Inhibition of SARS-CoV-2 replication in the lung with siRNA/VIPER polyplexes

Abstract

SARS-CoV-2 has been the cause of a global pandemic since 2019 and remains a medical urgency. siRNA-based therapies are a promising strategy to fight viral infections. By targeting a specific region of the viral genome, siRNAs can efficiently downregulate viral replication and suppress viral infection. However, to achieve the desired therapeutic activity, siRNA requires a suitable delivery system. The VIPER (virus-inspired polymer for endosomal release) block copolymer has been reported as promising delivery system for both plasmid DNA and siRNA in the past years. It is composed of a hydrophilic block for condensation of nucleic acids as well as a hydrophobic, pH-sensitive block that, at acidic pH, exposes the membrane lytic peptide melittin, which enhances endosomal escape. In this study, we aimed at developing a formulation for pulmonary administration of siRNA to suppress SARS-CoV-2 replication in lung epithelial cells. After characterizing siRNA/VIPER polyplexes, the activity and safety profile were confirmed in a lung epithelial cell line. To further investigate the activity of the polyplexes in a more sophisticated cell culture system, an air-liquid interface (ALI) culture was established. siRNA/VIPER polyplexes reached the cell monolayer and penetrated through the mucus layer secreted by the cells. Additionally, the activity against wild-type SARS-CoV-2 in the ALI model was confirmed by qRT-PCR. To investigate translatability of our findings, the activity against SARS-CoV-2 was tested ex vivo in human lung explants. Here, siRNA/VIPER polyplexes efficiently inhibited SARS-CoV-2 replication. Finally, we verified the delivery of siRNA/VIPER polyplexes to lung epithelial cells in vivo, which represent the main cellular target of viral infection in the lung. In conclusion, siRNA/VIPER polyplexes efficiently delivered siRNA to lung epithelial cells and mediated robust downregulation of viral replication both in vitro and ex vivo without toxic or immunogenic side effects in vivo, demonstrating the potential of local siRNA delivery as a promising antiviral therapy in the lung.

Keywords: Human precision-cut lung slices; Pulmonary delivery; RNA therapeutics; SARS-CoV-2; siRNA delivery.

Graphical abstract

Fig. 1

Stability of polyplexes at increasing concentration of (A) mucin and (B) lung surfactant was tested by modified SYBR gold assay after 20 min incubation. Free siRNA represents 100% siRNA release. (Mean ± SD, n03). Green square: siRNA release from PEI polyplexes. Blue circle: siRNA release from VIPER polyplexes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

In vitro downregulation activity of PEI and VIPER polyplexes in a H1299 cell line stably expressing EGFP. (A) GAPDH gene knockdown efficiency of PEI and VIPER polyplexes 24 h after transfection with polyplexes. Blank samples consisted of H1299GFP cells treated with 5% glucose only. Negative controls consisted of polyplexes encapsulating scrambled siRNA. Positive controls consisted of Lipofectamine2000 lipoplexes with 100 pmol siGAPDH. GAPDH expression was normalized with β-actin expression and quantified by qRT-PCR. Data points indicate mean ± SEM. (B) GFP knockdown was measured by flow cytometry 48 h after transfection with PEI and VIPER polyplexes with 100 pmol siGFP. Blank samples consisted of H1299GFP cells treated with 5% glucose only. Negative controls consisted of samples treated with polyplexes encapsulating scrambled siRNA. Positive controls consisted of Lipofectamine2000 (LF) lipoplexes with 100 pmol siGFP. Data points indicated mean ± SD (n = 3). One-Way ANOVA, ***p < 0.005.

Fig. 3

Evaluation of cytotoxicity following incubation with PEI and VIPER polyplexes. (A) Evaluation of cellular viability by MTT assay of H1299/GFP cells incubated with PEI and VIPER with different N/P ratios: 6, 10 and 15. Positive controls consisted of cells treated with 20% DMSO. Data points indicate mean ± SD (n = 3). One-Way ANOVA, ns = not significant. (B) Assessment of membrane integrity by measuring lactate dehydrogenase release of cells incubate with PEI and VIPER polyplexes at different N/P ratios: 6, 10, 15. Cells treated with lysis buffer correspond to 100% LDH release. Data points indicate mean ± SD (n = 3). One-Way ANOVA, ns = not significant.

