研究背景

肥厚型心肌病（hypertrophic cardiomyopathy, HCM）是目前发病率最高的人类遗传性心肌病之一，也是诱发儿童及青年运动员猝死的常见病因。HCM的病理生理特征以心肌重塑为关键改变，具体表现为心腔缩小、心肌排列紊乱和显著纤维化，且无论是否伴随左心室流出道梗阻，患者均表现出典型的超动力心肌收缩和舒张功能障碍。因此，解除心肌异常收缩、延缓心肌重塑是预防及治疗HCM的重要目标。HCM的典型病理特征主要由肌节蛋白相关的编码基因（如MYH7或MYBPC3）突变引起，然而HCM的临床治疗现以负性肌力药物或手术干预改善症状为主要方式，尚缺乏基因分子水平的有效治疗手段。

研究发现，心肌细胞中特异性表达的miR-208a在心肌肥厚与纤维化进程中发挥重要作用。然而，既往研究利用同源重组或反义寡核苷酸抑制miR-208a的策略存在明显的安全风险及重复给药的局限性。随着CRISPR基因编辑技术的不断发展，碱基编辑器（base editing，BE）作为一种安全、有效的基因组编辑工具，显示出了治疗难治性遗传病的重大潜力。

研究目标

利用腺相关病毒血清型9（adeno-associated virus serotype 9, AAV9）搭载靶向miR-208a-3p种子序列的腺嘌呤碱基编辑工具，实现小鼠心肌miR-208a在体突变及功能抑制，探究miR-208a精准编辑对于HCM病理表型的逆转效果及潜在分子机制，同时对该策略进行安全性评估。

研究方法

（1）miR-208a碱基编辑系统构建、筛选与体外验证：设计、筛选特异性靶向miR-208a种子序列的高效向导RNA（single guide RNA，sgRNA）；构建miR-208a突变体过表达系统并利用双荧光素酶报告基因对miR-208a成熟突变体进行功能性验证；（2）在体编辑miR-208a：构建并利用带有人类心肌肌钙蛋白T（cTnT）启动子的AAV9向HCM小鼠（Myh6 ^R404Q/+和Mybpc3 ^R946X/R946X）体内递送腺嘌呤碱基编辑器变体（ABE8e-SpRY）及sgRNA；20周后检测小鼠心肌组织miR-208a编辑效率、转录组表达水平；（3）HCM表型评估：利用超声心动图周期性评估HCM小鼠心脏重塑及心功能；检测小鼠心肌肥厚相关分子mRNA及蛋白表达水平；通过多种组织染色评估小鼠HCM表型改善程度；（4）潜在机制及安全性分析：通过转录组高通量测序，开展miRNAs及RNA转录组学分析，探究miR-208a改善HCM表型的潜在机制；验证ABE8e-SpRY-sgRNA4编辑系统的安全性。

研究结果

（1）miR-208a碱基编辑系统构建、筛选与体外验证：sgRNA-4可在体外实现稳定的miR-208a编辑，编辑范围覆盖种子序列全部腺嘌呤，DNA水平检测最高A>G效率可达25%，miR-208a转录组水平显著下降约40%（p<0.05）；编辑策略可在细胞中产生15种miR-208a突变成熟体且均丧失对靶基因mRNA抑制功能。

（2）在体编辑miR-208a：双AAV9搭载的ABE8e-SpRY-sgRNA4可有效实现小鼠心肌miR-208a在体编辑，RNA水平检测最高A>G效率可达50%，心肌组织miR-208a转录组水平显著下降约80%（p<0.0001）；编辑策略可在心肌细胞中产生15种miR-208a突变成熟体。

（3）HCM表型评估：该编辑策略显著延缓了小鼠心肌肥厚进程，17周时左室前壁舒张期厚度（mm）降低12.8%（1.099±0.138 vs. 1.260±0.165，p=0.0327），左室后壁厚度（mm）降低约21.4%（0.912±0.058 vs. 1.161±0.168，p=0.0010）；同时治疗恢复了小鼠部分心功能（射血分数：64.83±7.96% vs 72.00±5.73%，p=0.0473；短缩分数：35.00±5.73% vs. 41.31±4.68%，p=0.0204）；此外，治疗后心肌组织中肌节蛋白MYH7表达水平显著下降68%（1.009±0.259 vs. 3.153±0.504，p<0.0001）；病理检测显示miR-208a突变显著减轻了左室壁的厚度，改善了心肌纤维化程度、心肌细胞大小和紊乱程度。

（4）潜在机制及安全性分析：miR-208a可调控多条与HCM发生相关的信号通路，包括肌节蛋白合成、钙稳态调节及线粒体能量代谢；miR-208a突变可协同miR-499抑制异常肌节蛋白合成，减少ATP过度消耗从而缓解心肌超动力收缩状态；该编辑策略未引发显著脱靶效应，且未引起额外的心律失常。

研究结论

本研究利用ABE8e-SpRY碱基编辑技术首次在体内精准编辑miR-208a，并验证了其在遗传性HCM治疗中的潜力。该编辑策略通过解除miR-208a对下游靶基因的抑制，调控肌节蛋白代谢、钙稳态及线粒体功能，有效减少心肌超动力收缩，延缓心肌肥厚与纤维化进程，改善心脏舒张功能。此外，miR-208a的精准编辑策略具有较高的安全性，未引发显著脱靶效应及额外心律失常。本研究为HCM及其他遗传性心血管疾病提供了一种精准、高效且安全的基因编辑干预策略，推动miRNA靶向的基因治疗向临床转化。

Background

Hypertrophic cardiomyopathy (HCM) is one of the most prevalent hereditary cardiomyopathies and a leading cause of sudden cardiac death in children and young athletes. The pathological hallmark of HCM is myocardial remodeling, characterized by ventricular chamber reduction, cardiomyocyte disarray, and significant fibrosis. Regardless of the presence of left ventricular outflow tract obstruction, patients exhibit hypercontractile myocardium and impaired diastolic function. Therefore, alleviating abnormal myocardial contraction and delaying myocardial remodeling are critical goals for HCM prevention and treatment. The characteristic pathological features of HCM are primarily attributed to mutations in sarcomeric protein-coding genes, such as MYH7 or MYBPC3. However, current clinical management primarily relies on negative inotropic agents or surgical interventions to alleviate symptoms, lacking effective molecular-level therapeutic strategies targeting the underlying genetic causes.

