Schmit Pauline F, Pacouret Simon, Zinn Eric, Telford Elizabeth, Nicolaou Fotini, Broucque Frédéric, Andres-Mateos Eva, Xiao Ru, Penaud-Budloo Magalie, Bouzelha Mohammed, Jaulin Nicolas, Adjali Oumeya, Ayuso Eduard, Vandenberghe Luk H
Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA 02114, USA.
INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France.
Mol Ther Methods Clin Dev. 2019 Nov 26;17:107-121. doi: 10.1016/j.omtm.2019.11.014. eCollection 2020 Jun 12.
Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.
腺相关病毒（AAV）变体文库的构建和筛选已成为一种强大的方法，用于鉴定基因治疗应用中的新型衣壳。对于大多数文库而言，庞大的群体多样性需要多重生产，即将包含反向末端重复序列（ITR）的质粒变体文库一起转染到细胞中以产生病毒文库。这个过程可能会受到交叉包装和嵌合现象的干扰，即病毒颗粒由来自不同文库成员的基因组和衣壳单体组成。在这里，我们使用旨在评估衣壳组成和包装保真度的新型检测方法，研究了简化的最小文库中交叉包装和嵌合现象的发生率。我们发现，当以高质粒水平产生时，AAV文库变体容易出现交叉包装和衣壳嵌合形成，尽管程度低于重组环境。我们还提供了实验证据，表明输入文库DNA的稀释显著提高了衣壳单体的同质性，并增加了AAV文库中衣壳与基因组的相关性。最后，我们确定，类似的稀释方法用于筛选时可产生更高质量的文库。总之，这些发现定量地描述了AAV文库中交叉包装和嵌合现象的发生率，并建立了在后续筛选中最小化相关噪声的条件。
