Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.
Gene Ther. 2023 Aug;30(7-8):575-580. doi: 10.1038/s41434-021-00301-6. Epub 2021 Nov 8.
Immune responses to Cas proteins have been demonstrated recently and these may prove to be an impediment to their clinical use in gene editing. To make meaningful assessments of Cas9 immunogenicity during drug development and licensure it is imperative the reagents are free of impurities that could affect in vitro assessments of immunogenicity. Here we address the issue of endotoxin levels in laboratory grade Cas9 proteins used to measure T-cell memory responses. Many of these reagents have not been developed for immunogenicity assays, are or microbial origin and carry varying levels of endotoxin. The use of these reagents, off the shelf, without measuring endotoxin levels is likely to introduce incorrect estimates of the prevalence of memory T-cell responses in research studies. We demonstrate wide variation in endotoxin levels in Cas9 proteins from seven suppliers. Different lots from the same supplier also contained varying levels of endotoxin. ELISPOT assays showed similar large variations in the interferon-γ signals. Finally, when we carried out endotoxin depletion in four Cas9 proteins with strong signals in the ELISPOT assay, we found dampening of the interferon-γ signals.
最近已经证明了对 Cas 蛋白的免疫反应，这可能会成为其在基因编辑临床应用中的障碍。为了在药物开发和许可过程中对 Cas9 的免疫原性进行有意义的评估，至关重要的是试剂中不含可能影响体外免疫原性评估的杂质。在这里，我们解决了用于测量 T 细胞记忆反应的实验室级 Cas9 蛋白中的内毒素水平问题。这些试剂中的许多都不是为免疫原性测定而开发的，它们可能来自微生物，并且带有不同水平的内毒素。在没有测量内毒素水平的情况下，使用这些现成的试剂，可能会导致研究中对记忆 T 细胞反应的流行率的估计不正确。我们展示了来自 7 个供应商的 Cas9 蛋白中内毒素水平的广泛差异。同一供应商的不同批次也含有不同水平的内毒素。ELISPOT 检测显示干扰素-γ信号存在类似的大差异。最后，当我们在 ELISPOT 检测中具有强烈信号的 4 种 Cas9 蛋白中进行内毒素耗尽时，我们发现干扰素-γ信号减弱。
