Bustamante-Jaramillo Luisa F, Yue Lei, Fingal Joshua, Rydell Gustaf, Johansson Maria, Edreira Tomas, Müller Oliver J, Hille Susanne, Müller Martin, Gallardo Franck, Chen Qingxin, Blondot Marie-Lise, Kann Michael
Department of Infectious Diseases, Institute of Biomedicine, Gothenburg University, Gothenburg, Sweden.
Department of Chemistry and Molecular Biology, Gothenburg University, Gothenburg, Sweden.
iScience. 2025 May 8;28(6):112624. doi: 10.1016/j.isci.2025.112624. eCollection 2025 Jun 20.
Adeno-associated virus (AAVs) with a self-complementary genome (sc) comprising a gene of interest are used in gene therapy. Their efficiency is limited but the molecular factors contributing to this restriction are poorly understood. We utilized scAAV2 containing a fluorescent protein-binding anchor sequence on its genome allowing visualization of released genomes by time-lapse microscopy. Pairing this technique with capsid staining, we showed that scAAV2 genome release was initiated by a partial genome exposure, triggered by elevated calcium levels while the capsids interacted with proteins of the nuclear pore. Genome release occurred subsequently requiring Rad52 decamerization in the vicinity of the host chromatin. A fraction of the released genomes was degraded by Mre11, an essential factor for chromatin stability, and cellular DNA double-strand breaks. These steps were key-factors limiting transduction, suggesting that temporary modulation of DNA damage-response-proteins is a promising way to increase scAAV efficiency in therapy.
具有包含感兴趣基因的自我互补基因组（sc）的腺相关病毒（AAV）被用于基因治疗。它们的效率有限，但导致这种限制的分子因素却知之甚少。我们利用了scAAV2，其基因组上含有荧光蛋白结合锚定序列，通过延时显微镜观察可以看到释放的基因组。将该技术与衣壳染色相结合，我们发现scAAV2基因组的释放是由部分基因组暴露引发的，这是由钙水平升高触发的，同时衣壳与核孔蛋白相互作用。随后发生基因组释放，这需要在宿主染色质附近的Rad52解聚。一部分释放的基因组被Mre11降解，Mre11是染色质稳定性和细胞DNA双链断裂的必需因子。这些步骤是限制转导的关键因素，这表明临时调节DNA损伤反应蛋白是提高scAAV治疗效率的一种有前景的方法。
