Structurally Mapping Antigenic Epitopes of Adeno-associated Virus 9: Development of Antibody Escape Variants
Structurally Mapping Antigenic Epitopes of Adeno-associated Virus 9: Development of Antibody Escape Variants
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DOI：
10.1128/jvi.01251-21
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发表时间：
2022-02-01
影响因子：
5.4
通讯作者：
Agbandje-McKenna, Mavis
中科院分区：
文献类型：
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作者：
Emmanuel, Shanan N.;Smith, J. Kennon;Agbandje-McKenna, Mavis
腺相关病毒（AAV）用作治疗性基因递送的载体。AAV 9载体已经被FDA批准为Zolgensma，用于治疗脊髓性肌萎缩症，并且正在临床试验中评估用于治疗亲神经性和亲肌肉性疾病。AAV介导的基因递送的一个主要障碍是在40%至80%的一般人群中存在预先存在的中和抗体。这些预先存在的抗体可以通过病毒中和和适合治疗的患者队列的大小来降低治疗功效。在本研究中，使用冷冻电子显微镜和图像重建来确定五种抗AAV 9单克隆抗体（MAb）（ADK 9、HL 2368、HL 2370、HL 2372和HL 2374）在衣壳表面上的表位。其中三个，ADK 9，HL 2370和HL 2374，结合到或接近二十面体3重轴，HL 2368结合到2/5重壁，HL 2372结合到5重轴周围的区域。伪原子建模使得能够映射和鉴定衣壳上的抗体接触氨基酸，包括S454和P659。这些表位与先前定义的细小病毒抗原位点重叠。对相互作用至关重要的衣壳氨基酸通过诱变来确认，随后通过生物化学测定来测试能够逃避亲本MAb的识别和中和的重组AAV 9（rAAV 9）变体。这些变体保留了亲本向性，并且与AAV 9相比具有相似或改善的转导效率。这些工程改造的rAAV 9变体可以通过避免预先存在的循环中和抗体来扩大适合AAV 9介导的基因递送的患者群体。使用重组腺相关病毒（rAAV）作为治疗基因的递送载体变得越来越流行，特别是在FDA批准分别基于血清型AAV 2和AAV 9的Luxturna和Zolgensma之后。然而，一般人群中的高滴度抗AAV中和抗体使患者免于治疗。本研究的目的是通过创建不被预先存在的中和抗体识别的AAV变体载体来规避这个问题。在AAV 9上绘制五种不同单克隆抗体（MAb）的抗原表位以概括多克隆应答，使得能够合理设计逃逸变体，同时对细胞嗜性和基因表达的破坏最小。这项研究，其中包括四个新开发的，现在可商购的单克隆抗体，提供了一个平台的rAAV 9载体的工程，可用于提供基因与预先存在的AAV抗体的患者。
Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma, for the treatment of spinal muscular atrophy and are being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of preexisting neutralizing antibodies in 40 to 80% of the general population. These preexisting antibodies can reduce therapeutic efficacy through viral neutralization and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction were used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs), ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound to or near the icosahedral 3-fold axes, HL2368 bound to the 2/5-fold wall, and HL2372 bound to the region surrounding the 5fold axes. Pseudoatomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis, followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding preexisting circulating neutralizing antibodies. IMPORTANCE The use of recombinant adeno-associated viruses (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna and Zolgensma, based on serotypes AAV2 and AAV9, respectively. However, high-titer anti-AAV neutralizing antibodies in the general population exempt patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by preexisting neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with preexisting AAV antibodies.