MICA: Strategy for heart repair in Duchenne Muscular Dystrophy (DMD) using genetically engineered autologous Mesoangioblasts

NHS官方网站显示，英国约有7万人患有肌营养不良症(MD)或相关疾病。Duchenne肌营养不良症(DMD)是最常见和最严重的MD形式之一。DMD是一种遗传性疾病，由于缺乏dystrophin蛋白的表达，导致骨骼肌进行性萎缩，导致儿童进行性行走能力丧失、肌肉无力和肌肉萎缩。在最严重的情况下，心肌细胞也会受到影响，这会导致心力衰竭，这是DMD患者过早死亡的最常见原因。尽管应用类固醇(虽然有严重的不良反应)、矫正手术、辅助机械通气和支持心功能的药物改善了患者的病程和生活质量，但所有MD仍然缺乏有效的治疗方法。许多基于干细胞或基因治疗的方法已进入临床实验，目的是修复DMD心脏，但没有一种方法达到显著的临床疗效。这些疗法的有限成功既是由于将治疗输送到心脏的困难，也是由于患者的免疫系统对供体细胞或病毒载体的激活。为了克服这些限制，我为骨骼肌开发了一种细胞介导的外显子跳过策略，目前正在曼彻斯特的一项临床试验(EudrAct n.2019-001825-28)中进行测试，该策略基于移植自体中血管成血管细胞(MAb)，这是血管相关的肌源性前体细胞。使用表达小核糖核酸(SnRNA)的慢载体对单抗进行遗传校正，该载体旨在诱导dystrophin外显子51上的外显子跳过，并产生一个简短但功能正常的dystrophin版本。这种方法的新颖性是基于SnRNA沿着再生的肌肉纤维扩散的能力，然后纠正驻留的营养不良的邻近核，导致外显子跳跃。然而，单抗只能用于治疗骨骼肌，因为它们不能自然分化为心肌：出于这个原因，MD患者的心脏将保持不被治疗。在这个项目中，我将解决这个问题。众所周知，成纤维细胞可以转化为心肌细胞，但与单抗相比，它们不能系统地输送，因为它们不能穿过血管壁。这使得心脏转化的成纤维细胞成为仅治疗心肌梗死等局部病变的良好候选者，而不是治疗进行性和广泛性心肌病。我的初步数据显示，通过瞬时过度表达特定的心脏相关基因，单抗可以在大约10-15天内转化为心肌细胞。因此，我假设这个时间窗口将允许通过心导管在整个心脏内输送这些单抗转化的心肌细胞。本项目旨在评价单抗转化的心肌细胞移植对DMD小鼠模型心脏结构和功能的改善。此外，我将量化经过基因校正的单抗转化的心肌细胞与常驻心肌细胞的融合程度，以及随后通过跳过外显子在相邻细胞核中的矫正率来量化Dstrophin的产生。这一策略的核心要素已经用于DMD骨骼肌的临床实验，但不适用于心脏。该项目将测试细胞介导的外显子跳过策略对心脏的适用性。该项目的成功结果证明了这一策略的有效性，将为未来在DMD患者的心脏和一般心脏病中进行这一策略的临床测试铺平道路。

The NHS official website indicates that around 70,000 people have Muscular Dystrophy (MD) or a related condition in the UK. Duchenne Muscular Dystrophy (DMD) is the most common and one of the most severe forms of MD. DMD is an inherited disease due to lack of expression of the dystrophin protein, causing a progressive waste of skeletal muscle which lead to progressive loss of ambulation, muscle weakness and muscle wasting in children. In the most severe cases, also heart muscle cells are affected, and this result in heart failure, the most common cause of premature death in DMD patients. All MDs still lack an effective therapy, even though administration of steroids (although with severe adverse effects), corrective surgery, assisted ventilation and drugs to support cardiac function have improved both the duration and the quality of life for patients.Many approaches based on either stem cells or gene therapy have entered clinical experimentation with the aim of repairing DMD heart, but none has reached significant clinical efficacy. The limited success of these therapies is due both to the difficulties of delivering the treatment to the heart and to the activation of the immune system of the patients towards donor cells or viral vectors. To overcome these limitations, I have developed a cell mediated exon skipping strategy, for skeletal muscle, currently being tested in a clinical trial in Manchester (EudrAct n.2019-001825-28), based upon transplantation of autologous Mesoangioblasts (Mabs), which are vessel-associated myogenic progenitors. Mabs are genetically corrected using a lentivector expressing a small nuclear RNA (snRNA) designed to induce exon-skipping on dystrophin exon 51 and to generate a short but functional version of dystrophin. The novelty of this approach is based on the ability of snRNA to diffuse along the regenerating muscle fibre and then correcting the resident dystrophic neighbouring nuclei inducing exon skipping. However, Mabs can be used only for the treatment of skeletal muscle as they cannot naturally differentiate into cardiac muscle: for this reason, the heart of the MD patients would remain untreated. In this project I will address this problem. It is well known that fibroblasts can be converted to cardiac muscle cells but, in comparison with Mabs, they cannot be delivered systematically, due to their inability to cross the vessel walls. This makes cardiac-converted fibroblasts good candidates to treat only localised lesions like myocardial infarct but not to treat progressive and widespread cardiomyopathies. My preliminary data show that Mabs can be converted to cardiomyocytes by transient over-expression of specific cardiac related genes in around 10-15 days. I therefore hypothesize that this time window will allow to deliver these Mabs-converted cardiomyocytes by cardiac catheterization in the whole heart. This project aims to evaluate the structural and functional amelioration of the heart in a mouse model of DMD after transplantation of Mabs-converted cardiomyocytes. Moreover, I will quantify the extent of the fusion of genetically corrected Mabs-converted cardiomyocytes with resident cardiomyocytes and subsequently the rate of correction, by exon-skipping, in the neighbouring nuclei quantifying the amount of dystrophin produced.The core element of this strategy is already in clinical experimentation for DMD skeletal muscle but not for the heart. This project will test the applicability of the cell mediated exon-skipping strategy to the heart. The succesful outcome of this project, demonstrating the efficacy of this strategy, will lead the way for a future clinical testing of this strategy in the heart of DMD patients and in heart diseases in general.