Volume 11, Issue 2, Pages 257-266 (February 2005) Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model  Gawiyou Danialou, Alain S. Comtois, Stefan Matecki, Josephine Nalbantoglu, George Karpati, Renald Gilbert, Pascale Geoffroy, Sandra Gilligan, Jean-Francois Tanguay, Basil J. Petrof  Molecular Therapy  Volume 11, Issue 2, Pages 257-266 (February 2005) DOI: 10.1016/j.ymthe.2004.09.016 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 (A) Time course of changes in IM pressure within the rat tibialis anterior (TA) and gastrocnemius (GAST) during intraarterial injections. Values for IM pressure were significantly higher in the TA at all time points shown after injection. (B) Quantification of β-gal expression in rat hind limb muscles after pDNA administration into the femoral artery. Both the total number of β-gal-expressing fibers (open bars) and the amount of β-gal protein (filled bars) were significantly greater in the GAST compared to the TA. (C) Relationship between peak IM pressure and the number of β-gal-expressing fibers in rat TA and GAST muscles. Note the significant negative correlation between IM pressure and number of β-gal-expressing fibers, irrespective of the use of histamine. Symbols represent the following: circles, GAST; squares, TA; open symbols, without histamine; filled symbols, with histamine. Molecular Therapy 2005 11, 257-266DOI: (10.1016/j.ymthe.2004.09.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 (A) Schematic illustration of experimental setup in the pig (BP, arterial blood pressure; CVP, central venous pressure). (B) Representative physiological recordings of IM pressure and hemodynamic variables during 25 ml/kg volume injection into the pig femoral artery. Double-headed arrow indicates the onset of the injection. Molecular Therapy 2005 11, 257-266DOI: (10.1016/j.ymthe.2004.09.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Differential effects of intraarterial volume delivery on IM pressure and arterial blood pressure during 12.5 (n = 2), 16 (n = 1), 19 (n = 1), and 25 (n = 2) ml/kg volume administration into the pig femoral artery. Molecular Therapy 2005 11, 257-266DOI: (10.1016/j.ymthe.2004.09.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Quantification of (A) total number of β-gal-expressing fibers and (B) β-gal protein, after pDNA administration into the pig femoral artery using an injection volume of either 12.5 or 25 ml/kg. Four muscles were sampled from the anterior compartment (TA; EDL, extensor digitorum longus; PT, peroneus tertius; PL, peroneus longus) as well as the posterior compartment (GAST; SOL, soleus; TC, tibialis caudalis; FDL, flexor digitorum longus) of the pig hind limb. Data represent the average values pooled from all regions (proximal, middle, and distal) of each muscle. Molecular Therapy 2005 11, 257-266DOI: (10.1016/j.ymthe.2004.09.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Representative photomicrographs of histochemical staining for β-gal in GAST, SOL, EDL, and TA at the two different injection volumes (12.5 and 25 ml/kg). Magnification is identical for all photomicrographs; scale bar, 1 mm. Molecular Therapy 2005 11, 257-266DOI: (10.1016/j.ymthe.2004.09.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Number of β-gal-expressing fibers quantified within proximal, middle, and distal muscle regions under optimized conditions (12.5 ml/kg injection volume). In most cases, there was higher transfection within the more proximal regions of targeted muscles. Molecular Therapy 2005 11, 257-266DOI: (10.1016/j.ymthe.2004.09.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 (A) Creatine kinase and (B) lactate dehydrogenase activities at 24, 48, 72, and 120 h after injection of either 12.5 or 25 ml/kg volume into the pig femoral artery. The mean data are expressed as a percentage of baseline values obtained for each animal immediately prior to the intraarterial injection. Data include pigs that received intraarterial volume injections without pDNA, which did not differ in their enzyme leakage responses. Molecular Therapy 2005 11, 257-266DOI: (10.1016/j.ymthe.2004.09.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions