Improved Noninvasive In Vivo Tracking of AAV-9 Gene Therapy Using the Perchlorate-Resistant Sodium Iodide Symporter from Minke Whale.

The sodium iodide symporter (NIS) is widely used as a reporter gene to noninvasively monitor the biodistribution and durability of vector-mediated gene expression via gamma scintigraphy, single-photon emission computed tomography (SPECT), and positron-emission tomography (PET). However, the approach is limited by background signal due to radiotracer uptake by endogenous NIS-expressing tissues. In this study, using the SPECT tracer pertechnetate (99mTcO4) and the PET tracer tetrafluoroborate (B18F4), in combination with the NIS inhibitor perchlorate, we compared the transport properties of human NIS and minke whale (Balaenoptera acutorostrata scammoni) NIS in vitro and in vivo. Based on its relative resistance to perchlorate, the NIS protein from minke whale appeared to be the superior candidate reporter gene. SPECT and PET imaging studies in nude mice challenged with NIS-encoding adeno-associated virus (AAV)-9 vectors confirmed that minke whale NIS, in contrast to human and endogenous mouse NIS, continues to function as a reliable reporter even when background radiotracer uptake by endogenous NIS is blocked by perchlorate.

Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS).

The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na+ ions and I- substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) 125I- uptake of 35 hNIS variants.

Noninvasive 3-dimensional imaging of liver regeneration in a mouse model of hereditary tyrosinemia type 1 using the sodium iodide symporter gene.

Cell transplantation is a potential treatment for the many liver disorders that are currently only curable by organ transplantation. However, one of the major limitations of hepatocyte (HC) transplantation is an inability to monitor cells longitudinally after injection. We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to visualize transplanted HCs in a rodent model of inherited liver disease: hereditary tyrosinemia type 1. Wild-type C57Bl/6J mouse HCs were transduced ex vivo with a lentiviral vector containing the mouse Slc5a5 (NIS) gene controlled by the thyroxine-binding globulin promoter. NIS-transduced cells could robustly concentrate radiolabeled iodine in vitro, with lentiviral transduction efficiencies greater than 80% achieved in the presence of dexamethasone. Next, NIS-transduced HCs were transplanted into congenic fumarylacetoacetate hydrolase knockout mice, and this resulted in the prevention of liver failure. NIS-transduced HCs were readily imaged in vivo by single-photon emission computed tomography, and this demonstrated for the first time noninvasive 3-dimensional imaging of regenerating tissue in individual animals over time. We also tested the efficacy of primary HC spheroids engrafted in the liver. With the NIS reporter, robust spheroid engraftment and survival could be detected longitudinally after direct parenchymal injection, and this thereby demonstrated a novel strategy for HC transplantation. This work is the first to demonstrate the efficacy of NIS imaging in the field of HC transplantation. We anticipate that NIS labeling will allow noninvasive and longitudinal identification of HCs and stem cells in future studies related to liver regeneration in small and large preclinical animal models.

Oncolytic measles and vesicular stomatitis virotherapy for endometrial cancer.

OBJECTIVE: Current adjuvant therapy for advanced-stage, recurrent, and high-risk endometrial cancer (EC) has not reduced mortality from this malignancy, and novel systemic therapies are imperative. Oncolytic viral therapy has been shown to be effective in the treatment of gynecologic cancers, and we investigated the in vitro and in vivo efficacy of the Edmonston strain of measles virus (MV) and vesicular stomatitis virus (VSV) on EC. METHODS: Human EC cell lines (HEC-1-A, Ishikawa, KLE, RL95-2, AN3 CA, ARK-1, ARK-2, and SPEC-2) were infected with Edmonston strain MV expressing the thyroidal sodium iodide symporter, VSV expressing either human or murine IFN-ß, or recombinant VSV with a methionine deletion at residue 51 of the matrix protein and expressing the sodium iodide symporter. Xenografts of HEC-1-A and AN3 CA generated in athymic mice were treated with intratumoral MV or VSV or intravenous VSV. RESULTS: In vitro, all cell lines were susceptible to infection and cell killing by all 3 VSV strains except KLE. In addition, the majority of EC cell lines were defective in their ability to respond to type I IFN. Intratumoral VSV-treated tumors regressed more rapidly than MV-treated tumors, and intravenous VSV resulted in effective tumor control in 100% of mice. Survival was significantly longer for mice treated with any of the 3 VSV strains compared with saline. CONCLUSION: VSV is clearly more potent in EC oncolysis than MV. A phase 1 clinical trial of VSV in EC is warranted.

