Graduate School of Biomedical Engineering, Tohoku University, Aoba-ku, Sendai, Japan.
J Nucl Med. 2010 Jun;51(6):951-8. doi: 10.2967/jnumed.109.074443. Epub 2010 May 19.
The development of nonviral gene delivery systems is essential in gene therapy, and the use of a minimally invasive imaging methodology can provide important clinical endpoints. In the current study, we present a new methodology for gene therapy-a delivery system using nanobubbles and ultrasound as a nonviral gene delivery method. We assessed whether the gene transfer allowed by this methodology was detectable by PET and bioluminescence imaging.
Two kinds of reported vectors (luciferase and human Na/I symporter [hNIS]) were transfected or cotransfected into the skeletal muscles of normal mice (BALB/c) using the ultrasound-nanobubbles method. The kinetics of luciferase gene expression were analyzed in vivo using bioluminescence imaging. At the peak of gene transfer, PET of hNIS expression was performed using our recently developed PET scanner, after (124)I injection. The imaging data were confirmed using reverse-transcriptase polymerase chain reaction amplification, biodistribution, and a blocking study. The imaging potential of the 2 methodologies was evaluated in 2 mouse models of human pathology (McH/lpr-RA1 mice showing vascular disease and C57BL/10-mdx Jic mice showing muscular dystrophy).
Peak luciferase gene activity was observed in the skeletal muscle 4 d after transfection. On day 2 after hNIS and luciferase cotransfection, the expression of these genes was confirmed by reverse-transcriptase polymerase chain reaction on a muscle biopsy. PET of the hNIS gene, biodistribution, the blocking study, and autoradiography were performed on day 4 after transfection, and it was indicated that hNIS expression was restricted to the site of plasmid administration (skeletal muscle). Similar localized PET and (124)I accumulation were successfully obtained in the disease-model mice.
The hNIS gene was delivered into the skeletal muscle of healthy and disease-model mice by the ultrasound-nanobubbles method, and gene expression was successfully visualized with PET. The combination of ultrasound-nanobubble gene transfer and PET may be applied to gene therapy clinical protocols.
在基因治疗中，开发非病毒基因传递系统至关重要，而微创成像方法的应用则可以提供重要的临床终点。本研究提出了一种新的基因治疗方法，即利用纳米气泡和超声作为非病毒基因传递方法的传递系统。我们评估了该方法介导的基因转移是否可通过 PET 和生物发光成像检测到。
使用超声-纳米气泡方法将两种报道的载体（荧光素酶和人钠/碘同向转运体[hNIS]）转染或共转染到正常小鼠（BALB/c）的骨骼肌中。使用生物发光成像在体内分析荧光素酶基因表达的动力学。在基因转移峰值时，使用我们最近开发的 PET 扫描仪进行 hNIS 表达的 PET 扫描，之后进行（124）I 注射。通过逆转录酶聚合酶链反应扩增、生物分布和阻断研究确认成像数据。在 2 种人类病理模型（表现血管疾病的 McH/lpr-RA1 小鼠和表现肌肉疾病的 C57BL/10-mdx Jic 小鼠）中评估了这 2 种方法的成像潜力。
转染后 4 天观察到骨骼肌中荧光素酶基因活性的峰值。hNIS 和荧光素酶共转染后第 2 天，通过肌肉活检进行逆转录酶聚合酶链反应确认这些基因的表达。转染后第 4 天进行 hNIS 基因的 PET、生物分布、阻断研究和放射自显影，结果表明 hNIS 表达仅限于质粒给药部位（骨骼肌）。在疾病模型小鼠中成功获得了类似的局部 PET 和（124）I 积累。
超声-纳米气泡方法将 hNIS 基因递送至健康和疾病模型小鼠的骨骼肌中，并且成功地通过 PET 可视化了基因表达。超声-纳米气泡基因转导与 PET 的结合可能应用于基因治疗临床方案。
