Local Gene Delivery Research Articles

Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery.

Necrosis is a common feature of solid tumours that offers a unique opportunity for targeted cancer therapy as it is absent from normal healthy tissues. Tumour necrosis provides an ideal environment for germination of the anaerobic bacterium Clostridium from endospores, resulting in tumour-specific colonisation. Two main species, Clostridium novyi-NT and Clostridium sporogenes, are at the forefront of this therapy, showing promise in preclinical models. However, anti-tumour activity is modest when used as a single agent, encouraging development of Clostridium as a tumour-selective gene delivery system. Various methods, such as allele-coupled exchange and CRISPR-cas9 technology, can facilitate the genetic modification of Clostridium, allowing chromosomal integration of transgenes to ensure long-term stability of expression. Strains of Clostridium can be engineered to express prodrug-activating enzymes, resulting in the generation of active drug selectively in the tumour microenvironment (a concept termed Clostridium-directed enzyme prodrug therapy). More recently, Clostridium strains have been investigated in the context of cancer immunotherapy, either in combination with immune checkpoint inhibitors or with engineered strains expressing immunomodulatory molecules such as IL-2 and TNF-α. Localised expression of these molecules using tumour-targeting Clostridium strains has the potential to improve delivery and reduce systemic toxicity. In summary, Clostridium species represent a promising platform for cancer therapy, with potential for localised gene delivery and immunomodulation selectively within the tumour microenvironment. The ongoing clinical progress being made with C. novyi-NT, in addition to developments in genetic modification techniques and non-invasive imaging capabilities, are expected to further progress Clostridium as an option for cancer treatment.

P-108 - PAM and FUS-assisted local AAV gene delivery into the brain of Alzheimer’s disease murine models assessed by quantitative PET reporter gene

P-108 - PAM and FUS-assisted local AAV gene delivery into the brain of Alzheimer’s disease murine models assessed by quantitative PET reporter gene

Local delivery of adeno-associated viral vectors with electrospun gelatin nanofiber mats.

Adeno-associated viral (AAV) vectors play a significant role in gene therapy, yet the typical delivery methods, like systemic and local AAV injections, often lead to unintended off-target distribution and tissue damage due to injection. In this study, we propose a localized delivery approach for AAV vectors utilizing electrospun gelatin nanofiber mats, which are cross-linked with glutaraldehyde. The AAV vectors, which encoded a green fluorescent protein (GFP), were loaded onto the mats by immersing them in a solution containing the vectors. The amount of AAV vector loaded onto the mats increased as the vector concentration in the solution increased. The loaded AAV vector was steadily released into the cell culture medium over 3 days. The mats incubated for 3 days also showed the ability to transduce into the cells cultured on them. We evaluated the effectiveness of this delivery system by attaching the mats to mouse livers. GFP expression was visible on the surface of the liver beneath the attached mats, but not in areas in direct contact with the mats. These findings suggest that the attachment of AAV vector-loaded electrospun gelatin nanofiber mats to a target site present a promising solution for localized gene delivery while reducing off-target distribution.

Electrospun Scaffolds Enriched with Nanoparticle-Associated DNA: General Properties, DNA Release and Cell Transfection.

Biomaterial-mediated, spatially localized gene delivery is important for the development of cell-populated scaffolds used in tissue engineering. Cells adhering to or penetrating into such a scaffold are to be transfected with a preloaded gene that induces the production of secreted proteins or cell reprogramming. In the present study, we produced silica nanoparticles-associated pDNA and electrospun scaffolds loaded with such nanoparticles, and studied the release of pDNA from scaffolds and cell-to-scaffold interactions in terms of cell viability and pDNA transfection efficacy. The pDNA-coated nanoparticles were characterized with dynamic light scattering and transmission electron microscopy. Particle sizes ranging from 56 to 78 nm were indicative of their potential for cell transfection. The scaffolds were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, stress-loading tests and interaction with HEK293T cells. It was found that the properties of materials and the pDNA released vary, depending on the scaffold's composition. The scaffolds loaded with pDNA-nanoparticles do not have a pronounced cytotoxic effect, and can be recommended for cell transfection. It was found that (pDNA-NPs) + PEI9-loaded scaffold demonstrates good potential for cell transfection. Thus, electrospun scaffolds suitable for the transfection of inhabiting cells are eligible for use in tissue engineering.

Downregulated calmodulin expression contributes to endothelial cell impairment in diabetes.

Endothelial dysfunction, a central hallmark of cardiovascular pathogenesis in diabetes mellitus, is characterized by impaired endothelial nitric oxide synthase (eNOS) and NO bioavailability. However, the underlying mechanisms remain unclear. Here in this study, we aimed to identify the role of calmodulin (CaM) in diabetic eNOS dysfunction. Human umbilical vein endothelial cells and murine endothelial progenitor cells (EPCs) treated with high glucose (HG) exhibited downregulated CaM mRNA/protein and vascular endothelial growth factor (VEGF) expression with impeded eNOS phosphorylation and cell migration/tube formation. These perturbations were reduplicated in CALM1-knockdown cells but prevented in CALM1-overexpressing cells. EPCs from type 2 diabetes animals behaved similarly to HG-treated normal EPCs, which could be rescued by CALM1-gene transduction. Consistently, diabetic animals displayed impaired eNOS phosphorylation, endothelium-dependent dilation, and CaM expression in the aorta, as well as deficient physical interaction of CaM and eNOS in the gastrocnemius. Local CALM1 gene delivery into a diabetic mouse ischemic hindlimb improved the blunted limb blood perfusion and gastrocnemius angiogenesis, and foot injuries. Diabetic patients showed insufficient foot microvascular autoregulation, eNOS phosphorylation, and NO production with downregulated CaM expression in the arterial endothelium, and abnormal CALM1 transcription in genome-wide sequencing analysis. Therefore, our findings demonstrated that downregulated CaM expression is responsible for endothelium dysfunction and angiogenesis impairment in diabetes, and provided a novel mechanism and target to protect againstdiabetic endothelial injury.

Alginate-Chitosan Hydrogels Containing shRNA Plasmid for Inhibition of CTNNB1 Expression in Breast Cancer Cells.

The hydrogels prepared with alginate and chitosan polymers were prepared to deliver the shRNA-encoding plasmid (pshRNA) to MDA-MB-231 cells for the inhibition of β-catenin (CTNNB1), which was reported to be overexpressed in breast cancer. Polyion complex hydrogels prepared using sodium alginate and chitosan were characterized by Fourier transform infrared spectrometry (FTIR) analysis, scanning electron microscope (SEM) analysis, swelling, and degradation properties. After the release properties and serum stability of pshRNA-loaded hydrogels were determined, their cytotoxicity, transfection efficacy, and effects on CTNNB1 expression were investigated in MDA-MB-231 cells. All hydrogels were shown to protect pshRNA from the enzymatic activity of serum and to deliver pshRNA to cells efficiently. As a result of transfection studies, pshRNA-loaded hydrogels reduced CTNNB1 expression by up to 30.25%. Cell viability also decreased by 38% in cells treated with 2.5% (w/v) alginate-chitosan hydrogel containing pshRNA targeting CTNNB1. Alginate-chitosan hydrogels were shown to be a suitable matrix system for local gene delivery.

Non-Viral Gene Delivery to Hepatocellular Carcinoma via Intra-Arterial Injection.

