AAV vectors are promising delivery tools for human gene therapy. However, broad tissue tropism and pre-existing immunity against natural serotypes limit their clinical use. We identified two AAV capsid variants, AAV2-THGTPAD and AAV2-NLPGSGD, by in vivo AAV2 peptide display library screening in a murine model of pressure overload-induced cardiac hypertrophy. Both variants showed significantly improved efficacy in in vivo cardiomyocyte transduction compared with the parental serotype AAV2 as indicated by a higher number of AAV vector episomes in the nucleus and significant improved transduction efficiency. Both variants also outcompeted the reference serotype AAV9 regarding cardiomyocyte tropism, reaching comparable cardiac transduction efficiencies accompanied with liver de-targeting and decreased transduction efficiency of non-cardiac cells. Capsid modification influenced immunogenicity as sera of mice treated with AAV2-THGTPAD and AAV2-NLPGSGD demonstrated a poor neutralization capacity for the parental serotype and the novel variants. In a therapeutic setting, using the long non-coding RNA H19 in low vector dose conditions, novel AAV variants mediated superior anti-hypertrophic effects and revealed a further improved target-to-noise ratio, i.e., cardiomyocyte tropism. In conclusion, AAV2-THGTPAD and AAV2-NLPGSGD are promising novel tools for cardiac-directed gene therapy outperforming AAV9 regarding the specificity and therapeutic efficiency of in vivo cardiomyocyte transduction.

AAV 载体是用于人类基因治疗的有前途的递送工具。然而，广泛的组织嗜性和针对天然血清型的预先存在的免疫力限制了它​​们的临床应用。我们通过在压力过载诱导的心脏肥大小鼠模型中进行体内AAV2 肽展示文库筛选，鉴定了两种 AAV 衣壳变体 AAV2-THGTPAD 和 AAV2-NLPGSGD 。两种变体均显示出显着改善的体内功效心肌细胞转导与亲本血清型 AAV2 相比，细胞核中 AAV 载体附加体数量更多，转导效率显着提高。两种变体在心肌细胞趋向性方面也优于参考血清型 AAV9，达到相当的心脏转导效率，伴随着肝脏去靶向和非心脏细胞的转导效率降低。衣壳修饰影响免疫原性，因为用 AAV2-THGTPAD 和 AAV2-NLPGSGD 处理的小鼠血清显示对亲本血清型和新变体的中和能力较差。在治疗环境中，使用长链非编码 RNA H19在低载体剂量条件下，新型 AAV 变体介导了优异的抗肥大作用，并揭示了进一步改善的靶噪比，即心肌细胞趋向性。总之，AAV2-THGTPAD 和 AAV2-NLPGSGD 是用于心脏定向基因治疗的有前途的新工具，在体内心肌细胞转导的特异性和治疗效率方面优于 AAV9。