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. 2022 Jul 21;90(7):e0019622.
doi: 10.1128/iai.00196-22. Epub 2022 Jun 1.

RNA-Based Therapy for Cryptosporidium parvum Infection: Proof-of-Concept Studies

A Castellanos-Gonzalez  1 , A Sadiqova  1 , J Ortega-Mendez  1 , A C White Jr  1
Affiliations

RNA-Based Therapy for Cryptosporidium parvum Infection: Proof-of-Concept Studies

A Castellanos-Gonzalez et al. Infect Immun. .

Abstract

Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children, which is one of the major causes of death in children under 5 years old. Nitazoxanide is the only FDA-approved treatment for cryptosporidiosis. However, it has limited efficacy in immunosuppressed patients and malnourished children. Therefore, it is urgent to develop novel therapies against this parasite. RNA interference-mediated therapies are emerging as novel approaches for the treatment of infectious diseases. We have developed a novel method to silence essential genes in Cryptosporidium using single-stranded RNA (ssRNA)/Argonaute (Ago) complexes. In this work we conducted proof-of-concept studies to test the anticryptosporidial activity of these complexes by silencing Cryptosporidium parvum nucleoside diphosphate kinase (NDK) using in vitro and in vivo models. We demonstrated that a 3-day treatment with anti-sense NDK ssRNA/Ago decreased parasite burden by ~98% on infected cells. In vivo studies showed that ssRNA/Ago complexes encapsulated in lipid nanoparticles can be delivered onto intestinal epithelial cells of mice treated orally. In addition a cryptosporidiosis-mouse model showed that treatment with NDK ssRNA/Ago complexes reduced oocyst shedding in 4/5 SCID/beige mice during the acute phase of the infection. Our findings highlight the potential use of antisense RNA-based therapy as an alternative approach to cryptosporidiosis treatment.

Keywords: Argonaute; Cryptosporidium; NDK; cryptosporidiosis; gene silencing; siRNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
NDK silencing in Cryptosporidium. Oocysts were treated with sense NDK ssRNA (dark gray), antisense NDK ssRNA (white), sense NDK ssRNA/Ago (diagonal lines), and antisense NDK ssRNA/Ago (light gray). Samples were analyzed by qRT-PCR, and NDK mRNA expression was normalized compared to Cryptosporidium GAPDH and NDK expression compared to untreated controls using the ΔΔCT method. (*, P ≤ 0.05; **, P ≤ 0.001).
FIG 2
FIG 2
NDK ssRNA/Ago complexes localize to infected cells. HCT-8 cells were infected with fluorescent Cryptosporidium prestained with the vital dye CFSE (green). After 24 h of infection, cells were transfected with Cy-5 labeled ssRNA (red)/Ago complexes. (A) For fluorescence microscopy, HCT-8 cells were counterstained with DAPI (blue). White arrows show the localization of intracellular parasites. Colocalization is demonstrated with ssRNA-Cy5 (naked) and Ago/ssRNA-Cy5 (complexes). Scale bar = 40 μm. (B) For confocal microscopy, HCT-8 cells cultured on coverslips were treated as before. Nuclei were stained with DAPI (white asterisk [*]). The white arrow points to an intracellular parasite, which was transfected with ssRNA/Ago complexes. Scale bar = 5 μm.
FIG 3
FIG 3
In vitro anticryptosporidial activity of ssRNA/Ago by fluorescent quantitative microscopy. (A) Schematic illustration of anticryptosporidial assay. HCT-8 cells were infected with fluorescent-labeled Cryptosporidium for 2 h. After infection, cells were treated with anti-NDK ssRNA/Ago, sense-ssNDK/Ago (control), or untreated (Fig. S2A). (B) For fluorescence microscopy, HCT-8 cells were counterstained with propidium iodide (red), and parasites stained with vital dye CFSE (green). (C and D) After treatment, cells were fixed at 0 h and 24 h, and then the total number of parasites was evaluated by fluorescent quantitative microscopy (Fig. S2B).
FIG 4
FIG 4
Anticryptosporidial activity of ssRNA/Ago by qRT-PCR. HCT-8 cells were infected with Cryptosporidium. After 24 h, infected cells were treated daily with anti-NDK ssRNA (gray bar) or anti-NDK ssRNA/Ago complexes (black bars) or were mock-infected (white bars). The total number of parasites was calculated by qRT-PCR; results of triplicates are expressed as the percentage change from baseline. (*, P ≤ 0.05; **, P ≤ 0.005).
FIG 5
FIG 5
Intestinal cells of mice treated with labeled ssRNA. (A) Schematic illustration of mouse model. Mice treated orally with labeled ssRNA-FAM/Ago complexes encapsulated in lipid-based nanoparticles (LNP). (B) After 4 h, intestinal cells were obtained and cellular uptake was analyzed by fluorometry. Fluorescence intensity is measured in relative fluorescence units (RFU). Gray bar, mice treated with ssRNA/Ago complexes encapsulated in LNP; white bar, mice treated with ssRNA/Ago complexes without LNP; black bar, mice treated only with LNP. (*, P ≤ 0.05; **, P ≤ 0.001).
FIG 6
FIG 6
Parasite burden in stool samples of mice orally treated with ssRNA/Ago. Mice were infected with 1 × 106 parasites. After 4 days of infection, mice were treated orally with daily ssRNA/Ago complexes encapsulated in LPN. Parasite burden in 25 mg of stool was evaluated by qPCR. Black triangles, anti-NDK ssRNA/Ago complexes; white triangles, scramble ssRNA/Ago. *, P ≤ 0.05.
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