Computer-assisted Hydrodynamic Gene Delivery Takeshi Suda, Kieko Suda, Dexi Liu  Molecular Therapy  Volume 16, Issue 6, Pages 1098-1104 (June 2008) DOI: 10.1038/mt.2008.66 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Schematic presentation of computer-controlled hydrodynamic injection device (A/D, analog-to-digital converter). Molecular Therapy 2008 16, 1098-1104DOI: (10.1038/mt.2008.66) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Pressure profiles of hydrojector-assisted injection. (a) Experimental setup for computer-assisted hydrodynamic gene delivery into liver [arrowheads: clamping sites on the inferior vena cava (IVC)]. (b) Pressure profiles of hydrodynamic injection with the peak pressure set at 10, 15, or 20 mm Hg. (c), Pressure profiles of hydrodynamic injection with fixed peak pressure and different injection durations for reaching peak pressure. (d) Pressure profile of a prolonged injection at the peak pressure. (e) Pressure profile of an injection at peak pressure followed by a sustained injection at lower pressure. (f) Pressure profile of repeated injections with the same peak pressure and time intervals of 250 ms. Molecular Therapy 2008 16, 1098-1104DOI: (10.1038/mt.2008.66) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 Comparison of conventional (systemic) and computer-assisted (local) hydrodynamic gene delivery. (a) Comparison in respect of injection volume. (b) Comparison of levels of luciferase gene expression. (c, d) Comparison of densities of transfected cells in the liver. (e) Comparison of pressure profiles in the inferior vena cava (IVC) (blue line, computer-assisted injection; grey line, conventional hydrodynamic injection through the tail vein). RLU, relative light unit. Molecular Therapy 2008 16, 1098-1104DOI: (10.1038/mt.2008.66) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Hydrojector-assisted hydrodynamic gene delivery into muscle and kidney in rats. (a) Intrafemoral vein pressure during a continued injection (10 seconds) with programmed peak pressure at 350 mm Hg. (b) Comparison between conventional (systemic) and computer-assisted (local) hydrodynamic gene delivery in respect of levels of luciferase gene expression in muscle. (c) Intrarenal vein pressure of computer-assisted hydrodynamic injection into the right kidney with intravascular pressure set at 100 mm Hg. (d) Comparison between conventional (systemic) and computer-assisted (local) hydrodynamic gene delivery in respect of the levels of luciferase gene expression in kidney. The values shown on the bars in b and d represent the injection volumes presented as a percentage of total body weight. The data are presented as mean values ± SD, n = 3. RLU, relative light unit. Molecular Therapy 2008 16, 1098-1104DOI: (10.1038/mt.2008.66) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 Comparison of the levels of green fluorescent protein (GFP) expression in muscle and kidney. Fluorescence microscope images were taken (a,c) 4 days after computer-assisted hydrodynamic delivery, or (b,d) slow infusion of Ad-GFP vectors in (a,b) muscle, and (c,d) kidney. Immunohistochemical examination of the (e) nontargeted kidney sample, and targeted kidney sample (f) without or (g,h) with anti-GFP antibody staining. Clear positive signals are seen, both in the (g) medulla and (h) cortex of the surrounding glomerulus. Original magnifications are ×4 (a–d), ×10 (e,f), and ×40 (g,h). Molecular Therapy 2008 16, 1098-1104DOI: (10.1038/mt.2008.66) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 6 Image-guided hydrodynamic gene delivery to pig liver and kidney. (a,b) Fluoroscopic images of the pig liver and kidney showing the relative locations of the inserted balloon catheters [the injection balloon catheter (*), the occlusion balloon in portal vein (**), and the occlusion balloon in the IVC (***)]. The distribution of phase contrast medium injected through the injection catheter to verify obstruction of blood vessels by the balloon catheters is also indicated (****). (c) Lateral hepatic vein pressure profile during and soon after hydrodynamic injection with peak pressure set at 100 mm Hg. (d) Right renal vein pressure profile during and soon after hydrodynamic injection with peak pressure set at 240 mm Hg. (e) Photograph of hydrodynamically transfected pig liver in cross-section, showing the levels of luciferase gene expression at the approximate sites from which liver samples were collected. (f) Photograph of hydrodynamically transfected right kidney cut in half in the middle, showing the levels of luciferase gene expression at the sites from which tissue samples were collected. (g) Photograph of untreated left kidney cut in half in the middle, showing the background level of luciferase activity. The levels of luciferase gene expression were determined 6 hours after gene delivery. The data presented are from two pigs, one each for gene delivery to the liver and the kidney. The values in e–g represent luciferase activity in relative light unit/mg of extracted proteins. Molecular Therapy 2008 16, 1098-1104DOI: (10.1038/mt.2008.66) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 7 Assessment of tissue damage after hydrodynamic gene delivery using the computer-controlled injection device. Serum concentrations of tissue-specific marker enzymes in rats [(a) alanine aminotransferase (ALT) for liver, (b) creatinine (Crt) for kidney, and (c) creatinine phosphokinase (CPK) for muscle] at 6, 24, 48, 72, 120, and 168 hours after (closed circles) computer-assisted hydrodynamic gene delivery, or after (open circles) a slow (60 seconds) infusion of the same volume of saline. Open bars in each plot represent the serum concentration of marker enzyme in untreated animals. The data are presented as mean values ± SD, n = 3. Molecular Therapy 2008 16, 1098-1104DOI: (10.1038/mt.2008.66) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions