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New Tricks for an Aging Dog

2019; Lippincott Williams & Wilkins; Volume: 12; Issue: 7 Linguagem: Inglês

10.1161/circimaging.119.009452

1942-0080

James T. Thackeray, Pablo Bascuñana,

Muscle Physiology and Disorders

HomeCirculation: Cardiovascular ImagingVol. 12, No. 7New Tricks for an Aging Dog Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBNew Tricks for an Aging DogImaging Cardiovascular Gene Therapy with Nuclear Reporters James T. Thackeray, PhD and Pablo Bascuñana, PhD James T. ThackerayJames T. Thackeray James T. Thackeray, PhD, Department of Nuclear Medicine, Hannover Medical School, Carl Neuberg Str 1, 30625 Hannover, Germany. Email E-mail Address: [email protected] Department of Nuclear Medicine, Hannover Medical School, Germany. and Pablo BascuñanaPablo Bascuñana Department of Nuclear Medicine, Hannover Medical School, Germany. Originally published12 Jul 2019https://doi.org/10.1161/CIRCIMAGING.119.009452Circulation: Cardiovascular Imaging. 2019;12:e009452This article is a commentary on the followingNoninvasive 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 TomographySee Article by BoutagyCardiovascular imaging-based cell tracking and reporter gene technology were instrumental for demonstration of low retention of transplanted stem cells in ischemic striated muscle.1 Genetic manipulation of stem cells to express a positron emission tomography (PET) or single photon emission computed tomography (SPECT) reporter gene, for example, HSV-tk (herpes simplex virus-thymidine kinase) or NIS (sodium iodide symporter), allows for a longer imaging timeframe to delineate the fate of transplanted cells,1,2 demonstrating low engraftment of viable stem cells with paracrine effects primarily responsible for improved function. As cell therapy has evolved beyond direct cell implantation, the use of PET and SPECT reporter genes has precipitously declined in cardiology.In this issue of Circulation: Cardiovascular Imaging, Boutagy et al use the NIS reporter to noninvasively interrogate gene transfer in a model of peripheral artery disease.3 Application of an adeno-associated virus (AAV9) vector containing human NIS and GFP (green fluorescence protein) under a constitutive cytomegalovirus promoter to ischemic gastrocnemius muscle allowed visualization of NIS transfer to myocytes along the needle track up to 1 month after gene therapy. Quantification was aided by the U-Net deep learning algorithm to define the muscle boundaries. SPECT/computed tomography imaging-defined NIS expression was confirmed by ex vivo well counting and immunoblotting, revealing persistent NIS transfer to striated muscle. NIS gene transfer was enhanced in the presence of ischemia and by co-administration of neuramidinase, but no additive effect was observed. Histopathology confirmed GFP (co-transferred with NIS) colocalization with GLUT4 (glucose transporter-4) but not CD34 (cluster of differentiation 34), supporting selective transfer to striated muscle over endothelial cells.The technical capability to track effective gene therapy is necessary limited by the penetrance of the gene. While Boutagy et al3 demonstrate the transfer of the reporter gene, it is difficult to extrapolate these results to a therapeutic gene, for which in vivo transfer may be vastly different. Gene transfer of AAV9.NIS by intracoronary infusion of 1×1013 viral particles in healthy dogs was insufficient for SPECT detection of NIS. By contrast, epicardial vector administration led to a marked increase in radioiodine accumulation at 2 weeks after gene transfer, indicating the need for substantial dosing.4 Boutagy et al3 benefit from the high sensitivity of pinhole SPECT, which allows for detection of lower NIS expression than conventional SPECT or even PET. Scaling up the viral particle administration for systemic application and larger mammals poses an intimidating obstacle, as the dose required for a single muscle likely exceeds that of the entire mouse. The dose used in the present study is not excessive, rather at a similar order of magnitude used in some clinical studies,5,6 which may facilitate translation to human studies.Application of the SPECT reporter gene NIS to noninvasively assess gene therapy is an important repurposing of a cell labeling technique. Reporter gene imaging can be repurposed for tracking gene therapy, as has been evidenced in oncology, where NIS specifically is additionally used for delivery of radiation therapy via iodine-131.7The earliest incarnations of gene therapy were unsuccessful due in part to safety concerns and inability to effectively modulate the gene transfer. More recently, viral vectors such as the inactivated adeno-associated virus have been developed with an improved safety profile and effective transfer of therapeutic genes in a variety of disorders. Numerous ongoing clinical trials evaluate targeted gene therapies in peripheral vascular disease and cardiomyopathy, specifically, gene transfer of vascular endothelial growth factor-D to enhance angiogenesis, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase to improve contractility, or CXCL12 (chemokine [CXC motif] ligand-12) to modulate endogenous inflammatory and regenerative cell homing.8 A recent clinical study used perfusion PET with 15O-water to demonstrate increased myocardial perfusion reserve after VEGF-D (vascular endothelial growth factor-D) gene transfer in patients with refractory angina.9In peripheral artery disease, transfer of growth factors by intramuscular injection have been applied in phase II randomized control trials, with mixed results.8 Primary trials in peripheral artery disease have demonstrated a limited clinical benefit, which may relate to the restrictive end points of peak walking time, limb perfusion, and time to amputation or death.10–12 The principal limitations to gene therapy are postulated as dosing issues and lack of sustained gene transfer over time.8 Critical questions remain regarding