Engineering human artificial chromosomes containing the dystrophin locus for autologous cell therapy of Duchenne Muscular Dystrophy.

Duchenne肌营养不良症(DMD)是最常见和最严重的肌营养不良症(1/3500男性)。它影响骨骼肌和心肌，导致进行性运动障碍，被限制在轮椅上，最终导致完全瘫痪和辅助呼吸。死亡通常发生在生命的第三个十年，原因是心脏和/或呼吸衰竭。改善的医疗援助延长了患者的寿命，但仍然没有有效的治疗方法，类固醇是唯一可用的姑息治疗方法。在进入临床实验的几种新策略中，我们团队专注于细胞治疗，基于动脉内输注中血管母细胞(MAB)，这是一种与血管相关的干/祖细胞，在营养不良的小鼠和狗身上显示出疗效。虽然基于同种异体中血管母细胞移植的I/II期临床试验目前正在进行中，但Dystrophin基因的大小(2.4Mb)阻碍了病毒载体的使用。我们小组率先使用包含整个肌营养不良症基因座的人类人工染色体(HAC)治疗肌营养不良(DYS-HAC)，在营养不良的小鼠细胞中显示了这一策略的有效性。基于上述，我们现在计划开发一种最终的策略，使其能够转化到人类细胞并提高治疗效果。首先，我们将在DYS-HAC中插入一个包含可以绕过衰老(端粒酶和Bm1)的基因和一个自杀基因的“永生盒”作为安全装置。初步结果证实，这种盒式磁带在人类细胞中可以工作。DYS-HAC还将包含一个可诱导的MyoD(即连接到雌激素受体：MyoD-ER)，这是一种肌源性主控基因，将允许诱导肌肉分化(不同患者的情况有所不同)。将确定要包括的dystrophin的最佳拷贝数，以实现单个基因校正核的最佳表达水平，以补偿无法合成它的肌肉纤维驻留核。所有后续世代的HAC将被转移到人类营养不良的中血管母细胞中，然后将面临修复营养不良肌肉和改善疾病的能力的挑战。PI是细胞治疗领域的领导者；他和他的同事的专业知识将保证这个雄心勃勃但现实的项目获得成功的可能性很高，其结果可能导致快速临床翻译，同时为其他单基因疾病铺平道路，这些疾病的特征是非常大的基因突变。

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy (1/3500 males). It affects skeletal and cardiac muscles, leading to progressive loss of movements, confinement to a wheelchair and finally to total paralysis and assisted ventilation. Death occurs usually in the third decade of life because of cardiac and/or respiratory failure. Improved medical assistance has increased the lifespan of patients but still there is no efficacious therapy and steroids are the only palliative treatment available. Among several novel strategies that are entering clinical experimentation, our group focused on cell therapy, based upon intra-arterial infusion of mesoangioblasts (MABs), stem/progenitor cells associated to blood vessels that showed efficacy in dystrophic mice and dogs. Although a phase I/II clinical trial based upon allogeneic transplantation of mesoangioblasts is currently running, gene therapy of autologous cells would be preferable (no immune suppression and no need of HLA-matched donor) but the large size of the dystrophin gene (2.4Mb) hampers the use of viral vectors. Our group pioneered the use of human artificial chromosomes (HACs) containing the whole dystrophin locus for muscular dystrophy (DYS-HAC), showing efficacy of this strategy in dystrophic mouse cells. Based on the above, we now plan to develop a definitive strategy that would allow translation to human cells and boosting the therapeutic effect.First, we will insert into the DYS-HAC an "immortalizing cassette" containing genes that allow bypassing senescence (telomerase and Bm1) and a suicide gene as a safety device. Preliminary results confirm that this cassette works in human cells. The cassette is floxed and thus can be excised from the HAC before cells are infused into patients.The DYS-HAC will also contain an inducible MyoD (i.e linked to the Estrogen Receptor: MyoD-ER), a myogenic master gene that will allow induction of muscle differentiation (which is variable among different patients). The optimal number of copies of dystrophin to include will be determined to achieve the optimal level of expression for a single genetically corrected nucleus to compensate for the muscle fibre resident nuclei that cannot synthesize it. All the subsequent generations of HACs will be transferred in human dystrophic mesoangioblasts that will be then challenged for their ability to repair dystrophic muscle and ameliorate the disease.The PI is a leader in the field of cell therapy; his expertise, and that of his colleagues, will guarantee a high probability of success for this ambitious but realistic project, whose results may lead to rapid clinical translation and, at the same time, pave the way for other monogenic diseases, characterized by mutations of a very large gene.