腺相关病毒rAAV1增强HCN2/SkM1型生物起搏器长效性的实验研究

For decades, electronic cardiac pacing has been providing effective treatment for heart block in children, yet still important shortcomings remain. To meet the need for better alternatives, several strategies to create biological pacemakers have been explored. Biological pacemakers based on overexpression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are among the most promising. An advantage of overexpressing these channels to fabricate biological pacemakers lies in their regulation by cAMP, which contributes to automomic control of heart rates. However, function of HCN-based biological pacemakers still warrants improvement. For example, HCN2 delivered into the canine left bundle branch generates relatively slow beating rates interspersed with pauses sufficiently long to require electronic back-up pacing about 30% of the time. Hence, we proposed to create biological pacemakers having faster baseline and adrenergically stimulated rates and improved stability. In so doing, we considered baseline beating rates of 60-90 bpm and low-to-absent dependence on electronic back-up pacing as optimal biological pacing outcomes. In an effort to improve biological pacemaker performance, we recently developed a method that combines HCN2-dependent pacemaker function with the skeletal muscle sodium channel (SkM1). Here, HCN2 generates slow diastolic depolarization while overexpression of SkM1 hyperpolarizes action potential threshold thereby increasing pacemaker rate and improving stability. When injcetd into the left bunde branch of AV-blocked canine, adenoviral overexpression of HCN/SkM1 generated highly stable biological pacing with basal beating rates of 80 bpm, brisk automomic modulation, and a complete elimination of electronic back-up pacing. Local delivery into the ventricular conduction system of combined HCN2/SkM1 has thus emerged as a promising strategy. The next step in developing HCN2/SkM1-based pacemakers focuses on delivering long-term function...In this project we will move this concept forward by employing vectors based on Adeno-assoicated virus (AAV). Because the insert capacity of AAV is limited, SkM1 will be split into two parts via trans-splicing technology. HCN2 will be delivered from a separate vector. To test biological pacemaker function, these combined vectors will be injected in the left ventricular apex of guinea pig hearts. Next, ex vivo biological pacemaker function will be evaluated in Langendorff-perfused hearts. Pacemaker function will be assessed by measuring spontaneous beating rates, response to autonomic modulation, and vector orientation on the pseudo-electrocardiogram. Long-term persistence will be measured by comparing outcomes between one week and six months after construct injection. Demonstrating efficient and long-term stable biolgical pacing in this model will pave the way towards further large animal and clinical testing.

对于药物治疗无效的完全性房室传导阻滞患儿，目前只能接受电子起搏器植入治疗，但电子起搏器存在诸多缺陷。利用基因转染构建的生物起搏器，有望替代电子起搏器并避免其缺点。申请人的最新研究表明，腺病毒HCN2/SkM1移植动物体内后，可以产生符合生理需求的起搏心率（80次/分），且对肾上腺素类神经递质具有良好的反应性，并不再需要电子起搏器的备用起搏。但由于腺病毒自身特点等局限性，目前HCN2/SkM1生物起搏器工作时间较短，尚不能满足临床要求。针对上述问题，本项目拟选择腺相关病毒(rAAV1)为基因载体。由于腺相关病毒携带基因容量有限，我们拟采用转染分解技术将SkM1分成两部分，分别构建rAAV1-SkM1 I和rAAV2-SkM1 II后与rAAV1-HCN2共同转染，有效延长HCN2/SkM1型生物起搏器的作用时间。通过本项目，将完善生物起搏治疗的研究基础，加快其安全有效地应用于临床实践。

完全性房室传导阻滞，是指由窦房结发出的全部冲动在房室传导过程中受到阻滞的心脏传导系统疾病。对于药物治疗无效的完全性房室传导阻滞患儿，只能接受电子心脏起搏器植入治疗。但电子起搏器存在诸多缺陷，因此，探寻电子起搏器替代治疗手段愈发重要。前期实验完成了环核苷酸门控阳离子通道2（HCN2）与骨骼肌钠通道（SkM1）共同表达增强HCN2型生物起搏器起搏功能的实验研究，解决了生物起搏的效能“瓶颈”问题，使生物起搏应用于临床具备可行性。下一步研究重点集中于如何增强HCN2/SkM1生物起搏器起搏功能的长期稳定性。在本项目中，我们提出以腺相关病毒（AAV）作为基因载体改进HCN2/SkM1型生物起搏器，使其作用时间长期稳定。由于腺相关病毒容量有限，插入SkM1基因（5.4kb）时会出现空间不足。我们采用转染分解技术（Trans-splicing technology）。项目中，我们先后采用重组碱性磷酸酶AP和断裂蛋白将SkM1分解成为SkM1 I和SkM1 II两部分，分别构建rAAV1-SkM1 I和rAAV1-SkM1 II后进行转染，测试其在心肌内目的蛋白的表达。由于本项目实验周期较长，实验动物使用量大，我们采用豚鼠作为试验动物。将rAAV1-SkM1 I、rAAV1-SkM1 II和rAAV1-HCN2注射转染到豚鼠左心室心尖后，在不同的时间点，通过离体Langendorff心脏灌流和冷冻消融房室交界区的方法构建豚鼠离体心脏完全性房室传导阻滞模型，从而判断联合移植心肌后能否产生更为长期稳定的起搏心率。研究证实，Ssp DnaB内含子置于分离位点S1067前面，分解产生的rAA1-N-SkM1和rAA1-C-SkM1转染后可以在心肌内表达SkM1蛋白。rAAV1-SkM1 I、rAAV1-SkM1 II和rAAV1-HCN2联合转染后，能够在心肌内可以完整表达SkM1和HCN2，且能够提供长期的起搏心率，对神经递质有良好的反应性。项目成果完善了生物起搏治疗的理论基础，为下一步大动物实验和临床实践指明道路。