Department of Medicine, Section of Cardiovascular Medicine, Yale Translational Research Imaging Center (N.E.B., Z.W.Z., A.F., M.R.S., A.J.S.), Yale School of Medicine, New Haven, CT.
Department of Cellular and Molecular Physiology (S.R., N.C.), Yale School of Medicine, New Haven, CT.
Circ Cardiovasc Imaging. 2019 Jul;12(7):e009063. doi: 10.1161/CIRCIMAGING.119.009063. Epub 2019 Jul 12.
We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease.
AAV9-hNIS (2×10 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (TcO), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with TcO ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification.
As quantitated by standardized uptake value, there was a gradual temporal increase in TcO uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of TcO uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced TcO uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r=0.65; P<0.0001).
Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated TcO uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.
我们提出微单光子发射计算机断层扫描/计算机断层扫描成像 hNIS（人钠/碘同向转运体），以无创方式定量腺相关病毒 9（AAV9）介导的外周动脉疾病小鼠模型中的基因表达。
在单侧后肢缺血后，将 AAV9-hNIS（2×10 病毒基因组颗粒）注射到 C57Bl/6J 小鼠的非缺血或缺血比目鱼肌中±α-唾液酸酶 NA（神经氨酸酶）。对照非缺血肢体注射磷酸盐缓冲盐水或保持未注射。12 只小鼠在 AAV9-hNIS 注射后 28 天内连续注射放射性碘底物 pertechnetate（TcO）后进行微单光子发射计算机断层扫描/计算机断层扫描成像。24 只动物在 1 个月内的选定时间点处死，用于 1 个月的离体验证。42 只动物在第 10 天用 TcO±选择性 NIS 抑制剂高氯酸盐进行成像，以确定放射性示踪剂摄取的特异性。采集组织进行离体验证。使用改进的 U-Net 深度学习算法进行图像定量。
通过标准化摄取值定量，用 AAV9-hNIS 治疗的肌肉中 TcO 摄取呈逐渐增加的时间趋势。与仅用 AAV9-hNIS 治疗的非缺血肌肉相比，后肢缺血、NA 和后肢缺血加 NA 使 TcO 摄取增加 4-5 倍。高氯酸盐处理显著降低了 AAV9-hNIS 治疗肌肉中的 TcO 摄取，表明摄取具有特异性。成像结果与离体计数（r=0.9375；P<0.0001）和 NIS 蛋白免疫印迹分析（r=0.65；P<0.0001）高度相关。
微单光子发射计算机断层扫描/计算机断层扫描成像 hNIS 介导的 TcO 摄取可准确进行体内定量 AAV9 驱动的基因表达，该表达在缺血条件下或骨骼肌中神经氨酸酶去唾液酸化时增加。