Amalgamating oncolytic viruses to enhance their safety, consolidate their killing mechanisms, and accelerate their spread.

Oncolytic viruses are structurally and biologically diverse, spreading through tumors and killing them by various mechanisms and with different kinetics. Here, we created a hybrid vesicular stomatitis/measles virus (VSV/MV) that harnesses the safety of oncolytic MV, the speed of VSV, and the tumor killing mechanisms of both viruses. Oncolytic MV targets CD46 and kills by forcing infected cells to fuse with uninfected neighbors, but propagates slowly. VSV spreads rapidly, directly lysing tumor cells, but is neurotoxic and loses oncolytic potency when neuroattenuated by conventional approaches. The hybrid VSV/MV lacks neurotoxicity, replicates rapidly with VSV kinetics, and selectively targets CD46 on tumor cells. Its in vivo performance in a myeloma xenograft model was substantially superior to either MV or widely used recombinant oncolytic VSV-M51.

Neoadjuvant systemic oncolytic vesicular stomatitis virus is safe and may enhance long-term survivorship in dogs with naturally occurring osteosarcoma.

Osteosarcoma is a devastating bone cancer that disproportionally afflicts children, adolescents, and young adults. Standard therapy includes surgical tumor resection combined with multiagent chemotherapy, but many patients still suffer from metastatic disease progression. Neoadjuvant systemic oncolytic virus (OV) therapy has the potential to improve clinical outcomes by targeting primary and metastatic tumor sites and inducing durable antitumor immune responses. Here we describe the first evaluation of neoadjuvant systemic therapy with a clinical-stage recombinant oncolytic vesicular stomatitis virus (VSV), VSV-IFNß-NIS, in naturally occurring cancer, specifically appendicular osteosarcoma in companion dogs. Canine osteosarcoma has a similar natural disease history as its human counterpart. VSV-IFNß-NIS was administered prior to standard of care surgical resection, permitting microscopic and genomic analysis of tumors. Treatment was well-tolerated and a "tail" of long-term survivors (∼35%) was apparent in the VSV-treated group, a greater proportion than observed in two contemporary control cohorts. An increase in tumor inflammation was observed in VSV-treated tumors and RNA-seq analysis showed that all the long-term responders had increased expression of a T cell anchored immune gene cluster. We conclude that neoadjuvant VSV-IFNß-NIS is safe and may increase long-term survivorship in dogs with naturally occurring osteosarcoma, particularly those that exhibit pre-existing antitumor immunity.

Neoadjuvant systemic oncolytic vesicular stomatitis virus is safe and may enhance long-term survivorship in dogs with naturally occurring osteosarcoma.

Osteosarcoma is a devastating bone cancer that disproportionally afflicts children, adolescents, and young adults. Standard therapy includes surgical tumor resection combined with multiagent chemotherapy, but many patients still suffer from metastatic disease progression. Neoadjuvant systemic oncolytic virus (OV) therapy has the potential to improve clinical outcomes by targeting primary and metastatic tumor sites and inducing durable antitumor immune responses. Here we described the first evaluation of neoadjuvant systemic therapy with a clinical-stage recombinant oncolytic Vesicular stomatitis virus (VSV), VSV-IFNß-NIS, in naturally occurring cancer, specifically appendicular osteosarcoma in companion dogs. Canine osteosarcoma has a similar natural disease history as its human counterpart. VSV-IFNß-NIS was administered prior to standard of care surgical resection, permitting microscopic and genomic analysis of tumors. Treatment was well-tolerated and a 'tail' of long-term survivors (~35%) was apparent in the VSV-treated group, a greater proportion than observed in two contemporary control cohorts. An increase in tumor inflammation was observed in VSV-treated tumors and RNAseq analysis showed that all the long-term responders had increased expression of a T-cell anchored immune gene cluster. We conclude that neoadjuvant VSV-IFNß-NIS is safe and may increase long-term survivorship in dogs with naturally occurring osteosarcoma, particularly those that exhibit pre-existing antitumor immunity.

