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.