Footprint-free generation of autologous rejuvenated skeletal myocytes for sphincter muscle repair

压力性尿失禁（SUI）是尿失禁最常见的形式，尤其影响大量的老年妇女。在世界范围内，七分之一的女性患有SUI。因此，削弱或损坏的尿道关闭装置导致无法控制的尿液泄漏，这是由于体力消耗（如咳嗽、打喷嚏或进行运动）时的压力（应激）造成的。药物治疗和肌肉锻炼是标准的治疗方法。如果保守疗法不能满足病人的需要，手术干预是一种选择。但目前SUI方案的总体成功还不是最理想的。因此，以细胞为基础的尿道括约肌复合体修复是治疗SUI的一个有希望的选择。但是来自老年人的自体细胞可能产生有限的括约肌再生。因此，在这个项目中，我们的目标是产生再生的自体横纹肌细胞（SkMCs）用于SUI患者的个体化治疗。为此，我们通过外源性递送编码重编程因子的自我复制RNA (srRNA)，从人和猪尿路上皮细胞中生成iPSCs。iPSCs代表了恢复活力的细胞。随后，我们将生成的iPSCs分化为skmc。对获得的细胞进行SkMC特异性标记物表达、电生理特性和临床安全性分析。人类细胞的程序在非常接近良好生产规范（GMP）规定的条件下进行，以便于将该方法从实验室转换到床上。在一项临床前动物研究中，将生成猪ipsc衍生的SkMCs并将其注射到患有括约肌缺陷的母猪的括约肌中。在建立的SUI动物模型中分析术后21 - 90天的括约肌功能恢复情况。利用srrna成功地无足迹地生成自体SkMCs，可以使用自体再生细胞对SUI患者进行个性化治疗。由于生成的细胞是自体的，因此可以防止排斥反应。应用活化的自体SkMCs可以成为治疗老年SUI患者的一种改进的治疗策略。

Stress urinary incontinence (SUI) is the most frequent form of urinary incontinence and affects especially a significant number of elderly women. Worldwide, one in seven women is affected by SUI. Thereby, the weakened or damaged urethral closure apparatus leads to uncontrollable leakage of urine due to pressure (stress) during physical exertion such as coughing, sneezing, or performing exercise. Medication and muscle exercise are standard treatments. If the conservative regimen fails to meet the patient’s needs, surgical interventions are an option. But the overall success of the current SUI regimen is not yet optimal. Therefore, cell-based repair of the sphincter complex of the urethra is a promising option to treat SUI. But autologous cells from the elderly may yield limited sphincter regeneration. Thus, in this project, we aim to generate rejuvenated autologous striated muscle cells (SkMCs) for individualized therapy of SUI patients. To this end, we generate iPSCs from human as well as porcine urothelial cells by exogenous delivery of self-replicating RNA (srRNA) encoding the reprogramming factors. iPSCs represent rejuvenated cells. Subsequently, we differentiate the iPSCs generated into SkMCs. The cells obtained are analyzed for their expression of SkMC specific markers, electrophysiological characteristics, and clinical safety. Procedures with human cells are conducted under conditions very close to good manufacturing practice (GMP) regulations to facilitate the translation of this method from bench to bed. In a pre-clinical animal study, porcine iPSC-derived SkMCs will be generated and injected into the sphincter muscle of female pigs with sphincter deficiency. The functional recovery of the sphincter apparatus will be analyzed for 21 up to 90 days post-surgery in an established SUI animal model. The successful footprint-free generation of autologous SkMCs using srRNAs could enable the personalized treatment of patients suffering from SUI with autologous rejuvenated cells. As the generated cells are autologous, rejection reactions are prevented. The application of rejuvenated autologous SkMCs could become an improved curative strategy for the treatment of SUI in elderly patients.