A programmable dual-targeting siRNA scaffold supports potent two-gene modulation in the central nervous system.

Divalent short-interfering RNA (siRNA) holds promise as a therapeutic approach allowing for the sequence-specific modulation of a target gene within the central nervous system (CNS). However, an siRNA modality capable of simultaneously modulating gene pairs would be invaluable for treating complex neurodegenerative disorders, where more than one pathway contributes to pathogenesis. Currently, the parameters and scaffold considerations for multi-targeting nucleic acid modalities in the CNS are undefined. Here, we propose a framework for designing unimolecular 'dual-targeting' divalent siRNAs capable of co-silencing two genes in the CNS. We systematically adjusted the original CNS-active divalent siRNA and identified that connecting two sense strands 3' and 5' through an intra-strand linker enabled a functional dual-targeting scaffold, greatly simplifying the synthetic process. Our findings demonstrate that the dual-targeting siRNA supports at least two months of maximal distribution and target silencing in the mouse CNS. The dual-targeting divalent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g. Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting divalent siRNAs against each gene. This work enhances the potential for CNS modulation of disease-related gene pairs using a unimolecular siRNA.

A programmable dual-targeting di-valent siRNA scaffold supports potent multi-gene modulation in the central nervous system.

Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.