Abstract 719: Effectiveness of Therapeutic RNA Interference is Dependent on Genomic Target Selection and Emergence of Escape Mutants: Implications for Enteroviral Heart Disease
Background: We have shown previously that therapeutic RNA interference with short interfering RNA (siRNA) may limit coxsackieviral (CVB) heart disease ( Circulation 2005;111:1583–92). Aim of this study was the identification of major factors that limit early efficacy of antiviral siRNA therapy.
Methods and results: To assess therapeutic efficacy of siRNA depending on genomic target selection, siRNA were designed against distinct regions representing the entire CVB genome (5′UTR, VP1, VP2, 2A, 3C, 3D) and examined for their antiviral effectiveness in permissive human HeLa cells (Fig.A⇓). siRNA targeting the 3CD region were particularly protective for cell survival and inhibition of viral replication. Early effectiveness of single siRNA application was hampered by the emergence of viral escape mutants. Sequencing of siRNA resistant viral genomes revealed the occurrence of 1 or 2 point mutations at the siRNA binding site. Yet, mutated viruses remained fully susceptible to siRNA targeting different genomic regions. Thus, simultaneous administration of dual siRNA decreased the emergence of viral escape mutants. Delivery of a siRNA cocktail with 3 different siRNA almost completely suppressed the emergence of viral escape and further improved therapeutic effectiveness (Fig.B⇓).
Conclusion: The early effect of antiviral RNA interference is highly dependent on genomic target selection and impeded by the occurrence of viral escape mutations containing single or dual mutations at the siRNA binding site. Administration of at least three siRNA molecules targeting different regions is sufficient to significantly suppress viral escape, thus leading to improved outcome.