Burke Matthew J, Blatt Braiden M, Teixeira James A, Pérez-López Dennis O, Yue Yongping, Pan Xiufang, Hakim Chady H, Yao Gang, Herzog Roland W, Duan Dongsheng
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri, USA.
College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA.
J Cachexia Sarcopenia Muscle. 2025 Feb;16(1):e13681. doi: 10.1002/jcsm.13681.
Adeno-associated virus (AAV) 8 and 9 are in clinical trials for treating neuromuscular diseases such as Duchenne muscular dystrophy (DMD). Muscle consists of myofibres of different types and sizes. However, little is known about the fibre type and fibre size tropism of AAV in large mammals.
We evaluated fibre type- and size-specific transduction properties of AAV8 and AAV9 in 17 dogs that received systemic gene transfer (dose 1.94 ± 0.52 × 10 vg/kg; injected at 2.86 ± 0.30 months; harvested at 20.79 ± 3.30 months). For AAV8, two DMD dogs and three carrier dogs received an alkaline phosphatase (AP) reporter vector, and five DMD dogs received a four-repeat microdystrophin (uDys) vector. For AAV9, one normal and one DMD dog received the AP vector, and five DMD dogs received a five-repeat uDys vector. Association between AAV transduction and the fibre type/size was studied in three muscles that showed mosaic transgene expression, including the biceps femoris, teres major and latissimus dorsi.
Transgene expression was detected in 30%-45% of myofibres. In the AP reporter vector-injected dogs, neither AAV8 nor AAV9 showed a statistically significant fibre type preference. Interestingly, AP expression was enriched in smaller fibres. In uDys-treated DMD dogs, slow and fast myofibres were equally transduced. Notably, uDys-expressing myofibres were significantly larger than uDys-negative myofibres irrespective of the AAV serotype (p < 0.0001). In AAV8 uDys vector-injected dogs, the mini-Feret diameter was 15%, 16% and 23% larger in uDys-positive slow, fast and hybrid fibres, respectively; the cross-sectional area was 30%, 34% and 46% larger in uDys-positive slow, fast and hybrid fibres, respectively. In AAV9 uDys vector-injected dogs, the mini-Feret diameter was 12%, 13% and 25% larger in uDys-positive slow, fast and hybrid fibres, respectively; the cross-sectional area was 25%, 28% and 59% larger in uDys-positive slow, fast and hybrid fibres, respectively.
Our studies suggest that AAV8 and AAV9 transduce fast and slow myofibres at equivalent efficiency. Importantly, uDys therapy effectively prevented dystrophic myofibre atrophy. Our study provides important insight into systemic muscle AAV delivery in large mammals and supports further development of uDys gene therapy for DMD.
腺相关病毒（AAV）8型和9型正在进行治疗诸如杜氏肌营养不良症（DMD）等神经肌肉疾病的临床试验。肌肉由不同类型和大小的肌纤维组成。然而，关于大型哺乳动物中AAV的纤维类型和纤维大小嗜性知之甚少。
我们评估了17只接受全身基因转移的犬（剂量为1.94±0.52×10 vg/kg；在2.86±0.30个月时注射；在20.79±3.30个月时收获）中AAV8和AAV9的纤维类型和大小特异性转导特性。对于AAV8，两只DMD犬和三只携带者犬接受了碱性磷酸酶（AP）报告载体，五只DMD犬接受了四重复微肌营养不良蛋白（uDys）载体。对于AAV9，一只正常犬和一只DMD犬接受了AP载体，五只DMD犬接受了五重复uDys载体。在三块显示嵌合转基因表达的肌肉（包括股二头肌、大圆肌和背阔肌）中研究了AAV转导与纤维类型/大小之间的关联。
在30%-45%的肌纤维中检测到转基因表达。在注射AP报告载体的犬中，AAV8和AAV9均未显示出统计学上显著的纤维类型偏好。有趣的是，AP表达在较小的纤维中富集。在接受uDys治疗的DMD犬中，慢肌纤维和快肌纤维的转导效率相同。值得注意的是，无论AAV血清型如何，表达uDys的肌纤维明显大于uDys阴性肌纤维（p<0.0001）。在注射AAV8 uDys载体的犬中，uDys阳性慢肌纤维、快肌纤维和混合纤维的最小费雷特直径分别大15%、16%和23%；uDys阳性慢肌纤维、快肌纤维和混合纤维的横截面积分别大30%、34%和46%。在注射AAV9 uDys载体的犬中，uDys阳性慢肌纤维、快肌纤维和混合纤维的最小费雷特直径分别大12%、13%和25%；uDys阳性慢肌纤维、快肌纤维和混合纤维的横截面积分别大25%、28%和59%。
我们的研究表明，AAV8和AAV9以同等效率转导快肌纤维和慢肌纤维。重要的是，uDys疗法有效地预防了营养不良性肌纤维萎缩。我们的研究为大型哺乳动物全身肌肉AAV递送提供了重要见解，并支持uDys基因疗法用于DMD的进一步开发。
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