Ultrasound-mediated delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen size in a porcine interposition graft model

Abstract

Progressive saphenous vein graft (SVG) narrowing and occlusion remains a major limitation of coronary artery bypass grafting and is an important target for gene therapy. Ex vivo adenoviral gene transfer of tissue inhibitor of metalloproteinase 3 (TIMP-3) reduces adverse SVG remodelling postarterialization, but concerns remain over the use of viral vectors in patients. Ultrasound exposure (USE) in the presence of echocontrast microbubbles (ECM) substantially enhances nonviral gene delivery. We investigated the effects of ultrasound-enhanced gene delivery (UEGD) of TIMP-3 plasmid on vascular remodelling in porcine SVG. Maximal luciferase activity (3000-fold versus naked plasmid alone) and TIMP-3 transgene expression in porcine vascular smooth muscle cells in vitro was achieved using USE at 1 MHz, 1.8 mechanical index (MI), 6% duty cycle (DC) in the presence of 50% (v/v) BR14 ECM (Bracco). These conditions were therefore utilized for subsequent studies in vivo. Yorkshire White pigs received carotid interposition SVG that were untransfected or had undergone ex vivo UEGD of lacZ (control) or TIMP-3 plasmids. At 28 d postgrafting, lumen and total vessel area were significantly greater in the TIMP-3 group (10.171.2 and 25.572.2 mm 2 , respectively) compared to untransfected (6.3470.5 and 20.871.9 mm 2 ) or lacZ-transfected (6.170.7 and 19.771.2 mm 2 ) controls (Po0.01). These data indicate that nonviral TIMP-3 plasmid delivery by USE achieves significant biological effects in a clinically relevant model of SV grafting, and is the first study to demonstrate the potential for therapeutic UEGD to prevent SVG failure.

Gene therapy progress and prospects: Ultrasound for gene transfer

“…Indeed, we found that ex vivo transfection of saphenous vein segments using ultrasound yielded only 50-fold enhancements compared with the 3000-fold effects of ultrasound upon vascular smooth muscle cell transfection in vitro. 4 However, in vivo transfection is always less efficient than in vitro and several of the studies in Table 1 also report that the maximum efficiency of UEGT was similar to or better than viral or liposome-mediated transfection. Transgene expression post UEGT is transient, certainly when using standard promoters such as cytomegalovirus, generally peaking at 4-7 days and with rare exceptions is essentially gone by 21 days.…”

“…Table 2 summarizes some recent examples using animal models and therapeutic transgenes that have potential clinical relevance. 4,37,55,56,69,70 Although these and other in vivo studies provide proofof-concept that UEGT can indeed achieve therapeutically effective gene transfer, the delivery approaches used (e.g. direct injection of plasmid/MCB into the liver or left ventricular cavity, UEGT requiring temporary cessation of blood flow in target organs) are in many cases unattractive for clinical use, certainly if repeat administrations for chronic conditions are contemplated.…”

“…The development of ultrasound enhanced gene transfer (UEGT) began when a number of research groups speculated that the physical perturbations induced by an ultrasound beam would encourage plasmid DNA uptake and transgene expression. Although the early results were at best modest, achieving 10-to 30-fold increases in transfection efficiency in vitro, technical refinements have meant that enhancements of up to several thousand fold in vitro are now being observed, [1][2][3][4][5][6][7][8][9] sufficient to encourage studies of UEGT in vivo.…”

“…31 Cavitation 32 has received the most attention, as many scientists have shown that the deliberate addition of echocontrast MCB 33,34 (used to enhance clinical ultrasound imaging) to the transfection medium before USE markedly increases subsequent transgene expression in vitro. 4,7,23,28,[35][36][37] Cavitation may be subdivided into two categories (see Figure 1):…”

Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart

“…These qualities mean that BR14 may be a better agent in sonoporation-mediated transfection. Indeed, recent studies have indicated that BR14 effectively enhanced sonoporation-based gene transfection into hepatic cancer implant in mice [21] as well as into saphenous vein graft in porcine [22].…”