Key result
Hindlimb ischemia and neuraminidase desialylation increase 99mTcO4− uptake ~4-fold versus nonischemic AAV9-hNIS-treated muscle.
Why the study?
Noninvasive quantification of AAV9-mediated gene expression in skeletal muscle under ischemic conditions using hNIS imaging was not established in a murine model of peripheral artery disease.
Micro SPECT/CT imaging of hNIS allows for accurate in vivo quantification of AAV9-driven gene expression in skeletal muscle, which is enhanced by ischemia and neuraminidase desialylation.
Supports hNIS-SPECT for AAV gene expression monitoring in ischemic muscle; extends preclinical models but leaves open human translation.
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 (2×10 11 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 ( 99m TcO 4 − ), 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 99m TcO 4 − ± 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 99m TcO 4 − uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99m TcO 4 − uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99m TcO 4 − uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r 2 =0.9375; P <0.0001) and immunoblot analysis of NIS protein (r 2 =0.65; P <0.0001). Conclusions: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99m TcO 4 − uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.
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Boutagy et al. (2019) studied Peripheral artery disease (murine model) (n=78). AAV9-hNIS injection ± α-sialidase neuraminidase vs. Control nonischemic limbs injected with phosphate buffered saline or noninjected was evaluated on 99mTcO4− uptake quantitated by standardized uptake value (4- to 5-fold increase). Hindlimb ischemia and neuraminidase desialylation increased 99mTcO4− uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS.
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