Volume 12, Issue 3, Pages 555-561 (September 2005) Toward a Gene Therapy for Dominant Disease: Validation of an RNA Interference- Based Mutation-Independent Approach  Anna-Sophia Kiang, Arpad Palfi, Marius Ader, Paul F. Kenna, Sophia Millington-Ward, Gerry Clark, Avril Kennan, Mary O'Reilly, Lawrence C.T. Tam, Aileen Aherne, Niamh McNally, Pete Humphries, G. Jane Farrar  Molecular Therapy  Volume 12, Issue 3, Pages 555-561 (September 2005) DOI: 10.1016/j.ymthe.2005.03.028 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Titration curves from RT-PCR data comparing the relative efficiencies of shRNA (shMR3) and siRNA (siMR3) at targeting murine rhodopsin transcript expressed in COS-7 cells. Molecular Therapy 2005 12, 555-561DOI: (10.1016/j.ymthe.2005.03.028) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Expression of shMR3 and siMR3 in COS-7 cells 24 h posttransfection as shown by RPAs using 5′-end-labeled sense (SE PROBE) and antisense (AS PROBE) strands of siMR3. Lanes 1 and 18, molecular weight marker; 2 and 10, 5 μg shMR3; 3 and 11, 5 μg shPER2; 4 and 12, 0.05 μg (4 pmol) siMR3; 5 and 13, 0.25 μg (20 pmol) siMR3; 6 and 14, 0.5 μg (40 pmol) siMR3; 7 and 15, 2.5 μg (200 pmol) siMR3; 8 and 16, 5 μg (400 pmol) siMR3; 9 and 17, 5 μg (400 pmol) siPER2. β-Actin was used as an internal standard. Molecular Therapy 2005 12, 555-561DOI: (10.1016/j.ymthe.2005.03.028) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Immunostaining of electroporated retinal explants. Scale bars denote 20 μm. (A) Scanning laser micrograph of a section through a 14-day cultured retinal explant electroporated with EGFP-shMR3 at P0 and stained with rhodopsin antibodies. ROS, rod outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B–G) Dissociated retinal cells immunostained with rhodopsin antibodies (B, E), showing EGFP fluorescence (C, F), and DAPI stained (D, G), electroporated with nontargeting EGFP-shNT (B–D) or targeting EGFP-shMR3 (E–G). Molecular Therapy 2005 12, 555-561DOI: (10.1016/j.ymthe.2005.03.028) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Transcript and rhodopsin protein analyses of FACS-sorted dissociated retinal explant cells. (A) RT-PCR analysis of endogenous transcripts of rhodopsin, β-Pde, Eef2, Atp6, Ppia, and Pk3 targeted with EGFP-shMR3. (B) Percentage of EGFP-positive cells expressing murine rhodopsin protein following electroporation with EGFP-shNT and EGFP-shMR3. Molecular Therapy 2005 12, 555-561DOI: (10.1016/j.ymthe.2005.03.028) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 RT-PCR analyses of suppression and replacement of murine rhodopsin transcript. (A) Rhodopsin expression following cotransfections of rhodopsin-expressing constructs MR, MR1, HR, MR7, and MR + MR7 with shMR3 and control shCOL4 in COS-7 cells. (B) Rhodopsin expression in murine liver following systemic delivery of MR or MR7 with control (siPER2) or targeting (siMR3) siRNA. Molecular Therapy 2005 12, 555-561DOI: (10.1016/j.ymthe.2005.03.028) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 6. Suppression of murine rhodopsin by RNAi Fig. 6. Suppression of murine rhodopsin by RNAi. RT-PCR analysis shows residual rhodopsin expression following separate co-transfections of murine rhodopsin cDNA with each of five shRNAs (shMR1-shMR5) and an siRNA (siMR3) in cos-7 cells. Molecular Therapy 2005 12, 555-561DOI: (10.1016/j.ymthe.2005.03.028) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions