Simplex Virus Type-1 Thymidine Kinase Research Articles

Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.

Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.

A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation.

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. Although many tools are available to induce these systems, nature has provided other mechanisms to expand the split protein toolbox. Here, we show a novel method for reconstituting split proteins using magnetic stimulation. We found that the electromagnetic perceptive gene (EPG) changes conformation due to magnetic field stimulation. By fusing split fragments of a certain protein to both termini of the EPG, the fragments can be reassembled into a functional protein under magnetic stimulation due to conformational change. We show this effect with three separate split proteins: NanoLuc, APEX2, and herpes simplex virus type-1 thymidine kinase. Our results show, for the first time, that reconstitution of split proteins can be achieved only with magnetic fields. We anticipate that this study will be a starting point for future magnetically inducible split protein designs for cellular perturbation and manipulation. With this technology, we can help expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

New poly heterocyclic compounds based on pyrimidine-2-thiones: synthesis, evaluation of putative antiviral agents, DFT calculation, and molecular modeling

Owing to the enormous increase in viral infections in recent years, novel antiviral candidates are both eagerly awaited and unquestionably essential for the treatment of a variety of deadly and incapacitating viral disorders. As a part of our program in this area, a new series of thiazolopyrimidine, pyrimidothiazine, [1,2,4]triazolopyrimidine and N-substituted pyrimidinethione 6–17 have been synthesized in good yields (82–91%) through a facile method using new pyrimidinethione 4 as a building block and several reagents as halo acids/ketones or hydrazonyl halids in one framework. By using several spectroscopic methods, including IR, 1H NMR, 13C NMR, elemental analysis, and DFT calculations, the chemical structures of the novel derivatives were confirmed. The synthetic compounds' cytotoxicity and in vitro antiviral efficacy were examined against HSV-1. Results demonstrated that compounds 6, 7b, 14b,c, and 17 have been found to have significant antiviral activity against HSV-1. Additionally, after examining compounds (6 and 7b)' mechanisms of action, we discovered that compound 6 had a virucidal effect and a mild effect on replication at higher concentrations, whereas compound 7b had activity in all three modes of action, with the virucidal effect being the most potent, followed by the replication effect, and then adsorption. In order to comprehend these results, we investigated the thermal and electrical stability of the generated compounds, where their chemical and thermal properties, dipole moment, subtraction of the highest occupied molecular orbital from the lowest unoccupied molecular orbital (HOMO-LUMO gap), and molecular electrostatic potential were computed. We also, examined the binding mechanism, binding affinity, and non-bonding contacts of the generated derivatives using molecular docking against the Herpes simplex virus type-1 thymidine kinase (PDB ID: 2KI5). A hypothetical pharmacophore model was made using the Molecular Operating Environment (MOE) tool and 10 compounds with structural resemblances to those synthesized with known antiviral activity. The current work recommends compounds 6 and 7b as potential lead structures for future research and development of novel anti-HSV-1 medicines.

Split genome-based retroviral replicating vectors achieve efficient gene delivery and therapeutic effect in a human glioblastoma xenograft model

The murine leukemia virus-based semi-retroviral replicating vectors (MuLV-based sRRV) had been developed to improve safety and transgene capacity for cancer gene therapy. However, despite the apparent advantages of the sRRV, improvements in the in vivo transduction efficiency are still required to deliver therapeutic genes efficiently for clinical use. In this study, we established a gibbon ape leukemia virus (GaLV) envelopepseudotyped semi-replication-competent retrovirus vector system (spRRV) which is composed of two transcomplementing replication-defective retroviral vectors termed MuLV-Gag-Pol and GaLV-Env. We found that the spRRV shows considerable improvement in efficiencies of gene transfer and spreading in both human glioblastoma cells and pre-established human glioblastoma mouse model compared with an sRRV system. When treated with ganciclovir after intratumoral injection of each vector system into pre-established U-87 MG glioblastomas, the group of mice injected with spRRV expressing the herpes simplex virus type 1-thymidine kinase (HSV1-tk) gene showed a survival rate of 100% for more than 150 days, but all control groups of mice (HSV1-tk/PBS-treated and GFP/GCV-treated groups) died within 45 days after tumor injection. In conclusion, these findings sug-gest that intratumoral delivery of the HSV1-tk gene by the spRRV system is worthy of development in clinical trials for the treatment of malignant solid tumors. [BMB Reports 2022; 55(12): 615-620].

Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc.

Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. After 4weeks, 90% of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil ([18F]FEAU). This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

Human Wharton's jelly–derived mesenchymal stromal cells engineered to secrete Epstein-Barr virus interleukin-10 show enhanced immunosuppressive properties

Background aimsMesenchymal stromal cells (MSCs) modulate the immune response and represent a potential treatment for inflammatory and autoimmune diseases. We hypothesized that this feature could be potentiated by co-administering anti-inflammatory cytokines. In this article, we asked whether engineering of Wharton Jelly–derived human MSCs (WJ-hMSCs) to express an anti-inflammatory cytokine increases cell immunomodulatory properties without altering their native features. MethodsWe used Epstein-Barr virus–derived interleukin-10 (vIL-10), which shares some immunosuppressive properties with human IL-10 but lacks immunostimulatory activity. Engineering was accomplished by transducing WJ-hMSCs with a self-inactivating feline immunodeficiency virus–derived vector co-expressing vIL-10 and herpes simplex virus type-1 thymidine kinase (TK). TK was added to allow future tracking of WJ-hMSC in vivo by positron electron tomography (PET). ResultsThe results show that (i) expression of TK and/or vIL-10 does not change WJ-hMSC phenotypic and functional properties; (ii) vIL-10 is secreted, biologically active and enhances the immunosuppressing functions of WJ-hMSCs; (iii) v-IL10 and TK can be produced simultaneously by the same cells and do not interfere with each other. DiscussionWJ-hMSCs engineered to secrete vIL-10 could be a powerful tool for adoptive cell therapy of immune-mediated diseases, and therefore, additional studies are warranted to confirm their efficacy in suitable animal disease models.

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA–SCID) mice. TK-NOG mice were injected intraperitoneally with 6mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA–SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA–SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8weeks after injection) was similar in both TK-NOG mice and uPA–SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.

Transforming Thymidine into a Magnetic Resonance Imaging Probe for Monitoring Gene Expression

Synthetic chemistry has revolutionized the understanding of many biological systems. Small compounds that act as agonists and antagonists of proteins, and occasionally as imaging probes, have contributed tremendously to the elucidation of many biological pathways. Nevertheless, the function of thousands of proteins is still elusive, and designing new imaging probes remains a challenge. Through screening and characterization, we identified a thymidine analogue as a probe for imaging the expression of herpes simplex virus type-1 thymidine kinase (HSV1-TK). To detect the probe, we used chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI), in which a dynamic exchange process between an exchangeable proton and the surrounding water protons is used to amplify the desired contrast. Initially, five pyrimidine-based molecules were recognized as putative imaging agents, since their exchangeable imino protons resonate at 5-6 ppm from the water proton frequency and their detection is therefore less affected by endogenous CEST contrast or confounded by direct water saturation. Increasing the pK(a) value of the imino proton by reduction of its 5,6-double bond results in a significant reduction of the exchange rate (k(ex)) between this proton and the water protons. This reduced k(ex) of the dihydropyrimidine nucleosides fulfills the "slow to intermediate regime" condition for generating high CEST-MRI contrast. Consequently, we identified 5-methyl-5,6-dihydrothymidine as the optimal probe and demonstrated its feasibility for in vivo imaging of HSV1-TK. In light of these findings, this new approach can be generalized for designing specific probes for the in vivo imaging of a variety of proteins and enzymes.

Remnant living cells that escape cell loss in late-stage tumors exhibit cancer stem cell-like characteristics

A balance between cell proliferation and cell loss is essential for tumor progression. Although up to 90% of cells are lost in late-stage carcinomas, the progression and characteristics of remnant living cells in tumor mass are unclear. Here we used molecular imaging to track the progression of living cells in a syngeneic tumor model, and ex vivo investigated the properties of this population at late-stage tumor. The piggyBac transposon system was used to stably introduce the dual reporter genes, including monomeric red fluorescent protein (mRFP) and herpes simplex virus type-1 thymidine kinase (HSV1-tk) genes for fluorescence-based and radionuclide-based imaging of tumor growth in small animals, respectively. Iodine-123-labeled 5-iodo-2′-fluoro-1-beta-𝒟-arabinofuranosyluracil was used as a radiotracer for HSV1-tk gene expression in tumors. The fluorescence- and radionuclide-based imaging using the single-photon emission computed tomography/computed tomography revealed that the number of living cells reached the maximum at 1 week after implantation of 4T1 tumors, and gradually decreased and clustered near the side of the body until 4 weeks accompanied by enlargement of tumor mass. The remnant living cells at late-stage tumor were isolated and investigated ex vivo. The results showed that these living cells could form mammospheres and express cancer stem cell (CSC)-related biomarkers, including octamer-binding transcription factor 4, SRY (sex-determining region Y)-box 2, and CD133 genes compared with those cultured in vitro. Furthermore, this HSV1-tk-expressing CSC-like population was sensitive to ganciclovir applied for the suicide therapy. Taken together, the current data suggested that cells escaping from cell loss in late-stage tumors exhibit CSC-like characteristics, and HSV1-tk may be considered a theranostic agent for targeting this population in vivo.

