SUPPLEMENTARY INFORMATION

An ex vivo Gene Therapy Approach to Treat Muscular Dystrophy

Using inducible Pluripotent Stem Cells

Antonio Filareto1, Sarah Parker1, Radbod Darabi1, Luciene Borges1, Michelina Iacovino2, Tory

Schaaf1, Timothy Mayerhofer1, Jeffrey S Chamberlain3, James M. Ervasti4, R. Scott McIvor5,

Michael Kyba2 and Rita C.R. Perlingeiro1

1Lillehei Heart Institute, Department of Medicine, University of Minnesota, 312 Church Street

SE Minneapolis, MN 55455 USA, 2Lillehei Heart Institute, Department of Pediatrics, University

of Minnesota, 312 Church Street SE Minneapolis, MN 55455 USA, 3Department of Neurology,

University of Washington School of Medicine, K243b HSB, Box 357720 1959 N.E. Pacific Street

Seattle, WA 98195-7720, USA, 4Department of Biochemistry, Molecular Biology, and

Biophysics, University of Minnesota, 321 Church Street SE Minneapolis, MN 55455 MN, USA,

5Department of Genetics, Cell Biology and Development, University of Minnesota, 321 Church

St. SE Minneapolis, MN 55455, USA.

This PDF file includes: Supplementary Figures and respective legends (Fig S1-S9) Supplementary Table S1 Supplementary Table S2

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Supplementary Figure S1. Characterization of iPS cells derived from fibroblasts of dKO mice.

(a) Morphology and SSEA-1 expression by FACS of representative iPS clone. (b) Staining for alkaline phosphatase (AP). (c) Confocal microscopy confirms the presence of pluripotency markers in dKO iPS cells, as shown by the presence of SSEA-1 in the plasma membrane, and NANOG and OCT4 in the nuclei. ES cells were used a control. (d) Morphology of iPS-derived EBs at day 2 and 5 of differentiation. (e) Representative FACS profile for Flk-1 and PDGFαR expression in day 5 EBs of dKO iPS and ES cells for reference. (f) Normal karyotype of dKO iPS clone. (g) H&E staining shows the presence of various tissues in teratomas developed after injection of dKO iPS cells into immunodeficient mice H: heterogeneous tissue under low magnification; M: mesoderm cartilage and muscle-like; En: endoderm gut-like; Ec: ectoderm keratin pearl. (h-i) qPCR for pluripotency markers Oct4 and Nanog (h) as well as imprinted genes Gtl2 and Rian (i) at the undifferentiated stage (day 0), and during EB differentiation (day 2 to 6) of dKO iPS cells. ES cells were analyzed side-by-side as reference. Error bars represent S.E.M. from three replicates of three independent experiments.

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Supplementary Figure S2. Characterization of µUTRN-corrected iPS cells following transposition.

(a) Gene expression analysis of μUtrn in corrected iPS cells (green bar). Nanog is shown as a reference since it is highly expressed in undifferentiated cells (gray bar). Error bars represent S.E.M. from three replicates of three independent experiments. (b) Normal karyotype of corrected cells.

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Supplementary Figure S3. Generation of iPax3 iPS cells and non-dox controls.

(a) Schematic representation of the inducible system: the doxycycline-inducible TRE promoter drives the Pax3 gene and an ires-mCherry reporter. (b) Representative FACS profile of iPax3 iPS cells after three rounds of sorting. Doxycycline was added to the ES medium at 0.5 µg/ml 12h before sorting. mCherry+ cells are detected only in Pax3-induced cultures (plus dox). (c) Immunoblot of Pax3-induced (+ dox) and non-induced (no dox) iPax3 iPS cells. Pax3 is detected only in the presence of dox induction. (d) Flow cytometric analyses for Flk-1 and PDGFαR expression in non-induced (no dox) day 5 EBs from iPax3 control (uncorrected) (upper panel) and corrected-iPS (lower panel) cells. Fluorescence intensity for Flk-1 is indicated on the y axis, PDGFR, mCherry (Pax3), and GFP (μUTRN) are shown on the x axis. As expected, mCherry expression is not detected in these cultures. (e) Fusion index of corrected myotubes was scored by counting the number of nuclei present in myotubes divided by the total number of nuclei. Data are mean ± S.D. of 3 independent experiments. Five representative pictures were analyzed and the percentage of positive cells was calculated among total cells.

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Supplementary Figure S4. Characterization of μUTRN-corrected iPS-derived myogenic precursors.

