Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer

Purpose

We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1?? (HIF-1??-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively.

Procedures

The plasmid pUbi-hrl-pUbi-HIF-1??-VP2, which expresses both hrl and HIF-1??-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1??-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI).

Results

Strong correlations existed between the expression of hRL and each of HIF-1??-VP2, VEGF, and PlGF (r ²?>?0.83, r ²?>?0.82, and r ²?>?0.97, respectively). In vivo, both transplanted cells and HIF-1??-VP2 transgene expression were successfully imaged using BLI.

Conclusions

An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein.     

MR Reporter Gene Imaging of Endostatin Expression and Therapy

Purpose

The aim of this study is to monitor endostatin gene expression and therapy using transferrin receptor (TfR) as reporter gene and transferrin conjugate of ultrasmall supramagnetic iron oxide nanoparticle (Tf–USPIO) as magnetic resonance (MR) reporter probe.

Procedure

A retroviral plasmid (pLP-LNCX) encoding mouse endostatin and TfR was constructed, and packaged with a titer of 4?×?10⁷colony-forming units per millimeter. MDA-MB-231 breast tumors were established in BALB/c mice by subcutaneous injection of 2?×?10⁶ MDA-MB-231 cells. Mice were intratumorally injected with recombinant retrovirus and imaged with MR using Tf–USPIO. Western blot, Prussian blue, and immunohistochemical staining were performed to validate the magnetic resonance imaging results. The antitumor effect of retro-endostatin (ES)-TfR was also evaluated by intratumoral injection of the viral vector.

Results

The expression of both endostatin and TfR genes in MDA-MB-231 cells after retroviral transfection was confirmed by Western blot and flow cytometry. Tf–USPIO conjugate binds specifically to cells stably transfected with retro-ES-TfR. After intravenous injection of the Tf–USPIO conjugate, there was a more pronounced decrease in T2 relaxation time in tumors treated with retro-ES-TfR than in tumors treated with empty retrovirus retro-LNCX. The expression of ES gene significantly delayed the growth of MDA-MB-231 tumor and reduction of microvessel density and VEGF level as compared to those without viral transfection or transfected with empty retro-LNCX vector.

Conclusions

Endostatin therapeutic gene expression was visualized successfully using TfR reporter gene and Tf–USPIO MR reporter probe, which indicates that MR reporter gene imaging may be valuable in gene therapy to evaluate therapeutic gene expression and treatment efficacy.     

Non-invasive Reporter Gene Imaging of Cell Therapies,including T Cells and Stem Cells

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Non-invasive PET Imaging of PARP1 Expression in Glioblastoma Models

Purpose

The current study presents [¹⁸F]PARPi as imaging agent for PARP1 expression.

Procedures

[¹⁸F]PARPi was generated by conjugating a 2H-phthalazin-1-one scaffold to 4-[¹⁸F]fluorobenzoic acid. Biochemical assays, optical in vivo competition, biodistribution analysis, positron emission tomography (PET)/X-ray computed tomography, and PET/magnetic resonance imaging studies were performed in subcutaneous and orthotopic mouse models of glioblastoma.

Results

[¹⁸F]PARPi shows suitable pharmacokinetic properties for brain tumor imaging (IC₅₀?=?2.8?±?1.1 nM; logP_CHI?=?2.15?±?0.41; plasma-free fraction?=?63.9?±?12.6 %) and accumulates selectively in orthotopic brain tumor tissue. Tracer accumulation in subcutaneous brain tumors was 1.82?±?0.21 %ID/g, whereas in healthy brain, the uptake was only 0.04?±?0.01 %ID/g.

Conclusions

[¹⁸F]PARPi is a selective PARP1 imaging agent that can be used to visualize glioblastoma in xenograft and orthotopic mouse models with high precision and good signal/noise ratios. It offers new opportunities to non-invasively image tumor growth and monitor interventions.

     

Noninvasive Imaging of Cardiac Gene Expression and Its Future Implications for Molecular Therapy

Innovative approaches for cardiovascular molecular therapy are rapidly evolving, and translational efforts from experimental to clinical application are increasing. Gene and cell therapy hold promise for treatment of heart disease, but despite progress, some basic principles are still under development. Open issues are, e.g., related to the optimal method for delivery, to therapeutic efficacy, to time course and magnitude of gene expression, and to the fate of transplanted cells in target and remote areas. The use of reporter genes and labeled reporter probes for noninvasive imaging provides the methodology to address these questions by assessment of location, magnitude, and persistence of transgene expression in the heart and the whole body. Coexpression of a reporter gene allows for indirect imaging of the expression of a therapeutic gene of choice. Furthermore, reporter genes can be transferred to stem cells prior to transplantation for serial monitoring of cell viability using gene product imaging. Additionally, functional effects of therapy on the tissue level can be identified using established imaging approaches to determine blood flow, metabolism, innervation, or cell death. Measures of transgene expression can then be linked to physiologic effects and will refine the understanding of basic therapeutic mechanisms. Noninvasive gene-targeted imaging will thus enhance the determination of therapeutic effects in cardiovascular molecular therapy in the future.     

