Gene Silencing: A Therapeutic Approach to Combat Influenza Virus Infections

Madhu Khanna; Latika Saxena; Roopali Rajput; Binod Kumar; Rajendra Prasad

Future Microbiol. 2015;10(1):131-140.

Conclusion

Elucidation of molecular mechanisms of viral replication and signal transduction pathways involved in viral pathogenesis, have opened new avenues for the development of alternative therapeutic approaches.

The silencing effect of RNAi is highly specific and potent which makes it an attractive therapeutic strategy against viruses. RNAi-based sequence-specific therapeutic agents can maintain pace with the high rate of mutation of influenza virus and HIV, there is a great potential for such therapeutic agents in the near future. Nucleic acid-based therapeutics, especially siRNAs has also been utilized for both prevention and treatment of influenza virus infections. Although siRNA are cleared at a rapid rate but with proper modifications the life could be increased. Rzs and Dz are also being explored as additional mechanisms for gene silencing in many viruses including influenza and have shown their potential in controlling these viruses. Although, RNAi offers a promising antiviral strategy, there are obstacles that need to be overcome, which include toxicity and side effects of RNAi, delivery challenges and concerns that many viruses have evolved mechanisms to escape from RNAi.

Nevertheless, gene silencing based antiviral therapies have emerged as highly promising and versatile prospects as alternatives for treating viral infections in the near future. The aspect to be focused are the target region sequences, which must be conserved throughout all the known influenza A virus strains and efficient mode of delivery of the drug. Newer approaches include nanoparticle delivery of RNAi agents and other second generation transfection reagents.

There have been reports concerning the induction of immune responses by RNAi agents or suppression of RNAi mechanism by certain viral proteins. However, further studies have revealed that such responses are dependent on the viral strain and host cell type.

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Table 1. Mechanism gene silencing by different nucleic acids.
Gene silencing techniques	Description of mechanism	Ref.
siRNA	The siRNA associates with RISC leading to unwinding of the dsRNA, sense strand of which is cleaved by Argonaut2 protein. The antisense siRNA strand in the now activated RISC complex recognizes the sequence of target mRNA thereby cleaving it	[12]
Ribozyme	The catalytic activity by most of the ribozymes is done by phosphoryl transfer reactions. Each of the five types of Rz cleave the target site specifically by forming a 2′,3′-cyclic phosphate as a result of nucleophilic reaction between the 2′-O and the adjacent 3′-P of the nucleotide at the target site	[13]
miRNA	miRNA, primarily transcribed as pri-miRNA is processed into pre-miRNA by a microprocessor complex which consist of DGCR8. In presence of exportin 5, the pre-miRNA is transported from the nucleus to the cytoplasm where the Dicer enzyme processes it into its mature form as miRNA. The miRNA is taken up into RISC and the target gene is degraded via different mechanisms, viz., interaction of miRNA with the 3′-UTR, or 5′-UTR or coding regions of the target gene	[14,15]
DNAzyme	The two arms flanking the catalytic motif of the nucleozyme bind to the target sequence by complementary base-pairing and facilitate the breakage of phosphodiester bond between a purine and a pyrimidine in presence of metal ions	[16]

DGCR8: Drosha and DiGeorge syndrome critical region 8; dsRNA: Double-stranded RNA; pre-miRNA: Precursor miRNA; pri-miRNA: Primary miRNA; RISC: RNA-induced silencing complex; UTR: Untranslated region.

Table 2. Different gene segments of influenza A virus subtypes targeted for silencing the expression, using siRNA.
Target gene	Strain of influenza virus	Test animal/cell lines	Ref.
PB2	H1N1	In vitro	[31]
PB1	H1N1	In vitro and in vivo	[31,32]
PA	H1N1, H5N1, H7N7, H9N2	In vitro and in vivo	[31,32]
NP	H1N1, H5N1, H7N7, H9N2	In vitro and in vivo	[31,32]
M	H1N1	In vitro	[31,36]
NS	H1N1, H5N1	In vitro and in vivo	[31,33,35]