Rational Design of Therapeutic siRNAs: Minimizing Off-targeting Potential to Improve the Safety of RNAi Therapy for Huntington's Disease  Ryan L Boudreau, Ryan M Spengler, Beverly L Davidson  Molecular Therapy  Volume 19, Issue 12, Pages 2169-2177 (December 2011) DOI: 10.1038/mt.2011.185 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 The artificial microRNA (miRNA), miSCR, causes neurotoxicity in mouse brain. Wild-type mice were injected into the striatum with adeno-associated virus (AAV)-green fluorescent protein (GFP) (expresses GFP only) or AAV-miSCR-GFP (expresses both the artificial miRNA and GFP), and histological analyses were performed on brains harvested at 6 months post-treatment. Photomicrographs representing GFP autofluorescence and immunohistochemical staining of IbaI-positive microglia are shown. Bars = 200 and 50 µm for ×10 and ×40 images, respectively. Boxed inset represents region of ×40 image. Molecular Therapy 2011 19, 2169-2177DOI: (10.1038/mt.2011.185) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Overview of seed-related off-targeting: mechanism and probabilities. (a) Diagram depicting the expression and processing of an artificial microRNA (miRNA) to produce the mature small inhibitory RNA (siRNA) duplex. The antisense guide strand is loaded into RNA-induced silencing complex (RISC) and may direct on-target silencing (intended) and off-target silencing (unintended). (b) Cartoon highlighting the relationship between the frequencies of seed complement binding sites in the 3′-UTRome and the off-targeting potential for siRNAs. (c) The number of human mRNA 3′-UTRs containing a given hexamer was determined for all of the 4,096 possible hexamers and a binned distribution is shown. The probabilities that randomly selected siRNAs targeting human coding sequence (CDS) will contain seed complements in a given range (white and gray shading) are also presented. For example, there is only a 10% chance that a randomly selected siRNA contains a seed complement for a hexamer present in ~1,500 human 3′-UTRs or less. Note: the sequences tested in this article are placed above their respective ranges. Molecular Therapy 2011 19, 2169-2177DOI: (10.1038/mt.2011.185) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Selection and screening of huntingtin (htt)-targeting small inhibitory RNAs (siRNAs) with low off-targeting potentials. (a) Schematic outlining the selection of “safe” seed siRNAs with proper strand-biasing. (b) Plasmids expressing artificial microRNAs (miRNAs), harboring the indicated siRNA sequences, were transfected into HEK293 cells, and quantitative PCR (QPCR) analysis was performed 24 hours later to measure endogenous htt mRNA levels. U6 (promoter-only) and HD2.4 (a previously published htt RNAi sequence) serve as the negative and positive controls, respectively. Results are shown as mean ± SEM (N = 6, *P < 0.001, relative to U6). Molecular Therapy 2011 19, 2169-2177DOI: (10.1038/mt.2011.185) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Evaluation of microarray data for huntingtin (htt) silencing and off-targeting. HEK293 cells were transfected with U6 promoter-only or U6-driven artificial microRNA (miRNA) expression plasmids (n = 4 for each treatment), and RNA was harvested 72 hours later for microarray analysis. Two-way ANOVA was performed to detect differentially expressed genes among the treatment groups. (a) Htt mRNA levels determined by microarray (gray bars) were consistent with those measured by quantitative PCR (QPCR) (black bars) using the same RNA samples. (b) Hierarchical clustering and heat-maps were generated using differentially expressed genes (P < 0.0001, 827 genes) to visualize the relationships among the treatment groups. Interestingly, all of the “safe” seed sequences are more related to U6 than the remaining sequences predicted to have higher off-targeting potentials (boundary marked by white line). (c) Hierarchical clustering and heat-maps were generated using differentially expressed genes (P < 0.01, 985 genes) to visualize the relationships among the treatment groups. The impact of seed sequence on gene expression can be appreciated by the clustering of HD8.2 and 8.2mis which share the same seed. Notably, the predicted low off-targeting sequences (Safe, HDS1 and HDS2) are more similar to U6 and have smaller off-targeting signatures compared to both HD2.4 and HD8.2. Seed-related off-targeting was evaluated by (d) cumulative distribution and (e) motif discovery analyses. (d) Cumulative distribution plots for gene expression values are shown for transcripts containing (1 site or 2+ sites) or lacking (baseline) 3′-UTR seed complement binding sites for the indicated sequence and strand. A shift to the left indicates an increased likelihood of being downregulated. AS, antisense, S, sense. Kolmogorov–Smirnov (KS)-test P values are shown; N.S., no statistical significance (P > 0.1). (e) Motif discovery analyses identified an enrichment of seed complement binding sites in the 3′-UTRs of downregulated genes (>1.1-fold) unique to each treatment. Shown here are the examples of 8.2-124a and Terror; similar data for the remaining sequences supports that each mediates detectable seed-related off-targeting to some degree (Supplementary Figure S1). Molecular Therapy 2011 19, 2169-2177DOI: (10.1038/mt.2011.185) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Silencing efficacy and safety of HDS sequences in mouse brain. Wild-type mice were injected into the striatum with adeno-associated virus (AAV) viruses coexpressing artificial microRNAs (miRNAs) and green fluorescent protein (GFP). (a) At 3 weeks postinjection, GFP-positive striata were harvested and quantitative PCR (QPCR) analysis was performed to measure endogenous mouse huntingtin (htt) mRNA levels. Results are shown as mean ± SEM (n ≥ 3, *P = 0.001, relative to uninjected striata). (b) Brains from additional cohorts of injected mice were harvested at 6 months postinjection and histological analyses were performed to assess neurotoxicity. Photomicrographs representing GFP autofluorescence and immunohistochemical staining of IbaI-positive microglia are shown. Bars = 200 and 50 µm for ×10 and ×40 images, respectively. Boxed inset represents region of ×40 image. Molecular Therapy 2011 19, 2169-2177DOI: (10.1038/mt.2011.185) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions