Interfering RNA nanoparticles may be a potential therapy for COVID-19

The study shows that liRN based on lipid nanoparticles strongly suppresses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – the cause of coronavirus disease 2019 (COVID-19) – in cells as well as in mouse models.

COVID-19 cases continue to increase worldwide. Several vaccines have now been approved and are being administered. However, with the advent of new variants of the virus, questions have arisen about how effective the vaccines will be.

The genome in coronaviruses encodes ribonucleic acid (RNA), which makes them vulnerable to RNA interference, especially when delivered to the lungs. However, there are few therapies that target viral RNA.

Small interfering RNAs (siRNAs) are short double-stranded RNA molecules. They interfere with the expression of specific genes and prevent translation. SiRNA molecules can be delivered to the lungs through the nose or intravenously.

In a new study, researchers examined several siRNAs that target highly conserved SARS-CoV-2 regions and may inhibit virus replication. They reported their results in a paper published on bioRxiv * pretprint server.

SiRNA selection

The team designed siRNAs targeting viral RNA polymerase, helicase, and the 5 ‘untranslated leader region (5’UTR). The team characterized the SARS-CoV-2 genome for structural characteristics and sequence conservation and looked for sequences that were not present in the human transcriptome.

Using this information, they generated about 9,500 potential siRNA sequences using computational tools for siRNA design. Of these, 18 siRNAs were selected for further testing. In vitro testing of these candidates showed different responses, and about five showed the best reduction in virus gene expression, also called repression. Two of them can target both SARS-CoV and SARS-CoV-2.

siRNA can be stabilized by further chemical changes, allowing it to be stronger in vivo and show stronger virus repression. The authors chose siUTR3 for further chemical modification because its target site is in the highly conserved 5’UTR region, important for viral processing and expression of viral RNA.

Modification of 2 ‘O-methyl in siUTR3 increased serum stability and maintained the level of virus repression, but reduced the repressive power of SARS-CoV-2. Further optimization of modifications can lead to more stable and stronger siRNA for in vivo delivery.

Previous studies on RNA viruses have shown that individual siRNAs can lead to virus mutations, while combinations can reduce the occurrence of viral variants. So the team tested three powerful siRNAs alone and in combination. The combination showed the same level of virus repression as each of them alone, although their individual concentrations in the combination were lower.

SiRNA van

The team had previously designed “stealth” lipid nanoparticles (sLNPs) for intravenous delivery of siRNA to the lung. The thick mucus associated with COVID-19 is likely to interfere with aerosol delivery, and the intravenous method may prove useful. They modified this formulation to deliver SARS-CoV-2 siRNA to a mouse model expressing a human enzyme that converts angiotensin 2 (ACE2).

Mice infected with SARS-CoV-2 and injected sLNP-siRNA showed improved chances of survival, and these mice had lower weight loss compared to untreated mice. Although the virus was completely suppressed three days after siRNA administration, this effect was lost after six days, suggesting that repression is only transient and occurs a day or two after injection. Therefore, daily injections may be required during peak viral loads.

Intravenous administration of sLNP-siRNA repression COVID-19 in vivo.  (A) Females and males of mice aged 7 to 13 weeks K18-hACE.2 were intranasally infected with either PBS or 104 PFU / 20 μL SARS-CoV-2 (Australian strain VIC1, paragraph 4).  (A) Mice were treated intravenously with 1 mg / kg in 100 μL siRNA packaged in HFDM lipid nanoparticles (LNP) by retroorbital administration at -1 and 2 days post-infection (dpi).  At 3dpi and 6dpi, lung and brain tissues were collected and homogenized for immunoplac tests.  (BD) Mouse survival, probability of survival, body weight (weight change), and clinical outcome were assessed at the indicated days after infection (dpi).  (B) Weight loss> 15% was taken as the end point and mice were euthanized.  (C) Mice were weighed and scored daily to the experimental endpoint (6 dpi) for disease progression.  (D) Clinical outcome was assessed based on movement, behavior, and appearance.  Each data point represents an average of ± SEM 3 to 4 mice.  (E) The amount of infectious virus particles in lung tissues at 3 and 6 dpi was titrated by immunoplastic assays on Vero E6 cells, using an antibody specific for the SARS-CoV-2 N protein and expressed as PFU per gram of tissue.  Each data point represents a technical copy, with one mouse equivalent to 3 technical replicas, and the bars representing the mean ± SEM.  (F) Unexpected hierarchical cluster map of the heat map of immune gene expression in the lungs at 6dpi.  Each row is a gene, and each column is a treatment group.  The rows are normalized with a Z-score (green: low expression and red: high expression).  Value p of <0.0001 (****) is considered statistically significant when assessed by 2-way ANOVA (Dunnett’s post- test) when compared against siControl.

Intravenous administration of sLNP-siRNA repression COVID-19 in vivo. (A) Females and males of mice aged 7 to 13 weeks K18-hACE.2 were intranasally infected with either PBS or 104 PFU / 20 μL SARS-CoV-2 (Australian strain VIC1, paragraph 4). (A) Mice were treated intravenously with 1 mg / kg in 100 μL siRNA packaged in HFDM lipid nanoparticles (LNPs) by retroorbital administration at -1 and 2 days post-infection (dpi). At 3dpi and 6dpi, lung and brain tissues were collected and homogenized for immunoplac tests. (BD) Mouse survival, probability of survival, body weight (weight change), and clinical outcome were assessed at the indicated days after infection (dpi). (B) Weight loss> 15% was taken as the end point and mice were euthanized. (C) Mice were weighed and scored daily to the experimental endpoint (6 dpi) for disease progression. (D) Clinical outcome was assessed based on movement, behavior, and appearance. Each data point represents an average of ± SEM 3 to 4 mice. (E) The amount of infectious virus particles in lung tissues at 3 and 6 dpi was titrated by immunoplastic assays on Vero E6 cells, using an antibody specific for the SARS-CoV-2 N protein and expressed as PFU per gram of tissue. Each data point represents a technical copy, with one mouse equivalent to 3 technical replicas, and the bars representing the mean ± SEM. (F) Unexpected hierarchical cluster map of the heat map of immune gene expression in the lungs at 6dpi. Each row is a gene, and each column is a treatment group. The rows are normalized with a Z-score (green: low expression and red: high expression). A p <0.0001 (****) value is considered statistically significant when evaluated by bidirectional ANOVA (Dunnett’s posttest) compared to siControl.

The researchers did not see any immune dysregulation due to siRNA. They also further modified the formulation to ensure a reduction in any immune stimulation due to lipids.

They did this by reducing the amount of cationic lipid, which was found to activate the immune system. Decreasing the amount of this lipid in the formulation reduced the positive charge by almost half. Similar to the previous formulation, this formulation also showed effective repression of the virus and better survival of mice.

Both Modern and Pfizer vaccines use LNP as a means of delivery. Their success in the clinical translation of LNPs helps the idea that LNPs are safe to use as a means of delivering mRNA and RNAi. Building on this, this study shows that LNPs can also deliver siRNA and have the potential to be used as a COVID-19 treatment.

* Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered definitive, guide clinical practice / health-related behavior, or treat it as established information.

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