Comparison of Cell-Labeling Methods with 124I-FIAU and 64Cu-PTSM for Cell Tracking Using Chronic Myelogenous Leukemia Cells Expressing HSV1-tk and Firefly Luciferase

Cell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with ⁶⁴Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (⁶⁴Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated ¹²⁴I-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil (¹²⁴I-FIAU) were comparatively investigated in vitro and in vivo for tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tk and firefly luciferase gene in the K562 cell. K562-TL cells were labeled with ⁶⁴Cu-PTSM or ¹²⁴I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivo and ex vivo bioluminescence imaging (BLI) and tissue reverse transcriptase-polymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of ¹²⁴I-FIAU (95.2%±1.1%) was fourfold higher than ⁶⁴Cu-PTSM (23.6%±0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between ¹²⁴I-FIAU and ⁶⁴Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of ⁶⁴Cu-PTSM- and ¹²⁴I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of ¹²⁴I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The ⁶⁴Cu-PTSM-labeled cell-tracking method is more efficient than ¹²⁴I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and fast efflux of ¹²⁴I-FIAU in vivo. In spite of a high labeling yield and radiolabel retention of ¹²⁴I-FIAU in vitro, the in vivo cell-tracking method using ⁶⁴Cu-PTSM could be a useful method to evaluate the distribution and targeting of various cell types, especially, stem cells and immune cells.

An improved strategy for the synthesis of [¹⁸F]-labeled arabinofuranosyl nucleosides.

The expression of the herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene can be imaged efficaciously using a variety of 2'-[(18)F]fluoro-2'-deoxy-1-b-D-arabinofuranosyl-uracil derivatives [[(18)F]-FXAU, X=I(iodo), E(ethyl), and M(methyl)]. However, the application of these derivatives in clinical and translational studies has been impeded by their complicated and long syntheses (3-5h). To remedy these issues, in the study at hand we have investigated whether microwave or combined catalysts could facilitate the coupling reaction between sugar and nucleobase and, further, have probed the feasibility of establishing a novel approach for [(18)F]-FXAU synthesis. We have demonstrated that the rate of the trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed coupling reaction between the 2-deoxy-sugar and uracil derivatives at 90 °C can be significantly accelerated by microwave-driven heating or by the addition of Lewis acid catalyst (SnCl(4)). Further, we have observed that the stability of the α- and β-anomers of [(18)F]-FXAU derivatives differs during the hydrolysis step. Using the microwave-driven heating approach, overall decay-corrected radiochemical yields of 19%-27% were achieved for [(18)F]-FXAU in 120min at a specific activity of >22MBq/nmol (595Ci/mmol). Ultimately, we believe that these high yielding syntheses of [(18)F]-FIAU, [(18)F]-FMAU and [(18)F]-FEAU will facilitate routine production for clinical applications.

Accumulated HSV1-TK Proteins Interfere with Spermatogenesis through a Disruption of the Integrity of Sertoli-Germ Cell Junctions

Transgenic rats show spermatid-specific ectopic expression of the reporter gene, herpes simplex virus type1 thymidine kinase (HSV1-TK), in the testes and have demonstrated male infertility. However, the disruption of spermatogenesis and the underlying molecular mechanisms in these transgenic animals have not been well clarified. In this study, light and electron microscopic observations were performed to characterize the morphological changes in the testes. To explore the molecular mechanisms of male infertility in the HSV1-TK transgenic rat, cDNA microarray and quantitative real-time PCR analyses were performed. The seminiferous tubules of 3-month-old transgenic rats showed morphological alterations including seminiferous epithelial sloughing, vacuolization, and degeneration of spermatogenic cells, suggesting a failure of Sertoli-germ cell interaction. Components of the epididymal lumen from transgenic rats included abnormal spermatozoa, degenerating round spermatids and abnormal elongated spermatids indicating an appearance of direct impairment of spermiogenesis. cDNA microarray and real-time PCRanalyses revealed significant changes (P<0.05) in the gene expression level in six genes, testin, versican, mamdc1, fgf7, ostf1 and cnot7. Among them, testin drew most of our attention, since the testin gene is a sensitive marker for disruption of Sertoli-germ cell adhesion. Thus, our results suggest that the accumulation of HSV1-TK in the spermatids not only directly interferes with spermiogenesis but also disrupts spermatogenesis through a disruption of Sertoli-germ cell adhesions. It is important to explore the testicular actions of the HSV1-TK protein in transgenic experimental models and thereby gain clues to find an appropriate treatment for HSV-infected patients exhibiting human male infertility, as has been recently observed.

