1. Volume 23, Issue 4, Pages 627-637 (April 2015)
Intra-Amniotic rAAV-Mediated Microdystrophin Gene Transfer Improves Canine X- Linked Muscular Dystrophy and May Induce Immune Tolerance 
Hiromi Hayashita-Kinoh, Naoko Yugeta, Hironori Okada, Yuko Nitahara-Kasahara, Tomoko Chiyo, Takashi Okada, Shin'ichi Takeda 
Molecular Therapy 
Volume 23, Issue 4, Pages (April 2015)
DOI: /mt
Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Quantification of rAAV genome copy number and T-cell responses to the rAAV and transgene product in transduced dogs. (a) Quantitative PCR analysis of vector gene copy number/diploid genome in DNA samples extracted from the amnion and umbilical cords of transduced dogs. (b) IFN-γ mRNA expression in PBMCs stimulated with rAAV. PBMCs from 6-week-old transduced dogs were stimulated for 4 hours with rAAV9-microdystrophin. IFN-γ mRNA expression was determined by qRT-PCR (▵▵Ct method). Expression levels of IFN-γ in the untreated affected dog (10301MA) at same weeks old were used as the reference for calculating relative-fold increases. This DMD dog was delivered by rAAV-untreated mother. The RNA transcripts were normalized to endogenous 18S rRNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Clinical manifestations in the rAAV-transduced dogs. (a) Body weight changes in four dogs transduced with rAAV9-microdystrophin and rAAV-luciferase via amniotic fluid injection. (b) Clinical grading scale used to assess the transduced dogs. Clinical scores used to measure gait disturbance, mobility disturbance, limb or temporal muscle atrophy, drooling, macroglossia, and dysphagia are described (Supplementary Table S2).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Immunohistochemical detection of the transgene in the skeletal muscle. Immunohistochemical staining for luciferase and dystrophin is shown in cross-sections of muscle tissue from the negative control dog (6105MA), the intra-amniotic vector-transduced affected dog (6104MA) and the affected dog with intra-amniotic vector transduction followed by systemic AAV injection (6102MA). (a) Tibialis anterior and (b) diaphragm, at 2 years and 4 months of age. Scale bars = 100 μm. (c) Fibrillization of the diaphragm of the 6101MN, 6102MA, 6104MA, and 6105MA. Sirius Red staining of the diaphragm of the affected dogs. (d) Fibrotic areas of the both dogs are estimated by BZ-analyzer software (Keyence, Osaka, Japan) and analyzed by ImageJ software. *P < unpaired t-test.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Macroscopic findings and transgene expression in the hearts of transduced dogs. (a) Gross morphology of the heart from each dog. The bottom panels show a cross sectional view. Scale bar = 2 cm. White dotted lines show the position of cross dissection. White rectangles show the position of immunohistochemical sampling. LV; thickness of left ventricular wall and IVS; thickness of intraventricular septum. (b) Immunohistochemical analysis of the cardiac muscle tissue from each dog. Scale bar = 100 μm. (c) Summary of the 24-hour Holter ECG recordings for the transduced dogs at 65 weeks of age. Data shown are mean ± SEM. PAC, premature atrial contraction; PVC, premature ventricular contraction; SVT, supraventricular tachycardia.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 4
Macroscopic findings and transgene expression in the hearts of transduced dogs. (a) Gross morphology of the heart from each dog. The bottom panels show a cross sectional view. Scale bar = 2 cm. White dotted lines show the position of cross dissection. White rectangles show the position of immunohistochemical sampling. LV; thickness of left ventricular wall and IVS; thickness of intraventricular septum. (b) Immunohistochemical analysis of the cardiac muscle tissue from each dog. Scale bar = 100 μm. (c) Summary of the 24-hour Holter ECG recordings for the transduced dogs at 65 weeks of age. Data shown are mean ± SEM. PAC, premature atrial contraction; PVC, premature ventricular contraction; SVT, supraventricular tachycardia.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 5
Respiratory function of the AAV-transduced dogs. (a) SpO2 values measured at the earlobe of the transduced dogs at 70 weeks old for 2 minutes using a pulse oximeter. Bars show mean value. (b) Respiratory function in the untransduced and transduced dogs at 70 weeks old was monitored by whole-body plethysmography. Representative respiratory flow measurements from the transduced normal dog (6101MN), the untransduced affected dog (6105MA and 6104MA) and the transduced affected dog (6102MA) are shown. (c) The respiratory frequency (f) and tidal volume (TV) of the analyzed dogs is shown. Tidal volume was normalized to body weight (BW). Mean ± SEM.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions