RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism
RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism
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DOI：
10.1016/s1525-0016(03)00042-x
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发表时间：
2003-04-01
影响因子：
12.4
通讯作者：
Bartlett, JS
中科院分区：
文献类型：
--
作者：
Shi, WF;Bartlett, JS
重组腺相关病毒(AAV)已成为一种具有吸引力的载体系统，可用于多种基因治疗方案。然而，AAV载体的应用往往受到病毒主要附着受体--硫酸乙酰肝素蛋白多糖(HSPG)在所需靶细胞群上缺失的限制。为了实现HSPG非依赖的基因传递，几个研究小组已经证明，AAV的内源性趋向性可以通过改变病毒衣壳的基因来扩大。然而，这项正在开发的技术的参数还没有确定，也还没有确定这些修改后的载体是否真的通过这些工程相互作用感染细胞。在此之前，我们构建了一系列跨越AAV衣壳蛋白基因的插入突变体，并鉴定了可以耐受外源小肽插入的特定位点。在这里，我们描述了AAV衣壳基因中可用于插入整合素靶向多肽表位的一些位置。在这些位点上掺入含有Arg-Gly-Asp(RGD)的多肽使AAV能够独立于HSPG感染整合素表达细胞。显示这些多肽配体的突变型AAV载体可以生产出野生型滴度，并已被证明与靶向整合素受体特异性地相互作用，并通过这种相互作用介导感染。在Raji、K562和SKOV-3细胞中，表达整合素但不表达HSPG的细胞株的基因转移率显著增加，提示表达RGD多肽的rAAV载体可能在治疗以HSPG表达缺失为特征的肿瘤方面有很大的应用价值。我们还证明，由于它们的扩向性，这些新的载体能够在体内有效地转导AAV2耐药的肿瘤，这表明它们可能提供显著的治疗优势。
Recombinant adeno-associated virus (AAV) has become an attractive vector system for a number of gene therapy paradigms. However, the utility of AAV vectors is often limited by the absence of heparan sulfate proteoglycan (HSPG), the virus's primary attachment receptor, on the desired target cell population. In order to achieve HSPG-independent gene delivery, several groups have shown that the endogenous tropism of AAV can be expand by genetically altering the viral capsid. However, the parameters of this developing technology have yet to be defined and it has not yet been determined if these modified vectors actually infect cells via these engineered interactions. Previously we constructed a series of insertion mutants spanning the AAV capsid protein gene and identified specific sites that can tolerate the insertion of small exogenous peptides. Here we describe a number of sites within the AAV capsid gene that can be used for the insertion of integrin-targeting peptide epitopes. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide at these sites enables AAV to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying these peptide ligands can be produced to wild-type titer and have been shown to specifically interact with the targeted integrin receptors and mediate infection via this interaction. We report significant increases in gene transfer to Raji, K562, and SKOV-3 cell lines that express integrin, but little HSPG, suggesting that rAAV vectors displaying RGD peptides may be of great utility for treatment of neoplasms characterized by the deficiency of HSPG expression. We have also demonstrated that due to their expanded tropism, these novel vectors are capable of efficient transduction of AAV2-resistant tumors in vivo suggesting that they may offer significant therapeutic advantages.