Stable expression of calpain 3 from a muscle transgene in vivo:: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation
Stable expression of calpain 3 from a muscle transgene in vivo:: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation
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DOI：
10.1073/pnas.132269299
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发表时间：
2002-06-25
影响因子：
11.1
通讯作者：
Beckmann, JS
中科院分区：
文献类型：
--
作者：
Spencer, MJ;Guyon, JR;Beckmann, JS
肢带肌营养不良症2A型（LGMD 2A）是一种常染色体隐性疾病，可导致迟发性肌肉萎缩，是由于肌肉特异性蛋白酶钙蛋白酶3（0）突变所致。虽然LGMD 2A将是一个可行的候选基因治疗，C3在体外报告的不稳定性提出了问题的潜力，获得一个稳定的，高水平的表达C3的转基因在体内。我们已经产生了肌肉特异性过表达全长C3或C3亚型的转基因（Tg）小鼠，这是由选择性剪接产生的，以测试C3转基因是否可以在体内稳定表达。出乎意料的是，我们发现全长C3可以在体内高水平过表达，而没有毒性。此外，我们发现，Tg表达C3缺乏外显子6，胚胎表达的同种型，具有类似再生或发育肌肉的肌肉。Tg表达C3缺乏外显子15共享这种形态在比目鱼肌，但不是其他肌肉。炎症或肌肉膜损伤的测定表明，Tg肌肉没有退化，这表明未成熟的肌肉是由发育阻滞引起的，而不是退化和再生。这些研究表明，C3可以在体内稳定表达的转基因，并表明，选择性剪接C3亚型不应用于基因治疗应用，因为它们损害了适当的肌肉发育。
Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (0). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpresslon of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development.