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，尤其影响大量老年妇女。在全球范围内，七分之一的女性受到SUI的影响。因此，弱化或损坏的尿道闭合装置导致在诸如咳嗽、打喷嚏或进行锻炼的体力消耗期间由于压力（应力）而导致尿液的不可控制的泄漏。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患者的需要，手术干预是一种选择。但目前SUI方案的总体成功还不是最佳的。因此，以细胞为基础的尿道括约肌复合体修复是治疗SUI的一个有前途的选择。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）从人以及猪尿路上皮细胞产生iPSC。iPSC代表再生的细胞。随后，我们将产生的iPSC分化为SkMCs。分析获得的细胞的SkMC特异性标志物的表达、电生理学特征和临床安全性。使用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，以促进该方法从实验室到实验室的转化。在临床前动物研究中，将产生猪iPSC衍生的SkMC并将其注射到患有括约肌缺陷的雌性猪的括约肌中。将在已建立的SUI动物模型中分析术后21天至90天的括约肌功能恢复。使用srRNA成功地无足迹生成自体SkMC可以使得能够用自体再生细胞个性化治疗患有SUI的患者。由于产生的细胞是自体的，因此可以防止排斥反应。应用再生的自体SkMCs可能成为治疗老年患者SUI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.