Hepatocellular carcinoma (HCC) has limited treatment options, and modest survival after systemic chemotherapy or procedures such as transarterial chemoembolization (TACE). There is therefore a need to develop targeted therapies to address HCC. Gene therapies hold immense promise in treating a variety of diseases, including HCC, though delivery remains a critical hurdle. This study investigated a new approach of local delivery of polymeric nanoparticles (NPs) via intra-arterial injection for targeted local gene delivery to HCC tumors in an orthotopic rat liver tumor model. Poly(beta-amino ester) (PBAE) nanoparticles were formulated and assessed for GFP transfection in N1-S1 rat HCC cells in vitro. Optimized PBAE NPs were next administered to rats via intra-arterial injection with and without orthotopic HCC tumors, and both biodistribution and transfection were assessed. In vitro transfection of PBAE NPs led to >50% transfected cells in adherent and suspension culture at a variety of doses and weight ratios. Administration of NPs via intra-arterial or intravenous injection demonstrated no transfection of healthy liver, while intra-arterial NP injection led to transfection of tumors in an orthotopic rat HCC model. Hepatic artery injection is a promising delivery approach for PBAE NPs and demonstrates increased targeted transfection of HCC tumors compared to intravenous administration, and offers a potential alternative to standard chemotherapies and TACE. This work demonstrates proof of concept for administration of polymeric PBAE nanoparticles via intra-arterial injection for gene delivery in rats.

3D bioprinting of gene delivery scaffolds with controlled release

Localized gene delivery via engineered scaffolds offers spatiotemporal control of the gene vector release. Here, we explored the capability of digital light processing based bioprinting to fabricate 3D scaffolds in hydrogels for controlled gene delivery. We demonstrated the compatibility of the method with three representative hydrogel biomaterials for gene delivery. We further investigated the highly tunable release profile with these scaffolds by creating and combining distinct release mechanisms of diffusion and ion exchange. The efficacy of gene delivery of these scaffolds was validated in vitro using 293T cells. Results from this work could potentially facilitate the development of synergistic and personalized gene therapies.

Technical and engineering considerations for designing therapeutics and delivery systems.

The newly-emerged pathological conditions and increased rates of drug resistance necessitate application of the state-of-the-art technologies for accelerated discovery of the therapeutic candidates and obtaining comprehensive knowledge about their targets, action mechanisms, and interactions within the body including those between the receptors and drugs. Using the physics- and chemistry-based modern techniques for theranostic purposes, preparing smart carriers, local delivery of genes or drugs, and enhancing pharmaceutical bioavailability could be of great value against the hard-to-treat diseases and growing drug resistance. Besides the artificial intelligence- and quantum-based techniques, crystal engineering capable of designing new molecules with appropriate characteristics, improving the stability and bioavailability of poorly soluble drugs, and efficient carrier development could play a crucial role in manufacturing efficient pharmaceuticals and reducing the adverse events. In this context, identifying the structures and behaviors of crystals and predicting their characteristics are of great value. Electron diffraction by accelerated analysis of the chemicals and sensitivity to charge alterations, electromechanical tools for controlled delivery of therapeutics, mechatronics via fabrication of multi-functional smart products including the organ-on-chip devices for healthcare applications, and optomechatronics by overcoming the limitations of conventional biomedical techniques could address the unmet biomedical requirements and facilitate development of more effective theranostics with improved outcomes.

Lipoplex-Functionalized Thin-Film Surface Coating Based on Extracellular Matrix Components as Local Gene Delivery System to Control Osteogenic Stem Cell Differentiation.

A gene-activated surface coating is presented as a strategy to design smart biomaterials for bone tissue engineering. The thin-film coating is based on polyelectrolyte multilayerscomposed of collagen I and chondroitin sulfate, two main biopolymers of the bone extracellular matrix, which are fabricated by layer-by-layer assembly. For further functionalization, DNA/lipid-nanoparticles (lipoplexes) are incorporated into the multilayers. The polyelectrolyte multilayer fabrication and lipoplex deposition are analyzed by surface sensitive analytical methods that demonstrate successful thin-film formation, fibrillar structuring of collagen, and homogenous embedding of lipoplexes. Culture of mesenchymal stem cells on the lipoplex functionalized multilayer results in excellent attachment and growth of them, and also, their ability to take up cargo like fluorescence-labelled DNA from lipoplexes. The functionalization of the multilayer with lipoplexes encapsulating DNA encoding for transient expression of bone morphogenetic protein 2 induces osteogenic differentiation of mesenchymal stem cells, which is shown by mRNA quantification for osteogenic genes and histochemical staining. In summary, the novel gene-functionalized and extracellular matrix mimicking multilayer composed of collagen I, chondroitin sulfate, and lipoplexes, represents a smart surface functionalization that holds great promise for tissue engineering constructs and implant coatings to promote regeneration of bone and other tissues.

