Generation of a Novel Muscle-Tropic AAV Serotype
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摘要: 通过在AAV5衣壳的N573后插入一个寡肽PGPSPAD生成了一种改良的AAV血清型，称为AAVc1，在体外和体内均表现出比AAV5、AAV8和AAV9更好的肌肉感染性；恒河猴血清中针对AAVc1的中和抗体（Neutralizing antibodies，Nabs）滴度低于AAV9，这表明针对AAVc1的中和抗体的免疫应答低于AAV9。结果表明，新型血清型AAVc1应用到AAV基因治疗中优于野生型AAV血清型。Abstract: The paper generated a modified AAV serotype, named AAVc1, by inserting an oligopeptide PGPSPAD after N573 of the AAV5 capsid, which exhibited better muscle tropism than AAV5, 8 and 9 both in vitro and in vivo. The number of green fluorescence protein (GFP) positive C2C12 myoblast cells in AAVc1 group was 25% higher than AAV9 group, almost twice the AAV8 group and three times of the AAV5 group. The percentage of both type I and type II skeletal muscle fibers infected by AAVc1 was also 20-30% higher than those infected by AAV9, implicating better transduction efficiency of AAVc1 in murine muscles. Furthermore, we found that the neutralizing antibodies (Nabs) present in rhesus monkey sera against AAVc1 were less than those against AAV9, indicating less immune response against AAVc1 than AAV9. Altogether, these observations illuminate potential advantages of AAVc1 over wild type AAV serotypes for gene therapy.
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Key words:
- gene therapy /
- adeno-associated virus(AAV) /
- directed evolution /
- peptide display /
- muscle tropism
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图 1 从C2C12成肌细胞中筛选AAV5突变衣壳
Figure 1. Screen of AAV5 mutant capsids from C2C12 myoblast cell
图 2 AAVc1在C2C12成肌细胞中的强大转导效率
Figure 2. Robust transduction efficiency of AAVc1 in C2C12 myoblast cell
图 3 AAVc1在小鼠肌肉中的强大转导效率
Figure 3. Robust transduction efficiency of AAVc1 in mouse muscle. (a) Representative images of Tibial anterior (TA), soleus (SO) and gastrocnemius (GA) muscles infected with AAV carrying GFP gene. (b)-(c) Quantification of transduction efficiency, GFP positive muscle ratio (b) and fluorescence intensity (c)
表 1 AAV血清型效价的定量
Table 1. Quantification of AAV titers
Serotype Titer/(vg·mL−1) AAV5 AAV8 AAV9 AAVc1 qPCR 1.09×1012 1.54×1012 8.95×1011 1.28×1012 Silver stain 1.65×1012 3.25×1012 2.31×1012 1.93×1012 Full/empty 66.06% 47.38% 38.74% 66.32% 
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表 2 猴子血清中不同AAV血清型的中和抗体检测
Table 2. Detection of neutralizing antibodies of different AAV serotypes in monkey serum
Monkey ID Anti-AAV9 Anti-AAVc1 11559 1∶32 1∶2 13061 1∶8 1∶1 13073 1∶16 1∶2 13175 1∶16 1∶8 13343 1∶2 <1∶1 13653 1∶8 1∶2 13725 1∶32 1∶8 80043 1∶1 1∶1 100039 1∶16 1∶1 12347A 1∶1 <1∶1
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