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine.

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers.

Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P < 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity. IMPORTANCE Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers. We have critical virus reagent to test over 5 million samples, making our assay well suited for widespread monitoring of SARS-CoV-2-neutralizing antibodies, which can in turn be used to inform vaccine dosing schedules and answer fundamental questions regarding SARS-CoV-2 immunity.

Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity.

Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Enhanced noninvasive imaging of oncology models using the NIS reporter gene and bioluminescence imaging.

Noninvasive bioluminescence imaging (BLI) of luciferase-expressing tumor cells has advanced pre-clinical evaluation of cancer therapies. Yet despite its successes, BLI is limited by poor spatial resolution and signal penetration, making it unusable for deep tissue or large animal imaging and preventing precise anatomical localization or signal quantification. To refine pre-clinical BLI methods and circumvent these limitations, we compared and ultimately combined BLI with tomographic, quantitative imaging of the sodium iodide symporter (NIS). To this end, we generated tumor cell lines expressing luciferase, NIS, or both reporters, and established tumor models in mice. BLI provided sensitive early detection of tumors and relatively easy monitoring of disease progression. However, spatial resolution was poor, and as the tumors grew, deep thoracic tumor signals were massked by overwhelming surface signals from superficial tumors. In contrast, NIS-expressing tumors were readily distinguished and precisely localized at all tissue depths by positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging. Furthermore, radiotracer uptake for each tumor could be quantitated noninvasively. Ultimately, combining BLI and NIS imaging represented a significant enhancement over traditional BLI, providing more information about tumor size and location. This combined imaging approach should facilitate comprehensive evaluation of tumor responses to given therapies.

Ex Vivo Cell Therapy by Ectopic Hepatocyte Transplantation Treats the Porcine Tyrosinemia Model of Acute Liver Failure.

The effectiveness of cell-based therapies to treat liver failure is often limited by the diseased liver environment. Here, we provide preclinical proof of concept for hepatocyte transplantation into lymph nodes as a cure for liver failure in a large-animal model with hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. Autologous porcine hepatocytes were transduced ex vivo with a lentiviral vector carrying the pig Fah gene and transplanted into mesenteric lymph nodes. Hepatocytes showed early (6 h) and durable (8 months) engraftment in lymph nodes, with reproduction of vascular and hepatic microarchitecture. Subsequently, hepatocytes migrated to and repopulated the native diseased liver. The corrected cells generated sufficient liver mass to clinically ameliorate the acute liver failure and HT1 disease as early as 97 days post-transplantation. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes from lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Therefore, ectopic transplantation of healthy hepatocytes cures this pig model of liver failure and presents a promising approach for the development of cures for liver disease in patients.

Development and validation of IMMUNO-COV™: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2.