(a-b) Representative immunofluorescence staining of μUTRN-corrected iPS-derived myogenic precursors under proliferation (a) and differentiation (b) conditions for Pax3, Myf5, MyoD, and MHC (red). Cells are co-stained with DAPI (blue). Scale bar is 50 μm. These images are representative from three independent experiments. (c) Flow cytometric analysis of proliferating myogenic precursors at passage 3 (10 days after sorting for PDGFαR+Flk-1- cells) for M-CAD (M-cadherin), CD56 (neural cell adhesion molecule 1), VCAM1 (vascular cell adhesion molecule-1), SYND4 (syndecan-4), and CXCR4 (C-X-C chemokine receptor type 4 or CD184). Histograms show specific antibody staining (green line) versus isotype or secondary control staining (gray line). These plots are representative from 3 independent experiments.

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Supplementary Figure S5. Real time PCR analyses of corrected-μUTRN iPS-derived myogenic progenitors under proliferation and differentiation conditions.

Gene expression analysis of proliferating myogenic progenitors and their derivative myotubes. Transcripts are normalized to GAPDH. Error bars represent S.E.M. from three replicates of three independent experiments.

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Supplementary Figure S6. Transplantation analyses and control data.

(a) Immunofluorescence staining of wild-type (WT) control (Bl6) TA muscles using antibodies to Utrophin, -DG (-dystroglycan), α1-SYN (α1-syntrophin), and nNOS (red). Utrophin was localized solely at the neuromuscular junction, while the other members of the DAPC were detected along the sarcolemma. (b-c) Average CSA (b) and weight (c) on analyzed muscles. Error bars represent S.E.M. from a total of 13 transplanted. (d) Schematic diagram of experimental plan for assessing whether corrected myogenic precursors are able to replenish the satellite cell compartment. (e) Immunofluorescence staining of control PBS-injected TA muscles analyzed 1 week after re-injury. Arrowheads show the presence of host-derived newly formed myofibers as evidenced by the expression of embryonic MHC (green), and lack of utrophin (eMHC+/μUTRN-). DAPI is shown in blue. Scale bar is 50 μm.

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Supplementary Figure S7. Presence of μUTRN-corrected myofibers in the diaphragm.

(a) Representative images of one dKO mouse that displayed engraftment in the diaphragm muscle following the intravenous injection of corrected iPS-derived myogenic progenitors, as evidenced by staining with anti-utrophin antibody (red). Roman numbers within these images indicate the order of serial cuts, and ~200µm is the space between successive sections. (b) PBS-injected dKO mice showed no signal for utrophin. DAPI is shown in blue. Scale bar is 50 μm.

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Supplementary Figure S8. Control muscle cryosections of IV transplantation.

Representative images of TA (a), gastrocnemius lateralis (b), and peroneal (c) show no signal for Utrophin and α1-SYN (α1-syntrophin) in dKO mice that had received intravenous injection of PBS. DAPI is shown in blue. Scale bar is 50 μm.

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Supplementary Figure S9. Immunofluorescence analysis of different tissues following systemic delivery.

Representative images of heart (a), lung (b), and liver (c) show no signal for Utrophin in dKO mice that had received intravenous injection of μUTRN-corrected progenitors. DAPI is shown in blue. Scale bar is 50 μm.

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Supplementary Table S1. Quantification of μUTRN+ myofibers in dKO mice following systemic delivery.

Utrophin Positive fibers %

mice n° cells1 Dat2 Age3 TA GL PER Heart Lung Liver

#714 850,000 25 58 5.3 5.7 6.3 0 0 0 #708 850,000 25 58 7.9 6.2 2.5 0 0 0 #716 850,000 25 58 3.9 3.9 1.9 0 0 0 #660 640,000 21 67 5.0 3.1 1.1 0 0 0 #669 690,000 21 67 2.1 2.6 2.0 0 0 0

1Number of injected cells/150μl DMEM 10% FBS 2Days after transplantation 3Age of mice at the time of analysis

Percentage of μUTRN + myofibers in TA, gastrocnemius lateralis (GL), and peroneal (PER) muscles. Heart, Lung, and Liver were also analyzed and showed no staining. For each skeletal muscle in transplanted groups, 3-4 representative cross-sections at 1-2 mm intervals were counted. For PBS control groups and other tissues, we examined 20 random sections.

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Supplementary Table S2. Gene Expression Assay used for real time RT-PCR

Gene TaqMan Assay Provider

Gapdh Mm99999915 Applied Biosystems

Actin Mm00607939 Applied Biosystems

Nanog Mm02384862 Applied Biosystems

Oct3/4 Mm03053917 Applied Biosystems

Rian Mm01325839 Applied Biosystems

Gtl2 or Meg3 Mm00522599 Applied Biosystems

Pax3 Mm00435491 Applied Biosystems

Myf5 Mm00435125 Applied Biosystems MyoD Mm00440387 Applied Biosystems MyoG Mm00446194 Applied Biosystems

Myf6 or MRF4 Mm00435126 Applied Biosystems MyHC1 Mm01332493 Applied Biosystems Desmin Mm00802455 Applied Biosystems

μUtrophin Assay was customized by Applied Biosystems using the sequence we indicated between exon 16 and exon

53.