Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.
Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.
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神经氨酸酶介导的去唾液酸化增强了骨骼肌中 AAV9 介导的基因表达。
DOI：
10.1002/jgm.3049
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Annex,BrianH
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文献类型：
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作者：
Zhu,Hongling;Wang,Tao;JohnLye,Robert;French,BrentA;Annex,BrianH
在全身递送后，AAV 9介导的基因表达在缺血性肌肉中与非缺血性肌肉相比显著增加，这表明AAV 9是治疗外周动脉疾病的有吸引力的载体。缺血增强表达的潜在机制包括：（i）血管通透性增加和（ii）内源性AAV 9受体的“暴露”。在本研究中，我们的目的是重建AAV 9在体内的缺血诱导，使用局部注射组胺（以增加血管通透性）和神经氨酸酶（去唾液酸化细胞表面聚糖）MethodsBioassays进行肌肉注射后，组胺和神经氨酸酶的效果进行优化。然后在静脉内注射表达荧光素酶的AAV 9载体之前不久肌内注射组胺和/或神经氨酸酶。用生物发光成像连续评估荧光素酶表达。在研究结束时，组织收获荧光素酶活性和AAV 9基因组拷贝数的测定，旨在评估AAV介导的基因表达和transduction，respectively.ResultsIntramuscular injection的神经氨酸酶或神经氨酸酶加组胺显着增加转导和基因表达，而组胺单独几乎没有影响。预先注射神经氨酸酶可使AAV 9介导的基因递送增加4 - 9倍，荧光素酶活性增加60-100倍。注射神经氨酸酶的肌肉中的荧光素酶活性比任何脱靶组织（包括心脏、肝脏和脑）中的荧光素酶活性高> 100倍。这种策略可能在未来的人类基因治疗方案中被证明是有用的，作为一种快速有效的手段，选择性地将全身注射的AAV 9靶向肌肉的局部区域，从而降低脱靶组织中不良反应的可能性。
BackgroundFollowing systemic delivery, AAV9‐mediated gene expression is significantly increased in ischemic versus non‐ischemic muscle, suggesting that AAV9 is an attractive vector for treating peripheral arterial disease. Potential mechanisms underlying ischemia‐augmented expression include: (i) increased vascular permeability and (ii) “unmasking” of endogenous AAV9 receptors. In the present study, we aimed to reconstitute the ischemic induction of AAV9 in vivo, using local injection of histamine (to increase vascular permeability) and neuraminidase (to desialylate cell surface glycans).MethodsBioassays were performed to optimize the effects of histamine and neuraminidase after intramuscular injection. Histamine and/or neuraminidase were then injected intramuscularly shortly before intravenous injection of an AAV9 vector expressing luciferase. Luciferase expression was serially assessed with bioluminescence imaging. At the end of the study, tissues were harvested for assays of luciferase activity and AAV9 genome copy number aiming to assess AAV‐mediated gene expression and transduction, respectively.ResultsIntramuscular injection of either neuraminidase or neuraminidase plus histamine significantly increased both transduction and gene expression, whereas histamine alone had little effect. Pre‐injection with neuraminidase increased AAV9‐mediated gene delivery by four‐ to nine‐fold and luciferase activity by 60–100‐fold. Luciferase activity in neuraminidase‐injected muscle was > 100‐fold higher than in any off‐target tissue (including heart, liver and brain).ConclusionsThe ischemic induction of AAV9‐mediated gene expression in muscle can largely be reconstituted by pre‐injecting neuraminidase intranmuscularly. This strategy may prove useful in future human gene therapy protocols as a quick and efficient means to selectively target systemically injected AAV9 to localized regions of muscle, thus decreasing the potential for adverse effects in off‐target tissues.