Noninvasive In Vivo Quantification of Adeno-Associated Virus Serotype 9-Mediated Expression of the Sodium/Iodide Symporter Under Hindlimb Ischemia and Neuraminidase Desialylation in Skeletal Muscle Using Single-Photon Emission Computed Tomography/Computed Tomography

Circ Cardiovasc Imaging. 2019 Jul;12(7):e009063. doi: 10.1161/CIRCIMAGING.119.009063. Epub 2019 Jul 12.


Background: 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.

Methods: AAV9-hNIS (2×1011 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 (99mTcO4-), 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 99mTcO4- ± 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.

Results: As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001).

Conclusions: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.

Keywords: animals; genetic therapy; neuraminidase; peripheral artery disease; tomography, emission computed, single-photon.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Dependovirus / genetics*
  • Disease Models, Animal
  • Gene Expression Regulation / physiology*
  • Hindlimb / blood supply
  • Ischemia
  • Mice
  • Mice, Inbred C57BL
  • Muscle, Skeletal / diagnostic imaging
  • Muscle, Skeletal / metabolism*
  • Neuraminidase / metabolism*
  • Peripheral Arterial Disease / metabolism*
  • Saline Solution / administration & dosage
  • Single Photon Emission Computed Tomography Computed Tomography / methods*
  • Symporters / pharmacokinetics*


  • Saline Solution
  • Symporters
  • sodium-iodide symporter
  • Neuraminidase