Department of Ophthalmology, University Hospital, LMU Munich, Mathildenstr. 8, 80336 Munich, Germany.
Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany.
Cells. 2022 Jun 9;11(12):1881. doi: 10.3390/cells11121881.
AAV vectors are widely used in gene therapy, but the prevalence of neutralizing antibodies raised against AAV serotypes in the course of a natural infection, as well as innate and adaptive immune responses induced upon vector administration, is still considered an important limitation. In ocular gene therapy, vectors applied subretinally bear the risk of retinal detachment or vascular leakage. Therefore, new AAV vectors that are suitable for intravitreal administration for photoreceptor transduction were developed.
Here, we compared human immune responses from donors with suspected previous AAV2 infections to the new vectors AAV2.GL and AAV2.NN-two capsid peptide display variants with an enhanced tropism for photoreceptors-with the parental serotype AAV2 (AAV2 WT). We investigated total and neutralizing antibodies, adaptive and innate cellular immunogenicity determined by immunofluorescence staining and flow cytometry, and cytokine secretion analyzed with multiplex beads.
While we did not observe obvious differences in overall antibody binding, variants-particularly AAV2.GL-were less sensitive to neutralizing antibodies than the AAV2 WT. The novel variants did not differ from AAV2 WT in cellular immune responses and cytokine production in vitro.
Due to their enhanced retinal tropism, which allows for dose reduction, the new vector variants are likely to be less immunogenic for gene therapy than the parental AAV2 vector.
腺相关病毒 (AAV) 载体被广泛应用于基因治疗，但在自然感染过程中针对 AAV 血清型产生的中和抗体的流行率，以及载体给药后诱导的先天和适应性免疫反应，仍被认为是一个重要的限制因素。在眼部基因治疗中，视网膜下应用的载体有发生视网膜脱离或血管渗漏的风险。因此，开发了新的适合玻璃体内给药以进行光感受器转导的 AAV 载体。
在这里，我们比较了疑似先前 AAV2 感染的供体的人类免疫反应与新载体 AAV2.GL 和 AAV2.NN（两种对光感受器具有增强亲嗜性的衣壳肽展示变体）与亲本血清型 AAV2（AAV2 WT）。我们通过免疫荧光染色和流式细胞术检测总抗体和中和抗体、适应性和先天细胞免疫原性，并通过多重珠分析细胞因子分泌。
虽然我们没有观察到总抗体结合的明显差异，但与 AAV2 WT 相比，变体（尤其是 AAV2.GL）对中和抗体的敏感性较低。新型变体与 AAV2 WT 在体外的细胞免疫反应和细胞因子产生方面没有差异。
由于其增强的视网膜亲嗜性，允许减少剂量，新的载体变体在基因治疗方面的免疫原性可能低于亲本 AAV2 载体。
