Baatartsogt Nemekhbayar, Kashiwakura Yuji, Hayakawa Morisada, Kamoshita Nobuhiko, Hiramoto Takafumi, Mizukami Hiroaki, Ohmori Tsukasa
Department of Biochemistry, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan.
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan.
Mol Ther Methods Clin Dev. 2021 Jun 12;22:162-171. doi: 10.1016/j.omtm.2021.06.004. eCollection 2021 Sep 10.
Most gene therapy clinical trials that systemically administered adeno-associated virus (AAV) vector enrolled only patients without anti-AAV-neutralizing antibodies. However, laboratory tests to measure neutralizing antibodies varied among clinical trials and have not been standardized. In this study, we attempted to improve the sensitivity and reproducibility of a cell-based assay to detect neutralizing antibodies and to determine the detection threshold to predict treatment efficacy. Application of the secreted type of NanoLuc and AAV receptor-expressing cells reduced the multiplicity of infection (MOI) for AAV transduction and improved the sensitivity to detect neutralizing antibodies with a low coefficient of variation, whereas the detection threshold could not be improved by the reduction of MOI to <100. After human immunoglobulin administration into mice at various doses, treatment with high-dose AAV8 vector enabled evasion of the inhibitory effect of neutralizing antibodies. Conversely, gene transduction was slightly influenced in the mice treated with low-dose AAV8 vector, even when neutralizing antibodies were determined to be negative in the assay. In conclusion, we developed a reliable and sensitive cell-based assay to measure neutralizing antibodies against AAV and found that the appropriate MOI to detect marginal neutralizing antibodies was 100. Other factors, including noninhibitory antibodies, marginally influence transduction at low vector doses.
大多数系统性给予腺相关病毒（AAV）载体的基因治疗临床试验仅纳入没有抗AAV中和抗体的患者。然而，测量中和抗体的实验室检测在各临床试验中存在差异，且尚未标准化。在本研究中，我们试图提高基于细胞的检测方法检测中和抗体的灵敏度和可重复性，并确定预测治疗效果的检测阈值。分泌型纳米荧光素酶（NanoLuc）和表达AAV受体的细胞的应用降低了AAV转导的感染复数（MOI），并提高了检测中和抗体的灵敏度，变异系数较低，而将MOI降低至<100并不能提高检测阈值。在以不同剂量给小鼠注射人免疫球蛋白后，高剂量AAV8载体治疗能够规避中和抗体的抑制作用。相反，即使在检测中确定中和抗体为阴性，低剂量AAV8载体治疗的小鼠的基因转导也受到轻微影响。总之，我们开发了一种可靠且灵敏的基于细胞的检测方法来测量抗AAV中和抗体，并发现检测边缘中和抗体的合适MOI为100。包括非抑制性抗体在内的其他因素在低载体剂量下对转导有轻微影响。
