College of Veterinary Medicine, Chonbuk National University, Iksan Campus, 54596, Iksan, Republic of Korea.
Vet Res. 2021 Sep 30;52(1):125. doi: 10.1186/s13567-021-00995-x.
Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6 day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.
有效的 CRISPR/Cas9 质粒体内递送对于有效的治疗至关重要。在这里，我们研究了沙门氏菌作为质粒载体在体内治疗病毒诱导癌症中的可用性，使用马立克氏病病毒（MDV）作为鸡研究模型。构建了一种表达绿色荧光蛋白的 CRISPR/Cas9 质粒，针对 Marek's 病病毒的毒力基因 pp38。将治疗性质粒转化为携带 lon 和 sifA 基因缺失的沙门氏菌。将 5 组动物用磷酸盐缓冲盐水、载体对照或 MDV 感染前后的沙门氏菌或未感染的鸡作为对照进行腹腔内接种。通过观察疾病结果和外周血单核细胞中的病毒拷贝数来评估治疗效果。沙门氏菌递送质粒的效力在感染后 6 天为脾脏中的 13±1.7%和肝脏中的 8.0±1.8%。沙门氏菌处理组对 MDV 感染表现出显著的抗性。在 MDV 感染前用沙门氏菌处理的组中观察到最大效果。没有一只鸡完全康复；然而，结果表明，及时递送沙门氏菌可能对体内 CRISPR/Cas9 介导的针对高致病性 MDV 的遗传干扰有效。沙门氏菌在 CRISPR 系统中的使用为体内治疗提供了比传统体内基因递送方法更简单、更有效的平台，值得进一步发展。
