT. Following injection, mice underwent transverse aortic constriction (TAC) or sham surgery. Pressure overload was confirmed by measuring the blood flow velocity ratio between the right and left carotid arteries. Echocardiography was conducted at 4 and 8 weeks post-surgery, and hearts were harvested at 12-16 weeks. Ventricular measurements were taken using a VisualSonics Vevo 2100 Imaging System. All procedures were performed in a blinded manner and in accordance with ethical guidelines from the Lower Saxony State Office for Consumer Protection and Food Safety.

心肌重构与适应不良的转录调控网络密切相关，但识别并靶向具有逆转该进程潜力的关键因子仍是一项挑战。本研究采用基于网络的心脏单细胞转录组分析方法，对介导心力衰竭发生的转录因子活性进行量化计算。我们发现，Krüppel样因子15（KLF15）活性的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Cas9（dCas9）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，足以减轻病理性转录重编程，并在小鼠及人类心肌模型中维持心脏功能。我们还证实，在人类心肌细胞的广泛转化生长因子β（TGF-β）介导的应激反应中，KLF15是可靶向的下游因子，其可调控胚胎样转录重编程。最后，我们构建了可用于治疗的迷你型dCas9VPR变体，该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通过恢复应激诱导下调的核心基因表达，以预防心力衰竭的治疗策略，该策略对其他疾病场景亦具有广泛借鉴意义。

实验整体设计：通过将Myh6-dCas9VPR小鼠与Klf15敲除（knockout, KO）小鼠杂交，构建双转基因Myh6-dCas9VPR/Klf15 KO小鼠；以野生型C57BL/6N小鼠作为对照。选取4~6月龄的雌雄小鼠，通过尾静脉注射rAAV2/9-TRISPR-Klf15或rAAV2/9-TRISPR-NT载体。病毒注射后，对小鼠施行主动脉弓缩窄（transverse aortic constriction, TAC）手术或假手术。通过测量左右颈动脉的血流速度比值，确认压力超负荷模型构建成功。分别于术后4周和8周进行超声心动图检测，于术后12~16周采集心脏组织样本。使用VisualSonics Vevo 2100成像系统完成心室参数测量。所有实验操作均采用盲法进行，并符合下萨克森州消费者保护与食品安全局制定的伦理准则。