AAV13 Enables Precise Targeting of Local Neural Populations.

As powerful tools for local gene delivery, adeno-associated viruses (AAVs) are widely used for neural circuit studies and therapeutical purposes. However, most of them have the characteristics of large diffusion range and retrograde labeling, which may result in off-target transduction during in vivo application. Here, in order to achieve precise gene delivery, we screened AAV serotypes that have not been commonly used as gene vectors and found that AAV13 can precisely transduce local neurons in the brain, with a smaller diffusion range than AAV2 and rigorous anterograde labeling. Then, AAV13-based single-viral and dual-viral strategies for sparse labeling of local neurons in the brains of C57BL/6 or Cre transgenic mice were developed. Additionally, through the neurobehavioral test in the ventral tegmental area, we demonstrated that AAV13 was validated for functional monitoring by means of carrying Cre recombinase to drive the expression of Cre-dependent calcium-sensitive indicator. In summary, our study provides AAV13-based toolkits for precise local gene delivery, which can be used for in situ small nuclei targeting, sparse labeling and functional monitoring.

Phosphomimetic endothelial nitric oxide synthase-loaded microbubbles-mediated cardiac shock wave transfection therapy for angiogenesis in the ischemic myocardium

Abstract Background Cardiac shock wave therapy is a potential and effective remedy to promote revascularization in the ischemic myocardium of patients with refractory coronary heart disease (CHD). Although gene therapy holds great promise as a treatment for coronary heart disease, current clinical application is hampered by the development of safe and efficient systems for local gene delivery to a specific tissue or organ. Purpose In this study, we developed an experimental pulsed- focused electrohydraulic shock wave therapy system and investigated its effectiveness on myocardial angiogenesis in the hypercholesterolemic rabbit accompanied by phosphomimetic endothelial nitric oxide synthase-loaded microbubbles administration, wherein a high resolution diagnostic ultrasound is adjuncted with the therapy system, with a goal of increased safety. Methods Briefly, New Zealand white rabbits underwent 2% cholesterol rich diet injury for sixteen weeks. Histopathology results showed the formation of foam cells-rich soft atherosclerotic plaques with severe stenosis in all of the rabbits' coronary arteries. Then treatment group underwent extracorporeal electrohydraulic pulsed-focused shock waves (25kV, 0.5Hz, 150 pulses) accompanied by phosphomimetic endothelial nitric oxide synthase-loaded PESDA (Perfluorocarbon Exposed Sonicated Dextrose Albumin) microbubbles (100 ml/kg, 2.5x105 bubbles/ml) administration. Results Results from ultrasonography and histopathology showed a significant increase in the mean value for myocardial neovessels density in the treatment group compared with the control group (p<0.05). Conclusion Enhanced angiogenesis and sonoporation effect of electrohydraulic shock wave therapy, induced by inertial collapsed microbubbles, accompanied by phosphomimetic endothelial nitric oxide synthase-mediated shock wave transfection therapy, can cause to enhance the nitric oxide synthesis and VEGF, interleukin8 (IL8), stromal cell- derived factor 1 (SDF1) and matrix metalloproteinase 9 (MMP9) secretion in the hypercholesterolemic rabbits' coronary arteries, resulting in the differentiation and recruitment of endothelial progenitor cells (EPCs), promotion of revascularization in the ischemic myocardium and improvement of the cardiac function. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3β-hydroxysteroid dehydrogenase (3β-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3β-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17β-hydroxysteroid dehydrogenase (17β-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Electrospun-Fibrous-Architecture-Mediated Non-Viral Gene Therapy Drug Delivery in Regenerative Medicine.