We here describe the development and validation of IMMUNO-COV™, a high-throughput clinical test to quantitatively measure SARS-CoV-2-neutralizing antibodies, the specific subset of anti-SARS-CoV-2 antibodies that block viral infection. The test measures the capacity of serum or purified antibodies to neutralize a recombinant Vesicular Stomatitis Virus (VSV) encoding the SARS-CoV-2 spike glycoprotein. This recombinant virus (VSV-SARS-CoV-2-S-Δ19CT) induces fusion in Vero cell monolayers, which is detected as luciferase signal using a dual split protein (DSP) reporter system. VSV-SARS-CoV-2-S-Δ19CT infection was blocked by monoclonal α-SARS-CoV-2-spike antibodies and by plasma or serum from SARS-CoV-2 convalescing individuals. The assay exhibited 100% specificity in validation tests, and across all tests zero false positives were detected. In blinded analyses of 230 serum samples, only two unexpected results were observed based on available clinical data. We observed a perfect correlation between results from our assay and 80 samples that were also assayed using a commercially available ELISA. To quantify the magnitude of the anti-viral response, we generated a calibration curve by adding stepped concentrations of α-SARS-CoV-2-spike monoclonal antibody to pooled SARS-CoV-2 seronegative serum. Using the calibration curve and a single optimal 1:100 serum test dilution, we reliably measured neutralizing antibody levels in each test sample. Virus neutralization units (VNUs) calculated from the assay correlated closely (p < 0.0001) with PRNT EC50 values determined by plaque reduction neutralization test against a clinical isolate of SARS-CoV-2. Taken together, these results demonstrate that the IMMUNO-COV™ assay accurately quantitates SARS-CoV-2 neutralizing antibodies in human sera and therefore is a potentially valuable addition to the currently available serological tests. The assay can provide vital information for comparing immune responses to the various SARS-CoV-2 vaccines that are currently in development, or for evaluating donor eligibility in convalescent plasma therapy studies.

Characterization of dengue virus entry into HepG2 cells.

BACKGROUND: Despite infections by the dengue virus being a significant problem in tropical and sub-tropical countries, the mechanism by which the dengue virus enters into mammalian cells remains poorly described. METHODS: A combination of biochemical inhibition, dominant negative transfection of Eps15 and siRNA mediated gene silencing was used to explore the entry mechanism of dengue into HepG2 cells. RESULTS: Results were consistent with entry via multiple pathways, specifically via clathrin coated pit mediated endocytosis and macropinocytosis, with clathrin mediated endocytosis being the predominant pathway. CONCLUSION: We propose that entry of the dengue virus to mammalian cells can occur by multiple pathways, and this opens the possibility of the virus being directed to multiple cellular compartments. This would have significant implications in understanding the interaction of the dengue virus with the host cell machinery.

Dual-Isotope SPECT Imaging with NIS Reporter Gene and Duramycin to Visualize Tumor Susceptibility to Oncolytic Virus Infection.

Noninvasive dual-imaging methods that provide an early readout on tumor permissiveness to virus infection and tumor cell death could be valuable in optimizing development of oncolytic virotherapies. Here, we have used the sodium iodide symporter (NIS) and 125I radiotracer to detect infection and replicative spread of an oncolytic vesicular stomatitis virus (VSV) in VSV-susceptible (MPC-11 tumor) versus VSV-resistant (CT26 tumor) tumors in BALB/c mice. In conjunction, tumor cell death was imaged simultaneously using technetium (99mTc)-duramycin that binds phosphatidylethanolamine in apoptotic and necrotic cells. Dual-isotope single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed areas of virus infection (NIS and 125I), which overlapped well with areas of tumor cell death (99mTc-duramycin imaging) in susceptible tumors. Multiple infectious foci arose early in MPC-11 tumors, which rapidly expanded throughout the tumor parenchyma over time. There was a dose-dependent increase in numbers of infectious centers and 99mTc-duramycin-positive areas with viral dose. In contrast, NIS or duramycin signals were minimal in VSV-resistant CT26 tumors. Combinatorial use of NIS and 99mTc-duramycin SPECT imaging for simultaneous monitoring of oncolytic virotherapy (OV) spread and the presence or absence of treatment-associated cell death could be useful to guide development of combination treatment strategies to enhance therapeutic outcome.

New transgenic NIS reporter rats for longitudinal tracking of fibrogenesis by high-resolution imaging.