Recent studies have identified miR-208a, a cardiac-specific microRNA, as a key regulator of myocardial hypertrophy and fibrosis. However, previous attempts to inhibit miR-208a using homologous recombination or antisense oligonucleotides have been limited by safety concerns and the need for repeated administration. With advances in CRISPR-based genome editing, base editing (BE) has emerged as a precise, efficient, and safe genomic modification tool, demonstrating tremendous potential for treating refractory genetic diseases.

Objectives

This study aims to construct an adenine base editing system to precisely mutate and functionally suppress miR-208a in vivo using an adeno-associated virus serotype 9 (AAV9) mediated delivery. We seek to evaluate the therapeutic potential of miR-208a precise editing in reversing HCM-associated pathological phenotypes, and delineate the molecular mechanisms underlying its efficacy.

Methods

(1) Construction, screening, and validation of the miR-208a base editing system in vitro: Single guide RNAs (sgRNAs) targeting the seed sequence of miR-208a-3p were designed and screened for optimal editing efficiency. A miR-208a mutant overexpression system was established, and its functional impact was validated using a dual-luciferase reporter assay. (2) In vivo miR-208a editing: AAV9 vectors carrying a cardiac troponin T (cTnT) promoter-driven base editor variant (ABE8e-SpRY) and sgRNA were constructed and delivered into two HCM mouse models (Myh6 ^R404Q/+ and Mybpc3^R946X/R946X). After 20 weeks, miR-208a editing efficiency and transcriptomic changes in cardiac tissue were analyzed. (3) HCM phenotype assessment: Echocardiography was performed periodically to assess myocardial remodeling and cardiac function. Hypertrophic markers at the mRNA and protein levels were quantified in cardiac tissues, and histological evaluations were conducted to examine pathological changes. (4) Mechanistic and safety analyses: RNA-sequencing was performed to explore miRNA and transcriptomic alterations associated with miR-208a editing. Additionally, the safety of the ABE8e-SpRY-sgRNA4 editing system was evaluated.

Results

(1) In vitro construction, screening, and validation of the miR-208a base editing system: sgRNA-4 demonstrated stable and efficient miR-208a editing, with an adenine-to-guanine (A>G) conversion efficiency of up to 25% in the miR-208a seed sequence. miR-208a expression was significantly reduced by approximately 40% (p<0.05). Moreover, 15 distinct miR-208a mature variants were identified, all of which exhibited a complete loss of target mRNA suppression.

(2) In vivo miR-208a editing: AAV9-mediated delivery of ABE8e-SpRY-sgRNA4 efficiently edited the miR-208a seed sequence in mouse cardiomyocytes, achieving an A>G conversion rate of up to 50% at the RNA level. The transcriptomic abundance of miR-208a was significantly reduced by approximately 80% (p<0.0001), and 15 distinct miR-208a mutant variants were detected in cardiomyocytes.

(3) HCM phenotype assessment: miR-208a base editing significantly attenuated myocardial hypertrophy. At 17 weeks, left ventricular anterior wall thickness in diastole decreased by 12.8% (1.099±0.138 vs. 1.260±0.165 mm, p=0.0327), and left ventricular posterior wall thickness decreased by 21.4% (0.912±0.058 vs. 1.161±0.168 mm, p=0.0010). Cardiac function was partially restored, as evidenced by an increase in ejection fraction (64.83±7.96% vs. 72.00±5.73%, p=0.0473) and fractional shortening (35.00±5.73% vs. 41.31±4.68%, p=0.0204). Additionally, MYH7 protein levels were significantly reduced by 68% (1.009±0.259 vs. 3.153±0.504, p<0.0001). Histological analyses revealed a decrease in left ventricular wall thickness, improved cardiomyocyte alignment, and reduced myocardial fibrosis following miR-208a editing.

(4) Mechanistic and safety analyses: Transcriptomic analysis indicated that miR-208a editing modulates multiple pathways implicated in HCM pathogenesis, including sarcomere protein synthesis, calcium homeostasis, and mitochondrial energy metabolism. miR-208a mutations cooperated with miR-499 to suppress abnormal sarcomeric protein synthesis and reduce excessive ATP consumption, leading to a decrease in myocardial hypercontractility. Safety assessments demonstrated no significant off-target effects, and no additional risk of arrhythmia was observed following miR-208a editing.

Conclusion

This study, for the first time, achieves precise in vivo editing of miR-208a using ABE8e-SpRY base editing technology and validates its therapeutic potential for hereditary HCM. The miR-208a editing strategy effectively alleviates myocardial hypercontractility, attenuates hypertrophic remodeling and fibrosis, and improves diastolic function by restoring sarcomere protein balance, calcium homeostasis, and mitochondrial function. Furthermore, this strategy exhibits high specificity and safety, with no significant off-target effects or arrhythmic complications. These findings establish a novel, precise, and efficient gene-editing approach for the treatment of HCM and other hereditary cardiovascular diseases, advancing miRNA-targeted genome editing toward clinical application.