Clinical Laboratory, Center for Clinical Studies, Department of Clinical Diagnostics and Services, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland.
Viruses. 2021 Aug 18;13(8):1636. doi: 10.3390/v13081636.
Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/Cas9-assisted gene therapy, the cat's immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/Cas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/Cas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved and regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/Cas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.
猫白血病病毒（FeLV）是一种在世界范围内感染猫的逆转录病毒。高病毒载量与进行性感染和宿主死亡有关，这是由 FeLV 相关疾病引起的。相比之下，在具有退行性感染的猫中，可以观察到低病毒载量、有效的免疫反应和更好的临床结果。我们假设，通过使用 CRISPR/Cas9 辅助基因治疗降低进行性感染猫中的病毒载量，猫的免疫系统可能会允许将感染引导向退行性结果。为此，本研究评估了不同的腺相关病毒（AAV）在将基因编辑系统递送到猫细胞中的能力，随后研究了 CRISPR/Cas9 对 FeLV 前病毒内不同位点的靶向效率。测试了 9 种天然 AAV 血清型、2 种 AAV 杂交株和 Anc80L65（一种计算机预测的 AAV 祖先），以评估它们感染不同猫细胞系和猫原代细胞的潜力。AAV-DJ 显示出优越的感染效率，因此在随后的转导实验中使用。通过 T7 内切酶 1（T7E1）以及通过分解（TIDE）分析跟踪插入/缺失证实了使用靶向 12 个选定 FeLV 前病毒位点的 CRISPR/Cas9 系统引入双链断裂。在高度保守的 和 区域中发现了高达 80%的非同源末端连接（NHEJ）的最高百分比。使用 AAV-DJ 的后续转导实验证实了缺失形成，并显示一些靶标中的 FeLV p27 抗原显著减少。体外使用 CRISPR/Cas9 方法靶向 FeLV 前病毒是有效的。是否观察到的前病毒靶向程度足以使患有进行性 FeLV 感染的猫获得克服感染的手段，需要进一步在体内进行研究。
