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

Abstract

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.

Graphical Abstract

Figure 1.

Design considerations and validation of dual-targeting siRNAs in vitro. (A) Concurrent synthesis with a ‘symmetric’ configuration results in single targeting divalent siRNA. (B) Approaches to dual-targeting siRNAs that require specialized reagents: an orthogonal synthesis with protecting groups (left) or linear synthesis with reverse amidites (right). (C) 3′–5′ linear synthesis enables the incorporation of two unique siRNA sequences without specialized reagents. (D) Panel of siRNA used in vitro. Left: single targeting HTT- or non-targeting control siRNA. Middle: dual targeting siRNA with two arm positions covalently linked through TEG. Right: dual targeting siRNA with two arm positions covalently linked through HEG. (E) HeLa cells with mRNA measured via QuantiGene at 72 h post-treatment. Htt mRNA levels shown on left and Jak1 mRNA levels shown on right. (F) Design of dual targeting siRNA where antisense strands are linked with PO, rather than PS backbone modification (G) HeLa cells with Htt mRNA measured via QuantiGene at 72 h post-treatment.

Figure 2.

Structures of single- and dual-targeting divalent siRNA. Symmetric divalent siRNA are linked 3′–3′. Linear divalent siRNA are linked 5′ to 3′. All divalent siRNA are linked with TEG linker (medium gray bar). Lighter colors represent the sense strand, darker colors represent antisense strand. siRNAs have an antisense strand length of 20 nucleotides and a sense strand length of 16 nucleotides. Sequences and modification patterns are shown in Supplemental Table S1.

Figure 3.

Linear (5′–3′ sense linked) divalent siRNA performs equivalently to previously validated symmetric (3′–3′ sense linked) divalent siRNA. 10 nmol of divalent siRNA were delivered to wild-type mice via intracerebroventricular injection (5 nmol/5 μl per ventricle). At 1-month post-injection (A) Msh3 mRNA expression and (B) Htt mRNA expression were measured by QuantiGene assay. (C) MSH3 protein expression and (D) HTT protein expression were quantified from western blots. (E) MSH3 antisense strand accumulation was measured by peptide nucleic acid hybridization assay. M/MC- Medial cortex, S/Stri- striatum, T/Thal- thalamus, H/Hpx- hippocampus, P- posterior cortex. Graphs show the mean with standard deviation. Each dot is the average of technical duplicates from a single mouse. N = 5–6 mice/treatment group specifically: NTC symmetric = 6 mice, NTC linear = 5 mice, MSH3 symmetric = 6 mice, MSH3 linear = 6 mice, HTT symmetric = 6 mice, HTT linear = 6 mice. NTC is non-targeting divalent siRNA control. A, B, E: Statistics are two-way ANOVA with Tukey's multiple comparisons. C, D: Statistics are one-way ANOVA with Tukey's multiple comparisons. **** indicates P < 0.0001

Figure 4.

Dual targeting divalent siRNA reduces mRNA and protein expression of both targets regardless of antisense strand configuration in vivo. 10 nmol of divalent siRNA were delivered to wild-type mice via intracerebroventricular injection (5 nmol/5 μl per ventricle). At 1-month post-injection (A) Msh3 mRNA expression and (B) Htt mRNA expression were measured by QuantiGene assay. Statistics are two-way ANOVA with Tukey's multiple comparisons (C) A head-to-head comparison of dual-targeting scaffold version 1 and 2. Blue bars are Msh3 mRNA levels, red bars are Htt mRNA levels. (D) MSH3 or HTT protein quantified from western blots from the striatum or medial cortex. M/MC- Medial cortex, S/Stri- striatum, T/Thal- thalamus, H/Hpx- hippocampus, P- posterior cortex. Sample group size: NTC linear = 5 mice, MSH3 symmetric = 6 mice, MSH3 linear = 6 mice, HTT symmetric = 6 mice, HTT linear = 6 mice, dual version 1= 5 mice, dual version 2 = 6 mice. Graphs are the mean with standard deviation. Each dot is the average of technical duplicates from a single mouse. Ns = no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 5.

Dual-targeting divalent siRNA is effective in mouse CNS up to two months in vivo. Divalent siRNA targeting Msh3 and/or Htt were delivered to wild-type mice via bilateral intracerebroventricular injection. At 2 months post-injection (A) Striatal Htt (red) or Msh3 (blue) mRNA expression was measured following treatment with single-targeting, dual-targeting, or a mixture of single-targeting divalent siRNA. Statistics are one-way ANOVA with Tukey's multiple comparisons. (B) Striatal HTT (red) or MSH3 (blue) protein expression following treatment with single-targeting, dual-targeting, or mixture of single-targeting divalent siRNA. ‘NTC’ is non-targeting divalent siRNA control. ‘MSH3’ or ‘HTT’ is single-targeting divalent control. Dual-target is dual targeting version 2. ‘Mixture’ is a 1:1 combination of Msh3 and Htt single-targeting divalent siRNAs. Statistics are one-way ANOVA with Tukey's multiple comparisons. N = 5 mice/treatment group. Graphs are the mean with standard deviation. Each dot is the average of technical duplicates from a single mouse. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 6.

Dual-targeting siRNA works as well as divalent siRNA mixtures. Divalent siRNA targeting Msh3 and/or Htt were delivered to wild-type mice via bilateral intracerebroventricular injection. At 2-months post-injection (A) Msh3 mRNA expression was measured by QuantiGene assay. Statistics are two-way ANOVA with Tukey's multiple comparisons. (B) MSH3 protein expression in the striatum and medial cortex was quantified from western blot. Statistics are two-way ANOVA with Tukey's multiple comparisons. (C) Htt mRNA expression was measured by QuantiGene assay. Statistics are two-way ANOVA with Tukey's multiple comparisons. (D) HTT protein expression in the striatum and medial cortex was quantified from western blot. Statistics are two-way ANOVA with Tukey's multiple comparisons. ‘NTC’ is non-targeting divalent siRNA control. ‘MSH3’ or ‘HTT’ is single-targeting divalent control. Dual-target is dual targeting version 2. ‘Mixture’ is a 1:1 combination of Msh3 and Htt single-targeting divalent siRNAs. M/MC, medial cortex; S/Stri, striatum; T/Thal, thalamus; H/Hpx, hippocampus; P, posterior cortex. N = 5 mice/treatment group. Graphs are the mean with standard deviation. Each dot is the average of technical duplicates from a single mouse. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 7.

Dual-targeting siRNAs are programable and functional across sequences in vivo. (A) 10 nmol of divalent siRNA were delivered to wild-type mice via intracerebroventricular injection (5 nmol/5 μl per ventricle). NTC, dual-targeting, or APOE control structures used for the experiment. Sequences and modification patterns are shown in Supplemental Table S1. At 2 months post-injection (B) ApoE mRNA expression and (C) Jak1 mRNA expression were quantified by QuantiGene. Statistics are two-way ANOVA with Tukey's multiple comparisons. NTC is non-targeting divalent siRNA control. N = 5 mice/treatment group. Graphs are the mean with standard deviation. Each dot is the average of technical duplicates from a single mouse. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.