Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models
Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models
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DOI：
10.3389/fphys.2019.00168
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发表时间：
2019-03-05
影响因子：
4
通讯作者：
Entcheva, Emilia
中科院分区：
文献类型：
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作者：
Ambrosil, Christina M.;Sadanandal, Gouri;Entcheva, Emilia
腺相关病毒（aav）在长期、心脏特异性基因表达方面具有优势。然而，与心脏电生理和光遗传学相关的实验模型缺乏MV血清型特异性数据。我们的目标是确定最佳的MV血清型（1、6或9），以追求可扩展的啮齿动物和人类模型，特别是利用心脏电生理学和光遗传学的遗传修饰，以及阐明病毒摄取的机制。用含有eGFP或通道视紫红质-2 （ChR2）基因的AAVs 1、6和9与mCherry融合，在体外培养新生大鼠心室心肌细胞（nrvm）和人诱导的多能干细胞源性心肌细胞（hiPSC-CMs）。成年大鼠体内静脉注射含有ChR2-mCherry的AAV1和aav9。大鼠和人体外细胞的转基因表达谱显示，AAV1和6显著优于AAV9。相比之下，AAV9在成年大鼠心脏的全身递送可显著提高ChR2-mCherry的表达水平和光遗传反应性。我们追踪了病毒摄取AAV1/6（细胞表面唾液酸）和AAV9 （37/67 kDa层粘胶蛋白受体，LamR）的受体介质的机制。在体外用神经氨酸酶（NM）对nrvm和hiPSC-CMs进行去脂化处理，可显著降低aav1,6介导的基因表达，但有趣的是，hiPSC-CMs去脂化处理可增加aav9介导的基因表达。事实上，只有非常高的病毒剂量的AAV9-ChR2-mCherry结合NM处理才能在hiPSC-CMs中产生一致的光遗传反应。AAV9在体外和体内表现的差异可能与LamR在完整心脏（新生大鼠心脏以及成人和大鼠心脏）中的强劲表达有关，但在体外培养细胞（原代大鼠细胞和hiPS-CMs）中没有表达。LamR表达的动态性质及其对环境因素的依赖性在完整的成人心室组织中得到进一步证实。结合转基因表达和细胞表面受体的数据可以解释AAV1/6在体外和AAV9在体内对心脏传递的优先效率，以及它们作用的机制知识可以帮助指导心脏光遗传学的研究。更广泛地说，这些发现与体内心脏电生理异常的基因治疗以及通过病毒手段在体外应用（如疾病建模或高通量药物测试）对心肌细胞进行遗传修饰的未来努力有关。
Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, MV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal MV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stern cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.