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Targeted Knockdown of Genes in the Choroid Plexus
Published on: June 16, 2023
Intracranial administration of P gene siRNA protects mice from lethal Chandipura virus encephalitis
Satyendra Kumar1, Vidya A Arankalle
1National Institute of Virology, Pune, India.
Insights
RNA interference (RNAi) using short interfering RNA (siRNA) shows promise for treating Chandipura Virus (CHPV) encephalitis. This approach effectively reduced viral load and increased survival rates in mice, highlighting its therapeutic potential.
Area of Science:
- Virology
- Molecular Biology
- Neuroscience
Background:
- Chandipura Virus (CHPV) causes severe pediatric encephalitis in India with high mortality.
- Current treatment options for CHPV encephalitis are limited.
- RNA interference (RNAi) was explored as a potential therapeutic strategy.
Purpose of the Study:
- To evaluate the efficacy of synthetic short interfering RNA (siRNA) and short hairpin RNA (shRNA) against Chandipura Virus (CHPV).
- To assess the therapeutic potential of RNAi in a mouse model of CHPV encephalitis.
Main Methods:
- Synthetic siRNAs targeting P and M genes of CHPV were designed and tested in vitro.
- Quantitative assays (RT-PCR, plaque assay) were used to measure gene silencing and viral titer reduction.
- Efficacy was assessed in a mouse model of CHPV infection, including survival studies and viral load determination in brain tissue.
Main Results:
- P-2 siRNA demonstrated the highest efficiency in inhibiting P gene expression in vitro.
- Transfection with P-2, M-5, or M-6 siRNAs resulted in a 2-log reduction in viral titer post-infection.
- Simultaneous administration of P-2 siRNA in mice led to extended survival, with 70% survival achieved with a two-dose regimen.
Conclusions:
- Synthetic siRNA exhibits significant potential as a therapeutic agent for Chandipura Virus encephalitis.
- RNAi treatment effectively reduced viral load and neuropathology in a CHPV mouse model.
- The findings suggest siRNA therapy could be a viable treatment for rapid and fatal Chandipura encephalitis.
Background:
In parts of India, Chandipura Virus (CHPV) has emerged as an encephalitis causing pathogen in both epidemic and sporadic forms. This pediatric disease follows rapid course leading to 55-75% mortality. In the absence of specific treatment, effectiveness of RNA interference (RNAi) was evaluated.
Methods And Findings:
Efficacy of synthetic short interfering RNA (siRNA) or short hairpin RNA (shRNA) in protecting mice from CHPV infection was assessed. The target genes were P and M genes primarily because important role of the former in viral replication and lethal nature of the latter. Real time one step RT-PCR and plaque assay were used for the assessment of gene silencing. Using pAcGFP1N1-CHPV-P, we showed that P-2 siRNA was most efficient in reducing the expression of P gene in-vitro. Both quantitative assays documented 2 logs reduction in the virus titer when P-2, M-5 or M-6 siRNAs were transfected 2 hr post infection (PI). Use of these siRNAs in combination did not result in enhanced efficiency. P-2 siRNA was found to tolerate four mismatches in the center. As compared to five different shRNAs, P-2 siRNA was most effective in inhibiting CHPV replication. An extended survival was noted when mice infected intracranially with 100 LD50 CHPV were treated with cationic lipid complexed 5 microg P-2 siRNA simultaneously. Infection with 10LD50 and treatment with two doses of siRNA first, simultaneously and second 24 hr PI, resulted in 70% survival. Surviving mice showed 4 logs less CHPV titers in brain without histopathological changes or antibody response. Gene expression profiles of P-2 siRNA treated mice showed no interferon response. First dose of siRNA at 2 hr or 4 hr PI with second dose at 24hr resulted in 40% and 20% survival respectively suggesting potential application in therapy.
Conclusions:
The results highlight therapeutic potential of siRNA in treating rapid and fatal Chandipura encephalitis.
