1. Artificial MicroRNAs as siRNA Shuttles: Improved Safety as Compared to shRNAs In vitro and In vivo 
Ryan L Boudreau, Inês Martins, Beverly L Davidson 
Molecular Therapy 
Volume 17, Issue 1, Pages (January 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Design of hairpin-based RNAi vectors for fair comparison studies. shRNA and artificial miRNA vectors were designed to release the same siRNA sequences [either targeting SCA1 (shown) or eGFP] after processing by Drosha and/or Dicer. Processing sites (arrows) and proper loading of the intended antisense strand (bottom) as opposed to the unintended sense strand (top) were previously confirmed.10
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Effects of shRNA- and miRNA-based vectors on artificial miRNA biogenesis and function. (a) Cartoon of RNAi and RNAi luciferase reporter vectors. “TTTTT” designates the Pol-III termination signal. (b) HEK293 cells were transfected with miGFP and GFP RNAi luciferase reporter expression plasmids to establish baseline silencing levels (dotted line). To assess for disruptions in miGFP biogenesis and function, shSCA1 or miSCA1 competitors were added to the transfections at varying doses of RNAi:target. U6 serves as the promoter-only control. (c) Gene silencing assays were performed by co-transfecting HEK293 cells with SCA1 RNAi and RNAi reporter plasmids at varying doses of RNAi:target. (d,e) Reciprocal experiments evaluating the effects of GFP RNAi competitors (shGFP or miGFP) on miSCA1 activity, in parallel with GFP RNAi efficacy studies. All bar graphs represent mean values ± SEM (n = 3; ***, ** and NS indicate P < 0.001, P < 0.01, and no significance, P > 0.05, respectively). (f) Northern blot analyses assessing the processing of miGFP in the presence of SCA1 RNAi competitors. Blots were probed for either GFP (top blot-pair) or SCA1 (bottom blot-pair) RNAi transcripts.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Effects of shSCA1 and miSCA1 on miR-1 function in differentiating myoblasts. (a) Cartoons depicting the miR-1 luciferase reporter (contains a perfect complementary miR-1 target site in the 3′UTR of Firefly luciferase) and the RNAi-hrGFP dual expression vector used in C2C12 studies. (b) Disruption of endogenous miRNA biogenesis and function was assessed in C2C12 cells which show induced miR-1 expression after differentiation. Cells were co-transfected with RNAi and miR-1 luciferase reporter plasmids, and then differentiated. Each group (n = 4) was normalized to cells treated with siCheck2-alone (i.e., no miR-1 target), and the results are shown as mean values ± SEM. No RNAi (—) served as the empty-vector control. (c) RNAi plasmids co-expressing hrGFP were co-transfected into C2C12 cells, and these were differentiated and stained for myosin heavy-chain (MHC) to identify transfected differentiating myotubes (i.e., hrGFP+/MHC+). (d) The lengths of hrGFP+/MHC+ cells were measured, and the results (mean values ± SEM) are shown as fold-elongation relative to undifferentiating cells (n indicated in parentheses, **P < 0.01).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Effects of shSCA1 and miSCA1 on cell viability in vitro. C2C12 cells were transfected with RNAi plasmids co-expressing hrGFP, and differentiated. Photomicrographs depicting hrGFP expression at 24 and 72 h after treatment are shown. Cell viability was measured by CellTiter-96 AQueous MTS assay (Promega, n = 3, mean values ± SEM).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Effects of shSCA1 and miSCA1 on cerebellar Purkinje cell viability in vivo. (a) Diagram of the recombinant AAV2/1 viral vectors containing SCA1 RNAi and hrGFP expression cassettes. (b,c) Wild-type mice were injected with either AAV1-shSCA1 or AAV1-miSCA1 into the cerebellum, and histological analyses were performed 10 weeks later. Representative photomicrographs, captured at (b) low magnification (Bar = 500 µm) and (c) high magnification (Bar = 100 µm), depicting hrGFP autofluorescence and immunohistochemical staining of calbindin-positive Purkinje cells, are shown for each treatment group.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Silencing of mutant ataxin-1 in cerebellar Purkinje cells. SCA1 mice, expressing mutant human ataxin-1 in cerebellar Purkinje cells, were injected with either AAV1-miSCA1 or AAV1-shSCA1 into the cerebellum, and histological analyses were performed 7 weeks later. Representative photomicrographs depicting hrGFP autofluorescence and immunohistochemical staining for calbindin-positive Purkinje cells and mutant human ataxin-1 are shown. Silencing of nuclear localized mutant ataxin-1 is observed in hrGFP-positive regions (gray arrows) of miSCA1-treated cerebella relative to nontransduced areas (white arrowheads). By contrast, there is a lack of ataxin-1 staining in shSCA1-treated regions, resulting from the loss of Purkinje cells as observed by the overlapping loss of calbindin staining (gray arrows). Bar = 200 µm for all photomicrographs.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions