Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin
Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin
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DOI：
10.1038/mtm.2015.40
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发表时间：
2015-01-01
影响因子：
4.7
通讯作者：
Wilson, James M.
中科院分区：
文献类型：
--
作者：
Wang, Qiang;Lock, Martin;Wilson, James M.
最近基于腺相关病毒(AAV)的基因治疗的成功创造了对大规模AAV载体制造和纯化技术的需求，以用于临床试验和其他领域。在Rh纯化方案的开发过程中。10，胡。37，AAV8，rh.64R1、AAV3B和AAV9载体，基于广泛使用的亲和树脂AVB琼脂糖凝胶(GE)，我们发现在相同的条件下，不同的血清型对树脂的亲和力不同，其中AAV3B结合最好，AAV9最差。进一步分析发现，表面暴露的残基(AAV8VP1编号中的氨基酸编号为665)在高亲和力AAV血清型(AAV3B中的丝氨酸，rh)之间存在差异。10，和胡。37)和低亲和力(AAV8中的天冬酰胺，rh.64R1和AAV9)。残基位于一个表面暴露的可变表位内，两侧是高度保守的残基。AAV8，rh.表位的替换。64R1和AAV9与AAV3B的相应表位(SPAKFA)结合后，与AVB琼脂糖凝胶的亲和力显著增强，而体外效价并未降低。新鉴定的AVB结合表位的存在将有助于亲和树脂的选择，以纯化新的AAV血清型。这也表明载体工程有可能产生一种适用于多种AAV血清型的通用亲和层析纯化方法。
Recent successes of adeno-associated virus (AAV)-based gene therapy have created a demand for large-scale AAV vector -manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh. 10, hu. 37, AAV8, rh. 64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE), we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering) differs between the high-affinity AAV serotypes (serine in AAV3B, rh. 10, and hu. 37) and the low-affinity ones (asparagine in AAV8, rh. 64R1, and AAV9). The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh. 64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA) resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors' in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.