<em>In Vivo</em> Gene Transfer to the Rabbit Common Carotid Artery Endothelium

Abstract

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

Novel expression cassettes for increasing apolipoprotein AI transgene expression in vascular endothelial cells

“…To test if this SSRE increases APOAI expression in response to uid shear stress in vivo, we transferred the SSRE-4XETE-gApoAI-oPRE expression cassette into an HDAd vector and compared it to HDAd-4XETE-gApoAI-oPRE in vivo in rabbit carotid arteries, using a model of EC-speci c in vivo gene transfer. 28,29 Addition of the SSRE to 4XETE-gApoAI-oPRE did not alter APOAI expression (Fig. 1c; P=0.8 and 0.5 at 3 and 28 days, respectively).…”

“…After 1 week of acclimation to the animal facility, 16 male New Zealand White rabbits (3.0-3.5 kg, Western Oregon Rabbit Co., Philomath, OR) underwent surgical isolation of both common carotid arteries, followed by temporary occlusion, and local luminal infusion of either HDAd-SSRE-4XETE-gApoAI-oPRE (n=8 rabbits) or the control vector HDAd-4XETE-gApoAI-oPRE (n=8 rabbits; both vectors were infused at 2 10 11 viral particles/mL diluted in DMEM) as previously described. 29 Rabbits were randomized to each vector group using a random number generator. The operator was aware of group allocation but the person analyzing the samples was blinded to group allocation.…”

Novel expression cassettes for increasing apolipoprotein AI transgene expression in vascular endothelial cells

“…To test if this SSRE increases APOAI expression in response to uid shear stress in vivo, we transferred the SSRE-4XETE-gApoAI-oPRE expression cassette into an HDAd vector and compared it to HDAd-4XETE-gApoAI-oPRE in vivo in rabbit carotid arteries, using a model of EC-speci c in vivo gene transfer. (51,53) Addition of the SSRE to 4XETE-gApoAI-oPRE did not alter APOAI expression (Fig. 1c; P=0.8 and 0.5 at 3 and 28 days, respectively).…”

Spatial control of robust transgene expression in mouse artery endothelium under ultrasound guidance

“…However, there is still a lack of method for transgene expression in vascular endothelium. 2 Herein, an effective and specific strategy was established for noninvasive spatial control of transgene expression into the target region of the mouse artery without systemic spillover using an ultrasound and microbubble (MB) guided adeno-associated viral vector (UMGAAV) (Supplementary Fig. S1 ).…”

“…Taken together, our results established proof of concept for UMGAAV as a paradigm for spatial control of transgene expression in mouse arteries. Compared with the method that delivers viral gene vectors into arterial endothelium via surgery, 2 UMGAAV enables targeted noninvasive transgene expression and is applicable to several arteries that are inaccessible to surgery. In addition, the ultrasound imaging machine used for UMGAAV is a very common one that has been used in clinics.…”