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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; 12(7):e009063CC

Abstract

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.

Authors+Show Affiliations

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.Department of Radiology and Biomedical Imaging (X.P., J.A.O., J.W., A.J.S.), Yale School of Medicine, New Haven, CT.Department of Radiology and Biomedical Imaging (X.P., J.A.O., J.W., A.J.S.), Yale School of Medicine, New Haven, CT.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 Radiology and Biomedical Imaging (X.P., J.A.O., J.W., A.J.S.), Yale School of Medicine, New Haven, CT.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 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 Biomedical Engineering (B.A.F., B.H.A.), University of Virginia, Charlottesville. Division of Cardiovascular Medicine, Department of Medicine (B.A.F., B.H.A.), University of Virginia, Charlottesville.Department of Biomedical Engineering (B.A.F., B.H.A.), University of Virginia, Charlottesville. Division of Cardiovascular Medicine, Department of Medicine (B.A.F., B.H.A.), University of Virginia, Charlottesville.Department of Cellular and Molecular Physiology (S.R., N.C.), Yale School of Medicine, New Haven, CT.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 Radiology and Biomedical Imaging (X.P., J.A.O., J.W., A.J.S.), Yale School of Medicine, New Haven, CT.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31296047

Citation

Boutagy, Nabil E., et al. "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." Circulation. Cardiovascular Imaging, vol. 12, no. 7, 2019, pp. e009063.
Boutagy NE, Ravera S, Papademetris X, et al. 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;12(7):e009063.
Boutagy, N. E., Ravera, S., Papademetris, X., Onofrey, J. A., Zhuang, Z. W., Wu, J., ... Sinusas, A. J. (2019). 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. Circulation. Cardiovascular Imaging, 12(7), pp. e009063. doi:10.1161/CIRCIMAGING.119.009063.
Boutagy NE, et al. 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;12(7):e009063. PubMed PMID: 31296047.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Boutagy,Nabil E, AU - Ravera,Silvia, AU - Papademetris,Xenophon, AU - Onofrey,John A, AU - Zhuang,Zhen W, AU - Wu,Jing, AU - Feher,Attila, AU - Stacy,Mitchel R, AU - French,Brent A, AU - Annex,Brian H, AU - Carrasco,Nancy, AU - Sinusas,Albert J, Y1 - 2019/07/12/ PY - 2020/07/12/pmc-release PY - 2019/7/13/entrez PY - 2019/7/13/pubmed PY - 2019/7/13/medline KW - animals KW - genetic therapy KW - neuraminidase KW - peripheral artery disease KW - tomography, emission computed, single-photon SP - e009063 EP - e009063 JF - Circulation. Cardiovascular imaging JO - Circ Cardiovasc Imaging VL - 12 IS - 7 N2 - 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. SN - 1942-0080 UR - https://www.unboundmedicine.com/medline/citation/31296047/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 L2 - http://www.ahajournals.org/doi/full/10.1161/CIRCIMAGING.119.009063?url_ver=Z39.88-2003&amp;rfr_id=ori:rid:crossref.org&amp;rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -