Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Mol Ther. 2017 Sep 6;25(9):2150-2162. doi: 10.1016/j.ymthe.2017.05.013. Epub 2017 Jun 16.
Dysferlinopathy is an autosomal recessive muscular dystrophy characterized by the progressive loss of motility that is caused by mutations throughout the DYSF gene. There are currently no approved therapies that ameliorate or reverse dysferlinopathy. Gene delivery using adeno-associated vectors (AAVs) is a leading therapeutic strategy for genetic diseases; however, the large size of dysferlin cDNA (6.2 kB) precludes packaging into a single AAV capsid. Therefore, using 3D structural modeling and hypothesizing dysferlin C2 domain redundancy, a 30% smaller, dysferlin-like molecule amenable to single AAV vector packaging was engineered (termed Nano-Dysferlin). The intracellular distribution of Nano-Dysferlin was similar to wild-type dysferlin and neither demonstrated toxicity when overexpressed in dysferlin-deficient patient myoblasts. Intramuscular injection of AAV-Nano-Dysferlin in young dysferlin-deficient mice significantly improved muscle integrity and decreased muscle turnover 3 weeks after treatment, as determined by Evans blue dye uptake and central nucleated fibers, respectively. Systemically administered AAV-Nano-Dysferlin to young adult dysferlin-deficient mice restored motor function and improved muscle integrity nearly 8 months after a single injection. These preclinical data are the first report of a smaller dysferlin variant tailored for AAV single particle delivery that restores motor function and, therefore, represents an attractive candidate for the treatment of dysferlinopathy.
肌营养不良蛋白病是一种常染色体隐性遗传的肌肉疾病，其特征是运动能力逐渐丧失，这是由 DYSF 基因的突变引起的。目前尚无改善或逆转肌营养不良蛋白病的批准疗法。使用腺相关病毒 (AAV) 的基因传递是治疗遗传疾病的主要策略；然而，肌营养不良蛋白 cDNA 的大小为 6.2kB，无法包装到单个 AAV 衣壳中。因此，通过 3D 结构建模和假设肌营养不良蛋白 C2 结构域的冗余性，设计了一种 30%更小的、适合单个 AAV 载体包装的肌营养不良蛋白样分子（称为 Nano-Dysferlin）。Nano-Dysferlin 的细胞内分布与野生型肌营养不良蛋白相似，在肌营养不良蛋白缺陷型患者成肌细胞中过表达时均未显示出毒性。在年轻的肌营养不良蛋白缺陷型小鼠中肌肉内注射 AAV-Nano-Dysferlin 可显著改善肌肉完整性，并分别通过 Evans 蓝染料摄取和中央核纤维来减少肌肉更新 3 周后。系统给予 AAV-Nano-Dysferlin 可恢复年轻成年肌营养不良蛋白缺陷型小鼠的运动功能，并在单次注射近 8 个月后改善肌肉完整性。这些临床前数据首次报道了一种针对 AAV 单颗粒传递量身定制的较小肌营养不良蛋白变体，该变体可恢复运动功能，因此代表了肌营养不良蛋白病治疗的有吸引力的候选物。
