Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors

Abstract

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T 2 /T 2 * (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T 2 *-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging. Gene Therapy (2011) INTRODUCTIONThe development of non-invasive imaging methods that can reliably report on therapeutic cell transplantation and/or gene expression is a critical step in the establishment of gene therapy protocols, both for clinical and for research applications. Several molecular imaging modalities are available that enable non-invasive and repeated imaging of gene expression in targeted cells in living organisms, thereby reducing the number of laboratory animals and reducing the inter-animal variability at the preclinical level. Among these are radionuclide imaging techniques such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET), optical imaging methods including fluorescence imaging and bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and spectroscopy, ultrasound and X-ray-based methods (for a review, see the studies by Massoud and Gambhir 1 and Deroose et al. 2 ). Apart from optical imaging methods, molecular imaging technologies using these imaging modalities have the advantage of being translatable to a clinical setting.When comparing imaging modalities, BLI, PET and SPECT provide high sensitivity but low resolution, whereas MRI can reach near-cellular resolution, 3-6 which makes MRI ideally suited to provide information on the location and migration of targeted cells in vivo.

Human neural stem cell intracerebral grafts show spontaneous early neuronal differentiation after several weeks

“…The low sensitivity of Ferritin unfortunately does not support MRI's superior three-dimensional in vivo resolution, in line with previously reported limitations [37]. The Ferritin overexpression and accumulation of iron as ferrihydrite linked to neuronal differentiation induced only small susceptibility changes, detectable only by quantitative T2* mapping.…”

Evaluating the effectiveness of transferrin receptor‐1 (TfR1) as a magnetic resonance reporter gene

“…Taken together, these data indicate that, for all cell types tested to date, FC supplementation appears to be a much more effective means of increasing intracellular iron concentrations than does overexpressing TfR1 , suggesting that to track cells with MRI it might be sufficient to simply incubate cells in the presence of FC shortly before implantation, rather than undertaking the more involved procedure of genetically manipulating the cells so that they overexpress TfR1 . This is also the case for the ferritin reporters, where, similarly to TfR1 , overexpression does not always increase intracellular iron concentrations as effectively as supplementation with FC 17, 30, 31, 32. However, such an approach would only be suitable for short‐term tracking, as it would be expected that with time the iron load would return to baseline levels and the cells would no longer be detectable via MRI.…”

CHAPTER 11. Magnetic Resonance‐Based Cell Imaging Using Contrast Media and Reporter Genes

“…155 The feasibility of using FerrH as an MRI reporter by in situ viral vector mediated delivery to the brain has been shown for different viral vector systems. 141,156,157 Upon injection of a FerrH over-expressing vector into the mouse brain, significant hypointense MRI contrast is produced at the site of vector injection. 141,156,157 Depending on the viral system used, vector-based delivery of MR reporter genes can elicit unspecific contrast due to the vector itself at the site of injection.…”