Non-invasive Characterization of Focal Arrhythmia with Electromechanical Wave Imaging in Vivo

There is currently no established method for the non-invasive characterization of arrhythmia and differentiation between endocardial and epicardial triggers at the point of care. Electromechanical wave imaging (EWI) is a novel ultrasound-based imaging technique based on time-domain transient strain estimation that can map and characterize electromechanical activation in the heart in vivo. The objectives of this initial feasibility study were to determine that EWI is capable of differentiating between endocardial and epicardial sources of focal rhythm and, as a proof-of-concept, that EWI could characterize focal arrhythmia in one patient with premature ventricular contractions (PVCs) before radiofrequency (RF) ablation treatment. First, validation of EWI for differentiation of surface of origin was performed in seven (n?=?7) adult dogs using four epicardial and four endocardial pacing protocols. Second, one (n?=?1) adult patient diagnosed with PVC was imaged with EWI before the scheduled RF ablation procedure, and EWI results were compared with mapping procedure results. In dogs, EWI was capable of detecting whether pacing was of endocardial or epicardial origin in six of seven cases (86% success rate). In the PVC patient, EWI correctly identified both regions and surface of origin, as confirmed by results from the electrical mapping obtained from the RF ablation procedure. These results reveal that EWI can map the electromechanical activation across the myocardium and indicate that EWI could serve as a valuable pre-treatment planning tool in the clinic.     

Orthopedic Gene Therapy in 2008

Orthopedic disorders, although rarely fatal, are the leading cause of morbidity and impose a huge socioeconomic burden. Their prevalence will increase dramatically as populations age and gain weight. Many orthopedic conditions are difficult to treat by conventional means; however, they are good candidates for gene therapy. Clinical trials have already been initiated for arthritis and the aseptic loosening of prosthetic joints, and the development of bone-healing applications is at an advanced, preclinical stage. Other potential uses include the treatment of Mendelian diseases and orthopedic tumors, as well as the repair and regeneration of cartilage, ligaments, and tendons. Many of these goals should be achievable with existing technologies. The main barriers to clinical application are funding and regulatory issues, which in turn reflect major safety concerns and the opinion, in some quarters, that gene therapy should not be applied to nonlethal, nongenetic diseases. For some indications, advances in nongenetic treatments have also diminished enthusiasm. Nevertheless, the preclinical and early clinical data are impressive and provide considerable optimism that gene therapy will provide straightforward, effective solutions to the clinical management of several common debilitating disorders that are otherwise difficult and expensive to treat.     

Evaluation of Non-invasive Optogenetic Stimulation with Transcranial Functional Ultrasound Imaging

Optogenetics employs engineered viruses to genetically modify cells to express specific light-sensitive ion channels. The standard method for gene delivery in the brain involves invasive craniotomies that expose the brain and direct injections of viruses that invariably damage neural tissue along the syringe tract. A recently proposed alternative in which non-invasive optogenetics is performed with focused ultrasound (FUS)–mediated blood–brain barrier (BBB) openings has been found to non-invasively facilitate gene delivery for optogenetics in mice. Although gene delivery can be performed non-invasively, validating successful viral transduction and expression of encoded ion channels in target tissue typically involves similar invasive techniques, such as craniotomies in longitudinal studies and/or postmortem histology. Functional ultrasound imaging (fUSi) is an emerging neuroimaging technique that can be used to transcranially detect changes in cerebral blood volume following introduction of a stimulus. In this study, we implemented a fully non-invasive combined FUS–fUSi technique for performing optogenetics in mice. FUS successfully delivered viruses encoding the red-shifted channelrhodopsin variant ChrimsonR in all treated subjects. fUSi successfully identified stimulus-evoked cerebral blood volume changes preferentially in brain regions expressing the light-sensitive ion channels. Improvements in cell-specific targeting of viral vectors and transcranial ultrasound imaging will make the combined technique a useful tool for neuroscience research in small animals.     

Retroviral Integrations in Gene Therapy Trials

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient''s follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis.