Department of Neurology, AG Gene Therapy, Berlin Institute of Health, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Univerisität zu Berlin, Berlin, Germany.
Hum Gene Ther. 2022 Sep;33(17-18):977-989. doi: 10.1089/hum.2021.182. Epub 2022 Apr 28.
Recombinant adeno-associated virus (rAAV) has become the most widely used vector in the gene therapy field with hundreds of clinical trials ongoing and already several products on the market. AAV's physicochemical stability, and the various natural and engineered serotypes allow for targeting a broad range of cell types and tissue by diverse routes of administration. Progressing from early clinical studies to eventual market approval, many critical quality attributes have to be defined and reproducibly quantified, such as AAV stability, purity, aggregates, empty/full particles ratio, and rAAV genome titration. Droplet digital PCR (ddPCR) is becoming the tool of choice to perform absolute quantification of rAAV genomes. In the present study, we have identified critical parameters that could impact AAV titration and characterization accuracy, such as Poisson distribution confidence interval, primers/probe position, and potential aggregates. Our work presents how ddPCR can help to better characterize AAV vectors on the single particle level and highlights challenges that we are facing today in terms of AAV titration.
重组腺相关病毒（rAAV）已成为基因治疗领域中应用最广泛的载体，目前有数百项临床试验正在进行，已有多种产品上市。AAV 的物理化学稳定性，以及各种天然和工程化的血清型，允许通过多种给药途径靶向广泛的细胞类型和组织。从早期的临床研究到最终的市场批准，许多关键的质量属性必须被定义和可重复地量化，如 AAV 的稳定性、纯度、聚集物、空/满颗粒比和 rAAV 基因组滴定度。液滴数字 PCR（ddPCR）正在成为进行 rAAV 基因组绝对定量的首选工具。在本研究中，我们确定了可能影响 AAV 滴定和特性准确性的关键参数，如泊松分布置信区间、引物/探针位置和潜在的聚集物。我们的工作展示了 ddPCR 如何帮助更好地在单个颗粒水平上表征 AAV 载体，并强调了我们在 AAV 滴定方面目前面临的挑战。