Fibrogenesis is the underlying mechanism of wound healing and repair. Animal models that enable longitudinal monitoring of fibrogenesis are needed to improve traditional tissue analysis post-mortem. Here, we generated transgenic reporter rats expressing the sodium iodide symporter (NIS) driven by the rat collagen type-1 alpha-1 (Col1α1) promoter and demonstrated that fibrogenesis can be visualized over time using SPECT or PET imaging following activation of NIS expression by rotator cuff (RC) injury. Radiotracer uptake was first detected in and around the injury site day 3 following surgery, increasing through day 7-14, and declining by day 21, revealing for the first time, the kinetics of Col1α1 promoter activity in situ. Differences in the intensity and duration of NIS expression/collagen promoter activation between individual RC injured Col1α1-hNIS rats were evident. Dexamethasone treatment delayed time to peak NIS signals, showing that modulation of fibrogenesis by a steroid can be imaged with exquisite sensitivity and resolution in living animals. NIS reporter rats would facilitate studies in physiological wound repair and pathological processes such as fibrosis and the development of anti-fibrotic drugs.

Comparative Oncology Evaluation of Intravenous Recombinant Oncolytic Vesicular Stomatitis Virus Therapy in Spontaneous Canine Cancer.

Clinical translation of intravenous therapies to treat disseminated or metastatic cancer is imperative. Comparative oncology, the evaluation of novel cancer therapies in animals with spontaneous cancer, can be utilized to inform and accelerate clinical translation. Preclinical murine studies demonstrate that single-shot systemic therapy with a vesicular stomatitis virus (VSV)-IFNß-NIS, a novel recombinant oncolytic VSV, can induce curative remission in tumor-bearing mice. Clinical translation of VSV-IFNß-NIS therapy is dependent on comprehensive assessment of clinical toxicities, virus shedding, pharmacokinetics, and efficacy in clinically relevant models. Dogs spontaneously develop cancer with comparable etiology, clinical progression, and response to therapy as human malignancies. A comparative oncology study was carried out to investigate feasibility and tolerability of intravenous oncolytic VSV-IFNß-NIS therapy in pet dogs with spontaneous cancer. Nine dogs with various malignancies were treated with a single intravenous dose of VSV-IFNß-NIS. Two dogs with high-grade peripheral T-cell lymphoma had rapid but transient remission of disseminated disease and transient hepatotoxicity that resolved spontaneously. There was no shedding of infectious virus. Correlative pharmacokinetic studies revealed elevated levels of VSV RNA in blood in dogs with measurable disease remission. This is the first evaluation of intravenous oncolytic virus therapy for spontaneous canine cancer, demonstrating that VSV-IFNß-NIS is well-tolerated and safe in dogs with advanced or metastatic disease. This approach has informed clinical translation, including dose and target indication selection, leading to a clinical investigation of intravenous VSV-IFNß-NIS therapy, and provided preliminary evidence of clinical efficacy and potential biomarkers that correlate with therapeutic response. Mol Cancer Ther; 17(1); 316-26. ©2017 AACR.

Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models.

BACKGROUND: Autologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single-cell or spheroid hepatocytes. METHODS: Pig and mouse hepatocytes were isolated, labeled with zirconium-89 and returned to the liver as single cells or spheroids. Biodistribution was evaluated through positron emission tomography-computed tomography. Fumarylacetoacetate hydrolase-deficient pig hepatocytes were isolated and transduced with a lentiviral vector containing the Fah gene. Animals received portal vein infusion of single-cell or spheroid autologous hepatocytes after ex vivo gene delivery. Portal pressures were measured and ultrasound was used to evaluate for thrombus. Differences in engraftment and expansion of ex vivo corrected single-cell or spheroid hepatocytes were followed through histologic analysis and animals' ability to thrive off 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione. RESULTS: Positron emission tomography-computed tomography imaging showed spheroid hepatocytes with increased heterogeneity in biodistribution as compared with single cells, which spread more uniformly throughout the liver. Animals receiving spheroids experienced higher mean changes in portal pressure than animals receiving single cells (P < .01). Additionally, two animals from the spheroid group developed portal vein thrombi that required systemic anticoagulation. Immunohistochemical analysis of spheroid- and single-cell-transplanted animals showed similar engraftment and expansion rates of fumarylacetoacetate hydrolase-positive hepatocytes in the liver, correlating with similar weight stabilization curves. CONCLUSION: Ex vivo gene correction of autologous hepatocytes in fumarylacetoacetate hydrolase-deficient pigs can be performed using hepatocyte spheroids or single-cell hepatocytes, with spheroids showing a more heterogeneous distribution within the liver and higher risks for portal vein thrombosis and increased portal pressures.

