To establish an efficient, safe immunosuppressive regimen of adeno-associated vector (AAV)-mediated gene therapy for Duchenne muscular dystrophy (DMD), we evaluated the effect of tacrolimus (FK506) on skeletal muscle transduction with AAV8 and AAV9 vectors expressing the LacZ and microdystrophin (M3) genes labeled by FLAG. We utilized 3- to 4-year-old Macaca fascicularis, screened for neutralizing antibodies against AAV. 3 days before AAV injection and throughout the experiment, 0.06 mg/kg tacrolimus was intravenously administered. A viral suspension of 1 × 10¹³ viral genomes/muscle was intramuscularly injected bilaterally at the tibialis anterior and biceps brachii muscles, which were biopsied at 8, 16, 24, and 42 weeks after injection. Without tacrolimus, AAV8- and AAV9-mediated LacZ expression disappeared 8 and 16 weeks after transduction, respectively. With tacrolimus, AAV8/9-mediated LacZ expression persisted for at least 42 weeks after injection. At 42 weeks after AAV8CMVLacZ and AAV9CMVLacZ injection, nearly 50% and 17% of muscle fibers were positive for β-galactosidase, respectively. AAV8/9-mediated M3-FLAG expression lasted for up to 42 weeks using tacrolimus. No significant generalized toxicity was observed in any monkey. These results indicate that tacrolimus administration regulated the immune response to transgenes and truncated microdystrophin in normal primates and may enhance the benefits of AAV-mediated gene therapy for DMD.

为了建立一种有效，安全的腺相关载体（AAV）介导的基因疗法治疗杜氏肌营养不良（DMD）的免疫抑制方案，我们评估了他克莫司（FK506）对表达LacZ和AAV8的AAV8和AAV9载体对骨骼肌转导的作用。FLAG标记的微肌营养不良蛋白（M3）基因。我们利用3至4岁的猕猴，筛选了抗AAV的中和抗体。在注射AAV之前3天以及整个实验过程中，静脉内注射他克莫司0.06 mg / kg。1×10 ^13的病毒悬浮液分别在胫骨前肌和肱二头肌肌肉内肌肉注射病毒基因组/肌肉，在注射后第8、16、24和42周进行活检。没有他克莫司，AAV8和AAV9介导的LacZ表达分别在转导后8周和16周消失。对于他克莫司，注射后AAV8 / 9介导的LacZ表达持续至少42周。在注射AAV8CMV LacZ和AAV9CMV LacZ后的42周时，分别有近50％和17％的肌肉纤维对β-半乳糖苷酶呈阳性。AAV8 / 9介导的M3使用他克莫司，FLAG的表达持续长达42周。在任何猴子中均未观察到明显的普遍毒性。这些结果表明他克莫司的给药调节了正常灵长类动物对转基因和截短的微营养不良蛋白的免疫反应，并可能增强AAV介导的DMD基因治疗的益处。