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。

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.