Table 1 Challenges of small interfering RNA (siRNA) applications to viral infections and strategies to address them

Stability and Pharmacokinetics

siRNAs are vulnerable to degradation by nucleases and may show poor cellular uptake and tissue penetration, which can limit their therapeutic effectiveness

· Various siRNA delivery systems include nanocarriers, self-replicating RNA virus vectors, and viral vectors

· Modifications of siRNA molecules improve the stability and increase the half-life of siRNA

Delivery

· siRNAs have a large molecular weight of approximately 13 kDa and a highly anionic charge, which makes it difficult for them to cross cell membranes

· Unmodified siRNAs are not stable and can cause immune responses

· Use of plasmid and viral vectors as expression cassettes

· Encapsulation in synthetic vehicles such as cationic liposomes or nanoparticles and conjugation with cell-penetrating peptides or specific antibodies that target infected cells

· Increased efficiency of antiviral siRNA delivery may involve (1) the use of new targeting ligands and chemical probes that specifically bind to surface markers on infected cell populations, (2) increasing the efficacy of siRNA uptake into the cytoplasm, (3) developing materials with low toxicity to widen the antiviral therapeutic window, 4) designing materials with defined degradation products that can be metabolized for repeated dosing, (5) simplifying the formulation procedure, and (6) exploring delivery to organs other than the liver

Off-target effects

Synthetic (exogenous) siRNAs can potentially target unintended genes that share homology with the target viral gene, leading to off-target effects

· Use of siRNAs with high specificity and careful design of the siRNA sequences (analyses of homology and specificity during the design of siRNAs)

· Use of multiple siRNA duplexes to increase the certainty of observing the desired phenotype and expression pattern

· Identification of the lowest concentration at which antiviral siRNAs are capable of inducing effective gene silencing while preventing off-target effects

· Local drug delivery strategies to prevent the off-target accumulation of siRNAs

Immunogenicity

Synthetic siRNA can activate the innate immune system either by siRNA or its delivery vehicles

Proper siRNA design considerations as follows:

· Chemical modifications to the RNA backbone (ribose modifications)

· Choice of siRNA target sequence (avoidance of sequences prone to inducing inflammatory responses)

· Delivery methods (e.g., methods to prevent contamination by remnants of lipid nanoparticle delivery systems)

· Use of delivery modalities such as carriers that enhance the cellular internalization

· A single siRNA molecule binding to and regulating multiple mRNA copies

· High purity of RNA therapeutics reduce the chance of unwanted immune reactions

Antiviral Resistance

· Intrinsic suppressors of viral RNA silencing

· Mutation and emergence of viruses resistant to siRNAs

· RNA silencing suppressors do not affect RNAi induced by artificial (synthetic) siRNAs

· Combinations of multiple siRNAs targeting separate regions of the viral genome

· siRNAs that target highly conserved regions among various isolates

siRNA accessibility

RNAi efficiency is affected by the accessibility of the target RNA, which may be influenced by protein binding and the secondary RNA structure

Targets within the structured viral genome that are highly accessible should be considered