Gene-based therapy represents the latest advancement in medical biotechnology. The principle behind this innovative approach is to introduce genetic material into specific cells and tissues to stimulate or inhibit key signaling pathways. Although enormous progress has been achieved in the field of gene-based therapy, challenges connected to some physiological impediments (e.g., low stability or the inability to pass the cell membrane and to transport to the desired intracellular compartments) still obstruct the exploitation of its full potential in clinical practices. The integration of gene delivery technologies with electrospun fibrous architectures represents a potent strategy that may tackle the problems of stability and local gene delivery, being capable to promote a controlled and proficient release and expression of therapeutic genes in the targeted cells, improving the therapeutic outcomes. This review aims to outline the impact of electrospun-fibrous-architecture-mediated gene therapy drug delivery, and it emphatically discusses the latest advancements in their formulation and the therapeutic outcomes of these systems in different fields of regenerative medicine, along with the main challenges faced towards the translation of promising academic results into tangible products with clinical application.

Hypoxia-inducible lentiviral gene expression in engineered human macrophages

BackgroundHuman immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression...

Development of a fibrin-mediated gene delivery system for the treatment of cystinosis via design of experiment

Cystinosis is a rare disease, caused by a mutation in the gene cystinosin and characterised by the accumulation of cystine crystals. Advantages of biomaterial-mediated gene delivery include reduced safety concerns and the possibility to cure organs that are difficult to treat using systemic gene transfer methods. This study developed novel fibrin hydrogels for controlled, localised gene delivery, for the treatment of cystinosis. In the first part, fabrication parameters (i.e., DNA, thrombin, and aprotinin concentrations) were optimised, using a Design of Experiment (DOE) methodology. DOE is a statistical engineering approach to process optimisation, which increases experimental efficiency, reduces the number of experiments, takes into consideration interactions between different parameters, and allows the creation of predictive models. This study demonstrated the utility of DOE to the development of gene delivery constructs. In the second part of the study, primary fibroblasts from a patient with cystinosis were seeded on the biomaterials. Seeded cells expressed the recombinant CTNS and showed a decrease in cystine content. Furthermore, conditioned media contained functional copies of the recombinant CTNS. These were taken up by monolayer cultures of non-transfected cells. This study described a methodology to develop gene delivery constructs by using a DOE approach and ultimately provided new insights into the treatment of cystinosis.

Injectable “nano-micron” combined gene-hydrogel microspheres for local treatment of osteoarthritis

Sustained and controllable local gene therapy is a potential method for treating osteoarthritis (OA) through the delivery of therapeutic microRNAs (miRNAs) to targeted cells. However, direct injection of crude miRNAs for local gene therapy is limited due to its inadequate transfection efficiency, easy inactivation, and short half-life. Here, a multifunctional gene vector, arginine, histidine, and phenylalanine-modified generation 5 polyamidoamine (named G5-AHP), was employed to form G5-AHP/miR-140 nanoparticles by forming a complex with microRNA-140 (miR-140). Then, the nanoparticles were entrapped in hydrogel microspheres (MSs) to construct a “nano-micron” combined gene hydrogel to alleviate the degradation of articular cartilage. Monodisperse gelatin methacryloyl hydrogel MSs were produced under ultraviolet light using one-step innovative microfluidic technology. Evenly dispersed MSs showed better injectability in sustainable and matrix metalloproteinases (MMPs)-responsive degradation methods for local gene delivery. The G5-AHP/miR-140 nanoparticles released from the MSs exhibited high gene transfection efficacy and long-term bioactivity, facilitated endocytosis, and thus maintained the metabolic balance of cartilage matrix by promoting the expression of type II collagen and inhibiting the expression of a disintegrin and metalloproteinase with thrombospondin motifs-5 and MMP13 in chondrocytes. After injection of the “nano-micron” combined gene hydrogel into the articular cavity of the OA model, the gene hydrogel increased G5-AHP/miR-140 nanoparticle retention, prevented articular cartilage degeneration, and reduced osteophyte formation in a surgically induced mouse model of OA. The present study provides a novel cell-free approach to alleviate the progression of OA that shows potential for locally injected gene delivery systems.

