Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors
Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors
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DOI：
10.1089/104303401750476212
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发表时间：
2001-09-20
影响因子：
4.2
通讯作者：
Bartlett, JS
中科院分区：
文献类型：
--
作者：
Shi, WF;Arnold, GS;Bartlett, JS
重组腺相关病毒（AAV）载体在基因治疗领域引起人们的关注，因为它们能够介导治疗基因在多种组织中的有效转移和稳定表达。然而，AAV 介导的基因传递到特定细胞群通常会被硫酸乙酰肝素蛋白多糖 (HSPG)（病毒的主要细胞受体）的广泛分布所阻碍。相反，越来越多的细胞类型被发现不表达 HSPG，因此不是 AAV 介导的基因转移的不良靶标。为了解决这些问题，我们制定了对 AAV 载体进行物理修饰的策略，并实现高效、不依赖 HSPG 的受体靶向感染。我们首先生成了一系列 38 个病毒衣壳突变体，其中在 AAV 衣壳蛋白内的 25 个独特位点包含肽插入。突变病毒根据其表型进行表征，分为三类：I 类突变体（38 个中的 4 个）不组装颗粒；I 类突变体（38 个中的 4 个）不组装颗粒； II 类突变体（38 个中的 14 个）组装非感染性颗粒； III 类突变体（38 个中的 20 个）组装了完全感染性的颗粒。我们检查了 II 类突变体的 HSPG 结合特征，结果表明受体结合缺陷是其缺乏感染性的常见原因。发现突变 AAV 颗粒表面上外源肽表位的展示高度依赖于适当支架序列的包含。确定了最佳支架序列和五个用于插入靶向肽表位的优选位点。这些位点位于三个 AAV 衣壳蛋白中的每一个内，因此每个载体颗粒显示插入表位 3、6 或 60 次。生成了修饰的 AAV 载体，该载体展示了 15 个氨基酸的肽，该肽与人黄体生成素受体 (LH-R) 结合，并评估了它们将基因递送至携带受体的细胞系的能力。这些突变载体的效价与野生型载体基本相同。 LH-R 靶向载体能够以不依赖 HSPG 的方式转导卵巢癌细胞 (OVCAR-3)。此外，转导被证明是通过LH-R进行的，因此用黄体酮处理OVCAR-3细胞，以增加LH-R表达，从而增加LH突变体介导的基因转移。这项技术可能会对 AAV 载体用于人类基因治疗产生重大影响。
Recombinant adeno-associated virus (AAV) vectors are of interest in the context of gene therapy because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues. However, AAV-mediated gene delivery to specific cell populations is often precluded by the widespread distribution of heparan sulfate proteoglycan (HSPG), the primary cellular receptor for the virus. Conversely, an increasing number of cell types are being identified that do not express HSPG and are therefore poor targets for AAV-mediated gene transfer. To address these issues, we have developed strategies to physically modify AAV vectors and allow efficient, HSPG-independent, receptor-targeted infection. We began by generating a series of 38 virus capsid mutants containing peptide insertions at 25 unique sites within the AAV capsid protein. The mutant viruses were characterized on the basis of their phenotypes and grouped into three classes: class I mutants (4 of 38) did not assemble particles; class II mutants (14 of 38) assembled noninfectious particles; and class III mutants (20 of 38) assembled fully infectious particles. We examined the HSPG-binding characteristics of the class II mutants and showed that a defect in receptor binding was a common reason for their lack of infectivity. The display of foreign peptide epitopes on the surface of the mutant AAV particles was found to be highly dependent on the inclusion of appropriate scaffolding sequences. Optimal scaffolding sequences and five preferred sites for the insertion of targeting peptide epitopes were identified. These sites are located within each of the three AAV capsid proteins, and thus display inserted epitopes 3, 6, or 60 times per vector particle. Modified AAV vectors displaying a 15-amino acid peptide, which binds to the human luteinizing hormone receptor (LH-R), were generated and assessed for their ability to target gene delivery to receptor-bearing cell lines. Titers of these mutant vectors were essentially the same as wild-type vector. The LH-R-targeted vector was able to transduce ovarian cancer cells (OVCAR-3) in an HSPG-independent manner. Furthermore, transduction was shown to proceed via the LH-R and therefore treatment of OVCAR-3 cells with progesterone, to increase LH-R expression, accordingly increased LH mutant-mediated gene transfer. This technology may have a significant impact on the use of AAV vectors for human gene therapy.