Development of an Enzyme-Linked Immunosorbent Spot Assay for the Assessment of Adeno-Associated Virus Peptides to Examine Immune Safety.

IF 3.9 3区 医学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Human gene therapy Pub Date : 2024-07-01 Epub Date: 2024-02-22 DOI:10.1089/hum.2023.180
Sara Rose Krivoshik, Lindsey Dzielak, April R Masters, Jennifer Hall, Alison J Johnson
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Abstract

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.

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开发用于评估 AAV 多肽的 ELISpot 检测法,以检查免疫安全性。
基于腺相关病毒(AAV)的基因疗法有望成为治疗血友病 A 和脊髓性肌肉萎缩症等罕见遗传疾病的新型疗法。然而,由细胞毒性(CD8+)和辅助性(CD4+)T 细胞介导的细胞免疫反应可能会针对载体转导的细胞以及健康的免疫细胞,从而影响安全性和疗效。在这里,我们描述了基于干扰素-γ(IFNγ)和白细胞介素-2(IL-2)的酶联免疫吸附点(ELISpot)测定法的优化和可重复性,该测定法用于测量针对 AAV 多肽抗原的 T 细胞反应。为优化方法,从健康人供体中分离出外周血单核细胞(PBMC),并用市售的主要组织相容性复合体(MHC)I 类或 II 类特异性肽作为阳性对照进行刺激。根据已发表的 AAV8 和 AAV9 包膜蛋白序列设计肽池,然后用于评估 AAV 特异性 T 细胞反应的存在。我们的研究结果表明,AAV 多肽呈现后,IFNγ 和 IL-2 生成细胞明显增加。此外,根据 AAV 血清型和供体的不同,肽池反应的程度和特异性也存在差异。最后,通过使用单个肽,我们确定了 AAV9 帽状蛋白中能引起免疫原性反应的区域。这项工作显示了 ELISpot 在评估抗 AAV 免疫反应方面的适用性,并为如何设计新型重组 AAV 载体以降低免疫原性提供了启示。
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来源期刊
Human gene therapy
Human gene therapy 医学-生物工程与应用微生物
CiteScore
6.50
自引率
4.80%
发文量
131
审稿时长
4-8 weeks
期刊介绍: Human Gene Therapy is the premier, multidisciplinary journal covering all aspects of gene therapy. The Journal publishes in-depth coverage of DNA, RNA, and cell therapies by delivering the latest breakthroughs in research and technologies. Human Gene Therapy provides a central forum for scientific and clinical information, including ethical, legal, regulatory, social, and commercial issues, which enables the advancement and progress of therapeutic procedures leading to improved patient outcomes, and ultimately, to curing diseases.
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