N-carboxymethyl chitosan/sodium alginate composite hydrogel loading plasmid DNA as a promising gene activated matrix for in-situ burn wound treatment.

Improving the degree of vascularization through the regulation of wound microenvironment is crucial for wound repair. Gene activated matrix (GAM) technology provides a new approach for skin regeneration. It is a local gene delivery system that can not only maintain a moist environment, but also increase the concentration of local active factors. For this purpose, we fabricated the mVEGF165/TGF-β1 gene-loaded N-carboxymethyl chitosan/sodium alginate hydrogel and studied its effect on promoting deep second degree burn wound repair. The average diameter of the hydrogel pores was 100 μm and the porosity was calculated as 50.9%. SEM and CLSM images showed that the hydrogel was suitable for cell adhesion and growth. The NS-GAM could maintain continuous expression for at least 9 days in vitro, showing long-term gene release and expression effect. Deep second-degree burn wound model was made on the backs of Wistar rats to evaluate the healing effect. The wounds were healed by day 22 in NS-GAM group with the prolonged high expression of VEGF and TGF-β1 protein. A high degree of neovascularization and high expression level of CD34 were observed in NS-GAM group in 21 days. The histological results showed that NS-GAM had good tissue safety and could effectively promote epithelialization and collagen regeneration. These results indicated that the NS-GAM could be applied as a promising local gene delivery system for the repair of deep second-degree burn wounds.

Layer-by-Layer Biomimetic Microgels for 3D Cell Culture and Nonviral Gene Delivery

Localized release of nucleic acid therapeutics is essential for many biomedical applications, including gene therapy, tissue engineering, and medical implant coatings. We applied the substrate-mediated transfection and layer-by-layer (LbL) technique to achieve an efficient local gene delivery. In the experiments presented herein, we embeded lipoplexes containing plasmid DNA encoding for enhanced green fluorescent protein (pEGFP) within polyelectrolyte alginate-based microgels composed of poly(allylamine hydrochloride) (PAH), chondroitin sulfate (CS), and poly-l-lysine (PLL) with diameters between 70 and 90 μm. Droplet-based microfluidics was used as the main process to produce the alginate (ALG)-based microgels with discrete size, shape, and low coefficient of variation. The physicochemical and morphological properties of the polyelectrolyte microgels were characterized via optical microscopy, scanning electron microscopy (SEM), and zeta potential analysis. We found that polyelectrolyte microgels provide low cytotoxicity and cell-material interactions (adhesion, spreading, and proliferation). In addition, the microsystem showed the ability to load lipoplexes and a loading efficiency equal to 83%, and it enabled in vitro surface-based transfection of MCF-7 cells. This approach provides a new suitable route for cell adhesion and local gene delivery.

The sodium iodide symporter (NIS): novel applications for radionuclide imaging and treatment.

Cloning of the sodium iodide symporter (NIS) 25 years ago has opened an exciting chapter in molecular thyroidology with the characterization of NIS as one of the most powerful theranostic genes and the development of a promising gene therapy strategy based on image-guided selective NIS gene transfer in non-thyroidal tumors followed by application of 131I or alternative radionuclides, such as 188Re and 211At. Over the past two decades, significant progress has been made in the development of the NIS gene therapy concept, from local NIS gene delivery towards promising new applications in disseminated disease, in particular through the use of oncolytic viruses, non-viral polyplexes, and genetically engineered MSCs as highly effective, highly selective and flexible gene delivery vehicles. In addition to allowing the robust therapeutic application of radioiodine in non-thyroid cancer settings, these studies have also been able to take advantage of NIS as a sensitive reporter gene that allows temporal and spatial monitoring of vector biodistribution, replication, and elimination - critically important issues for preclinical development and clinical translation.