Digital Biology Group, Bio-Rad Laboratories, Inc., Pleasanton, California, United States of America.
PLoS One. 2023 Jan 25;18(1):e0280242. doi: 10.1371/journal.pone.0280242. eCollection 2023.
Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.
精确、可重现的 AAV 特征描述对于比较实验室间的临床前结果以及确定基因治疗应用的安全有效临床剂量至关重要。在这项研究中，我们系统地评估了许多参数，以制定一种简单而稳健的 ddPCR 方案来进行 AAV 特征描述。该方案使用含有 Pluronic-F68 和聚腺苷酸的低离子强度缓冲液将 AAV 稀释到 ddPCR 浓度范围内，并在进行包含 MspI 的 ddPCR 反应组装之前，将热衣壳裂解 10 分钟。一个关键发现是，缓冲液组成会影响 AAV 的 ITR 浓度，但不会影响双链质粒的 ITR 浓度，这在使用理论的、化学计量转换因子根据 ITR 浓度获得滴度时会产生影响。使用该方案，通过在整个 AAV 载体基因组中分布多个 ddPCR 检测，可以对 AAV 载体制剂进行更全面的分析。这些检测扩增 ITR、调控元件和 eGFP 转基因，以更有信心地估计载体基因组浓度，并对载体基因组身份进行高分辨率特征描述。此外，我们比较了两种基因组完整性分析方法，用于分析三种对照样本类型在每个样本的八个不同浓度下的情况。基因组完整性与样本浓度无关，当基于两个 DNA 靶标偶然共包封的双阳性液滴预期数量与阳性液滴的多余数量确定完整性时，可以获得预期值。基因组完整性高度可变，当使用双阳性液滴百分比计算基因组完整性时，会产生意外值。在组装 ddPCR 反应之前使用一分钟热衣壳裂解的方案，在缺乏限制酶的情况下使用非 ITR 检测，通过连锁分析在 ddPCR 里程碑实验中以双检测形式确定基因组完整性。
