Immunomodulatory effect and safety of TNF-α RNAi mediated by oral yeast microcapsules in rheumatoid arthritis therapy

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease that requires long-term treatment and monitoring. Inhibition of inflammatory gene expression by gene therapy is a significant breakthrough in RA treatment, but the lack of a safe and effective gene delivery system hinders its application. Since oral administration can significantly reduce wound infection caused by parenteral administration, it also has the advantages of high patient compliance and convenience. Therefore, oral administration may be the best option for the treatment of this chronic disease. In this study, we developed a novel oral drug system by delivering tumor necrosis factor-α (TNF-α) short hairpin RNA (shRNA) mediated by non-pathogenic yeast to evaluate its regulation of systemic immune inflammation and safety in RA. Non-pathogenic yeast can resist the destruction of the gastrointestinal acid-base environment and can be recognized by the intestinal macrophages and act on systemic inflammatory lesions. Oral administration of yeast-mediated TNF-α shRNA significantly reduced the expression of TNF-α predominant pro-inflammatory factors in intestinal macrophages and joint synovium, and up-regulated the expression of anti-inflammatory cytokine IL-10 and M2 macrophages, systematically regulating the inflammatory response. This yeast-mediated oral gene delivery system can not only significantly inhibit knee joint synovial inflammation, but also has no toxic effects on peripheral blood and major organs. Therefore, yeast-mediated oral delivery of TNF-α shRNA may be used as a novel gene therapy strategy to treat RA through immunomodulating the mononuclear phagocyte system from the intestine to the joint synovium, and ultimately regulating systemic and local immune inflammation, providing new ideas for the clinical treatment of RA.

Keywords: Gene therapy; Immunomodulation; Oral administration; Rheumatoid arthritis.

Graphical abstract

Scheme 1

Schematic diagram of oral gene therapy for RA with recombinant yeast/TNF-α shRNA. (A) By transfecting shRNA vectors expressing uracil into yeast lacking uracil and culturing in selective medium (uracil deficient), the yeast clones grown out indicate successful yeast/shRNA construction. Cultures were then expanded and enriched to collect yeast/shRNA. (B) Oral gavage yeast/shRNA to RA rats. (C) The yeast/shRNA is recognized and phagocytosed by intestinal macrophages in the small intestine and be involved in regulating immune responses. (D) Macrophage@yeast/shRNA and the anti-inflammatory cytokines secreted by macrophages are transferred to distal inflammatory joints through humoral circulation and improve RA symptoms by gene therapy and immune regulation at synovial tissue sites.

Fig. 1

Preparation of yeast/shRNA. (A) Construction of yeast/shRNA by LiAc method. The negative control shRNA plasmid shR-NC and TNF-a shRNA (TNF-a shR-1, -2, -3) that could express uracil were transfected into yeast, and separately cultured in selective medium SD^-uracil. The yeast^-uracil alone was the control group. (B) Monoclonal bacterial solution PCR. Monoclonal colonies in Petri dishes were subjected to bacterial solution PCR to verify that the shRNA vector was in the yeast (n ​= ​3).

Fig. 2

In vitro functional assays of recombinant yeast/shRNA. (A) Yeast fluorescent staining to get green-labelled gYeast. (B) Green fluorescently labelled gYeast was co-cultured with macrophage. After 5 ​h co-culture, the effect of macrophages on recombinant yeast was determined by fluorescence imaging. (C) LPS-induced macrophages were treated with yeast/shRNA to examine the immune regulation of yeast/TNF-α shRNA by RT-qPCR method (shR-NC refers to yeast/shRNA negative control; shR-1, -2, -3 respectively refer to yeast/TNF-α shRNA-1, -2, -3). (D) Cytokines expression in macrophages cell culture medium after yeast/TNF-α shRNA treatment (+LPS or -LPS, respectively refers to macrophages with or without LPS treatment; Ctrl refers to macrophages without any treatment; shR-NC refers to yeast/shRNA negative control; shR-TNF refers to yeast/TNF-α shRNA). (E) TNF-α expression in macrophages without LPS treatment was detected via western blot after yeast/TNF-α shRNA treatment. (F) TNF-α expression in macrophages with LPS treatment was detected via western blot after yeast/TNF-α shRNA treatment. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001 and n.s. means no significance (n ​= ​3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Yeast phagocytized by macrophages was enriched in inflammatory joints. After administration of near-infrared fluorescently labelled DirYeast to RA rats, whether yeast could be delivered to inflamed tissues (joints) was detected by the oddessy fluorescence imaging system. (A) Fluorescence imaging of hind limbs of RA rats. (B) Hind limb fluorescence quantification. (C) Fluorescence imaging of liver, lung and spleen (left is the fluorescent labelling DirYeast group and right is the control group) (n ​= ​3).

Fig. 4

RA symptoms in rat knuckles. After 12 days of oral administration of recombinant yeast, rats hind limbs and forepaws were collected for anatomical analysis. (A) Forelimb anatomical analysis. (B) Hind limb anatomical analysis. (C) Total RA score of fore and hind limbs in different groups. n.s (no significance). ∗P ​< ​0.05, ∗∗∗P ​< ​0.001 (n ​= ​6).

Fig. 5

Immunofluorescence staining of the small intestine and articular synovium. (A–B) The immunofluorescence staining of TNF-α, CD206 and IL-10 in the small intestine. (C–D) The immunofluorescence staining of TNF-α, CD206 and IL-10 in the articular synovium (n ​= ​6).

Fig. 6

Blood cell values were measured by blood routine tests. After 12 days of oral administration of recombinant yeast, whole blood samples from their tail veins were collected for blood routine tests. (A) RBC: red blood cell count; (B) WBC: white blood cell count; (C) PLT: platelet count; (D) Lymph: lymphocyte count; (E) Mon: monocyte count; (F) Gran: neutrophil count. (G) NLR: peripheral blood neutrophil-to-lymphocyte ratio; (H) PLR: platelet-to-lymphocyte ratio. n.s (no significance). ∗P ​< ​0.05, ∗∗p ​< ​0.01. (n ​= ​6). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Biosafety assessment of yeast/shRNA drug delivery systems on blood cell component analysis level. After 12 days of oral administration of recombinant yeast, whole blood samples from their tail veins. Blood cell components were used to assess the safety of this drug delivery system. HCT: hematocrit. MCV: mean corpuscular volume. MCHC: mean corpuscular hemoglobin concentration. HGB: hemoglobin. MPV: mean platelet volume. PDW: platelet distribution width. n.s (no significance) (n ​= ​6).

Fig. 8

Biosafety assessment of yeast/shRNA drug delivery systems on histological staining level. After 12 days of oral administration of recombinant yeast/shRNA, intestine, kidney, liver, lung and spleen of rats were collected for hematoxylin-eosin (H&E) staining. Compared with the control groups (NC-PBS and RA-PBS), no significant difference in tissue integrity, cell structure and morphology have been found in RA-shR NC and RA-shR TNF group (n ​= ​6).