Fig. 4

Evaluation of AF647-siRNA/VIPER polyplexes delivery to Calu-3 cells grown at the air-liquid interface. (A) Schematic of the air-liquid interface culture model. Figure created with BioRender.com (B,C) Mucus penetration of AF647-siRNA/VIPER polyplexes in Calu-3 monolayers 24 h after transfection. (B) XY and XZ viewing direction. (C) 3D reconstruction. Red color represents AF647.siRNA, green color to the mucus layer stained with AF488-labelel wheat germ agglutinin. (D,E) Cell uptake of AF647-siRNA/VIPER polyplexes 24 h after transfection analyzed by confocal light scanning microscopy. (D) XY and XZ viewing direction. (E) 3D reconstruction. Red color corresponds to AF647-siRNA, green color to actin stained with rhodamine phalloidin and blue color corresponds to nuclei stained with DAPI. (F) GAPDH gene knockdown efficiency PEI and VIPER polyplexes in Calu-3 monolayers grown under ALI conditions 24 h after transfection with siGAPDH and scrambled siRNA as negative control. Blank samples consisted of Calu-3 monolayers treated with 5% glucose only. Positive controls consisted of Lipofectaamine2000 lipoplexes with siGAPDH. GAPDH expression was normalized with β-actin expression and quantified via qRT-PCR. Data points indicate mean ± SEM (n = 3). One-Way ANOVA, *p < 0.05, **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Evaluation of siORF/VIPER polyplexes activity against wild-type SARS-CoV-2 infection in Calu-3 cells grown at the air-liquid interface. (A, B) The expression of ACE-2 receptor on the apical side of Calu-3 cells was confirmed by preparing sections of Calu-3 monolayers. Pink color represents AF944 staining of ACE-2 receptor, blue color represents nuclei staining with DAPI. (C) SARS-CoV-2 downregulation in Calu-3 monolayers after treatment with SARS-CoV-2 specific siRNAs O1 and O3/VIPER polyplexes and siLuc/VIPER polyplexes as negative control. Calu-3 cells were infected with wild type SARS-CoV-2 (MOI 0.1) 6 h after transfection with polyplexes. Data points indicate mean ± SEM (n = 3). Statistical significance was calculated using One-way Anova with Dunnett's multiple comparisons correction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Evaluation of siRNA/VIPER polyplexes against wild-type SARS-CoV-2 in human PCLS. PCLS were transfected with chemically modified versions of SARS-CoV-2 specific and luciferase control siRNA (see Table 1) formulated as VIPER polyplexes 6 h before infection with wild-type SARS-CoV-2 (MOI 1). The readout was performed 24 h after infection via qRT-PCR. Significant differences (*p = 0.019) were observed between the treatment with the unrelated luciferase vs. the O3* siRNA sequences. Data points indicate mean ± SEM (n = 3).

Fig. 7

In vivo siRNA/VIPER polyplexes distribution in lung epithelial cells. BALB/c mice were intratracheally administered with 2 nmol AF647-siRNA complexed with either VIPER or PEI polymers. Negative control consisted of animals that received 5% glucose only. Cellular uptake of VIPER and PEI polyplexes in different lung cell populations was quantified by flow cytometry. One-Way ANOVA, ns = not significant, *p < 0.05, **p < 0.01.

Fig. 8

In vivo cytokines release was measured in bronchoalveolar lavage fluid (BALF) by LEGENDplex ELISA technique. Values are given in pg/ml as mean ± SEM (n = 4). Value below detection limit were set as the value corresponding to the minimum detection limit. One-Way ANOVA, only non-significant differences were observed.