Preclinical Development of Oncolytic Immunovirotherapy for Treatment of HPVPOS Cancers.

Immunotherapy for HPVPOS malignancies is attractive because well-defined, viral, non-self tumor antigens exist as targets. Several approaches to vaccinate therapeutically against HPV E6 and E7 antigens have been adopted, including viral platforms such as VSV. A major advantage of VSV expressing these antigens is that VSV also acts as an oncolytic virus, leading to direct tumor cell killing and induction of effective anti-E6 and anti-E7 T cell responses. We have also shown that addition of immune adjuvant genes, such as IFNß, further enhances safety and/or efficacy of VSV-based oncolytic immunovirotherapies. However, multiple designs of the viral vector are possible-with respect to levels of immunogen expression and method of virus attenuation-and optimal designs have not previously been tested head-to-head. Here, we tested three different VSV engineered to express a non-oncogenic HPV16 E7/6 fusion protein for their immunotherapeutic and oncolytic properties. We assessed their profiles of efficacy and toxicity against HPVPOS and HPVNEG murine tumor models and determined the optimal route of administration. Our data show that VSV is an excellent platform for the oncolytic immunovirotherapy of tumors expressing HPV target antigens, combining a balance of efficacy and safety suitable for evaluation in a first-in-human clinical trial.

Synthesis of 18F-Tetrafluoroborate via Radiofluorination of Boron Trifluoride and Evaluation in a Murine C6-Glioma Tumor Model.

UNLABELLED: The sodium/iodide symporter (NIS) is under investigation as a reporter for noninvasive imaging of gene expression. Although (18)F-tetrafluoroborate ((18)F-TFB, (18)F-BF4 (-)) has shown promise as a PET imaging probe for NIS, the current synthesis method using isotopic exchange gives suboptimal radiochemical yield and specific activity. The aim of this study was to synthesize (18)F-TFB via direct radiofluorination on boron trifluoride (BF3) to enhance both labeling yield and specific activity and evaluation of specific activity influence on tumor uptake. METHODS: An automated synthesis of (18)F-TFB was developed whereby cyclotron-produced (18)F-fluoride was trapped on a quaternary methyl ammonium anion exchange cartridge, then allowed to react with BF3 freshly preformulated in petroleum ether/tetrahydrofuran (50:1). The resultant (18)F-TFB product was retained on the quaternary methyl ammonium anion exchange cartridge. After the cartridge was rinsed with tetrahydrofuran and water, (18)F-TFB was eluted from the cartridge with isotonic saline, passing through 3 neutral alumina cartridges and a sterilizing filter. Preclinical imaging studies with (18)F-TFB were performed in athymic mice bearing NIS-expressing C6-glioma subcutaneous xenografted tumors to determine the influence of specific activity on tumor uptake. RESULTS: Under optimized conditions, (18)F-TFB was synthesized in a radiochemical yield of 20.0% ± 0.7% (n = 3, uncorrected for decay) and greater than 98% radiochemical purity in a synthesis time of 10 min. Specific activities of 8.84 ± 0.56 GBq/µmol (n = 3) were achieved from starting (18)F-fluoride radioactivities of 40-44 GBq. An avid uptake of (18)F-TFB was observed in human NIS (hNIS)-expressing C6-glioma xenografts as well as expected NIS-mediated uptake in the thyroid and stomach. There was a positive correlation between the uptake of (18)F-TFB in hNIS-expressing tumor and specific activity. CONCLUSION: A rapid, practical, and high-specific-activity synthesis of the NIS reporter probe (18)F-TFB was achieved via direct radiofluorination on BF3 using an automated synthesis system. The synthesis of high-specific-activity (18)F-TFB should enable future clinical studies with hNIS gene reporter viral constructs.