viral load and repeated dosing strategies for effective gene therapy, which may be addressed using noninvasive imaging of a transferred reporter gene. While SPECT or PET reporter genes alone may not distinguish the expression pattern of a therapeutic gene, it can provide some insights into the breadth and duration of expression. Boutagy et al3 demonstrate persistent NIS expression along the injection track over 28 days, which is promising for the sustainability of gene expression.Moreover, the expanded rollout of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) microbial adaptive immune system has revolutionized genome editing technology. Use of CRISPR simplifies viral vector production and improves gene modulation efficiency, ultimately providing the opportunity for mutation correction at precise locations in the human genome to treat genetic causes of disease.13 Several studies have reported therapeutic effects in animal models after in vivo CRISPR gene editing,14,15 with the first CRISPR-based therapeutic clinical trial recently approved. Accordingly, CRISPR use for viral gene editing presages an exponential growth of gene therapy clinical trials, such that methods for in vivo monitoring may be of significant value. Indeed, CRISPR technology has been applied to insert reporter genes for monitoring cell-based therapies,16 avoiding gene insertion to the cell genome, which may be detrimental.While the innovative work of Boutagy et al bears potential to address some of these crucial open questions, some limitations of their study should be acknowledged. First, the in vivo image analysis was restricted to the whole muscle, which does not provide an indication of the gene penetrance or relation to successful gene transfer. While immune-histology and GFP fluorescence validated the localization of the gene expression, more extensive in vivo regional analysis would be necessary for translation of the technique to determine the appropriate dosing strategy and area of transfer.Second, it is relevant that the viral load used in this study was selected based on the ability to detect the signal by SPECT, which raises the spectre of applicability—namely, how valid is the transfer and expression of NIS as a surrogate for a therapeutic gene? Combination of NIS with a therapeutic gene in the viral vector would allow more accurate surrogate measurement of gene expression and would provide a more direct indication of effective gene transfer and stability. But such an approach is necessarily limited by the need for identical viral load. Accordingly, further studies would need to address the relationship between viral load and therapeutic efficacy in comparison with the reporter gene expression. Separate and inducible promoters could further fine-tune this measurement, allowing inactivation of the reporter after detection, while maintaining expression of the therapeutic gene.The lack of additive transfer enhancement by neuraminidase in the presence of ischemia raises questions of its efficacy. In healthy muscle, neuraminidase elevated the expression of NIS over the timecourse, as expected. The authors' supposition that both ischemic muscle and neuramidinase act via desialylation of cell surface glycans with a maximal threshold seems logical, though a dose response and mechanistic investigation would provide greater credence for this observation. Furthermore, it underscores the need to evaluate the efficiency of gene transfer and reporter imaging in compromised tissue, where the microenvironment may affect drug efficacy as well as nuclear imaging tracer delivery.Despite these limitations, Boutagy et al provide a thoughtful new application of an aging imaging method. The application of reporter gene imaging for tracking gene therapy is of growing interest. Both NIS and HSV-tk have relevant applications in oncology, where they may be used therapeutically for targeted radiation delivery or as suicide gene.7,17 New PET reporter-tracer combinations are being developed, with greater flexibility for target organs and lower background signal.18 Development of CRISPR gene editing foreshadows exponential growth of gene therapy. With growing application and limited outcomes measures, a reliable, noninvasive imaging-based surrogate would be valuable for research and clinical purposes. As such, the repurposing of established techniques like SPECT and PET reporter genes enables the continued evolution of nuclear cardiology to maintain its position at the cutting edge of novel therapies.DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.James T. Thackeray, PhD, Department of Nuclear Medicine, Hannover Medical School, Carl Neuberg Str 1, 30625 Hannover, Germany. Email thackeray.[email protected]deReferences1. Swijnenburg RJ, Schrepfer S, Cao F, Pearl JI, Xie X, Connolly AJ, Robbins RC, Wu JC. In vivo imaging of embryonic stem cells reveals patterns of survival and immune rejection following transplantation.Stem Cells Dev. 2008; 17:1023–1029. doi: 10.1089/scd.2008.0091CrossrefMedlineGoogle Scholar2. Terrovitis J, Kwok KF, Lautamäki R, Engles JM, Barth AS, Kizana E, Miake J, Leppo MK, Fox J, Seidel J, Pomper M, Wahl RL, Tsui B, Bengel F, Marbán E, Abraham MR. Ectopic expression of the sodium-iodide symporter enables imaging of transplanted cardiac stem cells in vivo by single-photon emission computed tomography or positron emission tomography.J Am Coll Cardiol. 2008; 52:1652–1660. doi: 10.1016/j.jacc.2008.06.051CrossrefMedlineGoogle Scholar3. 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Related articlesNoninvasive 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 TomographyNabil E. Boutagy, et al. Circulation: Cardiovascular Imaging. 2019;12 July 2019Vol 12, Issue 7 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/CIRCIMAGING.119.009452PMID: 31296046 Originally publishedJuly 12, 2019 Keywordsgenes, reportercell therapyEditorialsmolecular imagingdogssodium iodidePDF download Advertisement SubjectsGene TherapyPeripheral Vascular Disease