Preassembled complexes of hAgo2 and ssRNA delivered by nanoparticles: a novel silencing gene expression approach overcoming the absence of the canonical pathway of siRNA processing in the apicomplexan parasite Babesia microti, blood parasite of veterinary and zoonotic importance

Due to the lack of efficacy of the currently used chemical drugs, poor tick control, and lack of effective vaccines against <i>Babesia</i>, novel control strategies are urgently needed. In this regard, searching for anti-<i>Babesia</i> gene therapy may facilitate the control of this infection. Following this pattern, small interfering RNAs (siRNAs) are widely used to study gene function and hence open the way to control the parasite. However, the primary constraint of this approach is the lack of <i>Babesia</i> to RNA-induced silencing complex (RISC) enzymes, making siRNA impractical. In this study, we preassembled complexes with the human enzyme argonaute 2 (hAgo2) and a small interfering RNA (siRNA)<i>/</i>single-stranded RNA (ssRNA) against <i>B. gibsoni</i> and <i>B. microti</i> metabolite transporters. The assembled complexes were generated by developing a gene delivery system with chitosan dehydroascorbic acid nanoparticles. The delivery system effectively protected the loaded RNAi and targeted <i>Babesia-</i>infected RBCs with a relatively high internalization rate. The assembled complexes were successfully transfected into live parasites for specific slicing of <i>Babesia</i> targets. We demonstrated a reduction in the expression of target genes at the mRNA level. Furthermore, this silencing inhibited <i>Babesia</i> growth <i>in vitro</i> and <i>in vivo</i>. For the first time, we used this method to confirm the role of the assembled complexes in manipulating the noncanonical pathway of RNAi in <i>Babesia</i> parasites. This novel method provides a means of silencing <i>Babesia</i> genes to study their role in host–parasite interactions and as potential targets for gene therapy and control.

鉴于当前临床使用的化学药物疗效不足、蜱虫防控效果欠佳，且尚无针对巴贝斯虫（<i>Babesia</i>）的有效疫苗，因此亟需开发新型防控策略。有鉴于此，开发抗巴贝斯虫基因疗法或可助力该感染的防控工作。基于此思路，小干扰RNA（small interfering RNAs, siRNAs）被广泛应用于基因功能研究，从而为寄生虫防控开辟了新路径。然而该方法的核心局限在于：巴贝斯虫缺乏RNA诱导沉默复合体（RNA-induced silencing complex, RISC）相关酶类，导致siRNA技术难以实际应用。本研究中，我们针对吉布森巴贝斯虫（<i>B. gibsoni</i>）与微小巴贝斯虫（<i>B. microti</i>）的代谢物转运蛋白，预先组装了由人类Argonaute 2蛋白（hAgo2）与小干扰RNA（siRNA）/单链RNA（ssRNA）构成的复合体。我们通过构建壳聚糖-脱氢抗坏血酸纳米粒基因递送系统，成功制备了上述组装复合体。该递送系统可有效保护负载的RNAi分子，并以较高的内化效率靶向感染巴贝斯虫的红细胞（red blood cells, RBCs）。上述组装复合体可成功转染活的巴贝斯虫，实现靶标基因的特异性剪切。研究证实，靶标基因的mRNA水平表达量显著下调。此外，该基因沉默可在体外（<i>in vitro</i>）与体内（<i>in vivo</i>）均抑制巴贝斯虫的增殖。本研究首次利用该方法，证实了组装复合体可调控巴贝斯虫体内非经典RNAi通路的作用。该新型方法为巴贝斯虫基因沉默研究提供了技术手段，可用于解析巴贝斯虫与宿主的互作机制，同时也可为基因治疗与防控策略开发提供潜在靶标。