この研究は、動脈の細胞への血管遺伝子の形質導入および発現を無線周波（RF）エネルギーによって強化する治療法を、MRIガイド下で、MRI用の加熱ガイドワイヤを血管内加熱ビークルとして使用して実施することの実現可能性を検討した。まず、インビトロ実験で、血管細胞への遺伝子形質導入および発現を強化するためのRFエネルギーの有効性を評価した。細胞培養プレートで、緑色蛍光タンパク質遺伝子（gfp）を含むレンチウイルスの形質導入と発現の効率を、RF加熱条件を変えて評価した。次に、インビボ実験として、治療に用いる遺伝子である血管内皮増殖因子165（VEGF-165）を含むレンチウイルスベクターを、ブタ（正常およびアテローム性動脈硬化症のブタ：合計9頭）の全部で18本の大腿腸骨動脈（9つの動脈ペア）に遺伝子送達バルーンカテーテルを使用して転移した。遺伝子導入中、各ブタの1つの大腿腸骨動脈は、血管内MRイメージング下で加熱ガイドワイヤを介して転送されるRFエネルギーで約41℃に加熱され、反対側の動脈は加熱されなかった（対照）。6日目に、RF加熱動脈群と非加熱動脈群のVEGF-165形質導入および発現効率を比較するための定量的ウエスタンブロット分析用に18本の動脈を摘出した。その結果、ウエスタンブロット分析で、RF加熱群の正常動脈のVEGF-165の発現は、非加熱群の発現レベルの300 %に増強された；RF加熱群のアテローム性動脈硬化動脈での発現レベルは、非加熱群のレベルの986 %となった、と報告している。
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To evaluate the feasibility of radiofrequency-enhanced vascular gene transduction and gene expression by using a magnetic resonance imaging-heating guidewire as an intravascular heating vehicle during magnetic resonance imaging-guided therapy.
Gene therapy presents great potential for the treatment of artherosclerotic cardiovascular disease. A magnetic resonance imaging-guidewire was designed to fulfill three functions simultaneously: to serve as a receiver antenna for generating high-spatial-resolution magnetic resonance images of vessel walls, a conventional guidewire for guiding endovascular interventions (such as balloon angioplasty and vascular gene transfer) and a potential intravascular heating source for local heating in targeted vessel segments.
The investigation included in vitro evaluation of the use of radiofrequency energy to enhance gene transduction and gene expression in vascular smooth muscle cells, as well as in vivo validation in pig arteries.
For in vitro experiments, cells were seeded in each of four chambers of a cell culture plate and a green fluorescent protein gene-bearing lentivirus was added. One of the chamber was heated at approximately 41°C for 15 minutes by using an magnetic resonance imaging-heating guidewire.
For the in vivo experiments, a lentivirus vector bearing the therapeutic vascular endothelial growth factor 165 gene was transferred by using a gene delivery balloon catheter in 18 femoral-iliac arteries (nine artery pairs) in pigs (with and without atherosclerosis). During gene infusion, one artery in each pig was heated to approximately 41°C with radiofrequency energy transferred via the intravascular magnetic resonance imaging-heating guidewire, while the contralateral artery was not heated (control condition).
The MR imaging-heating guidewire was configured in such a way that simultaneous MR imaging and RF heating was possible with the same MR imaging-heating guide wire.
|ばく露時間||continuous for 15 min|
|Additional information||In vitro experiments|
|チャンバの詳細||Cell culture plate with four chambers in which smooth muscle cells were cultured.|
|ばく露装置の詳細||The MR imaging-heating guidewire was attached to the bottom of chamber 4 with the hot spot of the guidewire located at the center of the chamber bottom.|
|ばく露時間||approximately 20 min|
|Additional information||In vivo experiments|
|ばく露装置の詳細||A lentivirus vector bearing a therapeutic gene was transferred by using a gene delivery balloon catheter in 18 femoral-iliac arteries in domestic pigs and Yucatan pigs with atherosclerosis. During the gene infusion, one femoral-iliac artery in each pig was heated by RF exposure. The contralateral artery was not heated and served as control.|
|Additional information||Infusion was given for continuously for 8.5 at a flow rate of 10 mL/h or intermittently at a flow rate of 21 mL/h for 8 min (4 increments of 2 mins each with 2 min interval between them).|
In vitro experiment: Confocal microscopy revealed green fluorescent protein expression in the heated chamber that was 293% the level of expression in chamber 1 (non-heated).
In vivo experiment: Data of Western blot analysis showed vascular endothelial growth factor 165 gene expression for normal arteries in the radiofrequency-heated group that was 300% the level of expression in the non-heated group, and, for atherosclerotic arteries in the radiofrequency-heated group, 986% the level in the non-heated group.
Simultaneous monitoring and enhancement of vascular gene delivery and gene expression is feasible with the magnetic resonance imaging-heating guidewire.