Combined Flt3L/TK Gene Therapy Induces Immunological Surveillance Which Mediates an Immune Response Against a Surrogate Brain Tumor Neoantigen

Glioblastoma multiforme (GBM) is a primary brain tumor with a median survival of 14.6 months postdiagnosis. The infiltrative nature of GBM prevents complete resection and residual brain tumor cells give rise to recurrent GBM, a hallmark of this disease. Recurrent GBMs are known to harbor numerous mutations/gene rearrangements when compared to the primary tumor, which leads to the potential expression of novel proteins that could serve as tumor neoantigens. We have developed a combined immune-based gene therapeutic approach for GBM using adenoviral (Ads) mediated gene delivery of Herpes Simplex Virus Type 1-thymidine kinase (TK) into the tumor mass to induce tumor cells' death combined with an adenovirus expressing fms-like tyrosine kinase 3 ligand (Flt3L) to recruit dendritic cells (DCs) into the tumor microenvironment. This leads to the induction of specific anti-brain tumor immunity and immunological memory. In a model of GBM recurrence, we demonstrate that Flt3L/TK mediated immunological memory is capable of recognizing brain tumor neoantigens absent from the original treated tumor. These data demonstrate that the Flt3L/TK gene therapeutic approach can induce systemic immunological memory capable of recognizing a brain tumor neoantigen in a model of recurrent GBM.

A new pyrimidine-specific reporter gene: a mutated human deoxycytidine kinase suitable for PET during treatment with acycloguanosine-based cytotoxic drugs.

In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET. Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs. Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers ((18)F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine). The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with (18)F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

Therapeutic Efficacy of Human Hepatocyte Transplantation in a SCID/uPA Mouse Model with Inducible Liver Disease

BackgroundSevere Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice.Methodology/Principal FindingsIn vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32–87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.Conclusions/SignificanceExtensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

Optical imaging of reporter gene expression using a positron-emission-tomography probe

Reporter gene∕reporter probe technology is one of the most important techniques in molecular imaging. Lately, many reporter gene∕reporter probe systems have been coupled to different imaging modalities such as positron emission tomography (PET) and optical imaging (OI). It has been recently found that OI techniques could be used to monitor radioactive tracers in vitro and in living subjects. In this study, we further demonstrate that a reporter gene∕nuclear reporter probe system [herpes simplex virus type-1 thymidine kinase (HSV1-tk) and 9-(4-(18)F-fluoro-3-[hydroxymethyl] butyl) guanine ([(18)F]FHBG)] could be successfully imaged by OI in vitro and in vivo. OI with radioactive reporter probes will facilitate and broaden the applications of reporter gene∕reporter probe techniques in medical research.

Triple Suicide Gene Therapy Radiosensitizes Prostate Cancer Cells

The double suicide gene therapy strategy combining herpes simplex virus type-1 thymidine kinase (HSV-1 TK) and cytosine deaminase (CD) with ganciclovir (GCV) and 5-fluorocytosine (5-FC) had been carried out in a phase I clinical trail for prostate cancer. The objective of the present study was to ascertain whether co-expression of HSV-1 TK, CD, and uracil phosphoribosyltransferase (UPRT) in conjunction with GCV and 5-FC treatment, a triple suicide gene therapy strategy, would be more effective at killing and radiosensitizing prostate cancer cells than double suicide gene therapy.

Multimodal Imaging of Neural Progenitor Cell Fate in Rodents

For clinical application of stem cell-based therapies, noninvasive detection of applied stem cells is of high importance. We report on the feasibility of detecting implanted neural progenitor cells (NPCs) noninvasively and follow their fate and functional status by sequential multimodal molecular imaging and reporter gene technology. We investigated C17.2 cells stably expressing herpes simplex virus type 1-thymidine kinase (HSV-1-tk) and green fluorescent protein (gfp) (C17.2-tkIRESgfp = C17.2-TIG) or HSV-1-tk, gfp, and firefly luciferase (luc) (C17.2-lucIREStkgfp = C17.2-LITG) and determined the detection sensitivity of positron emission tomography (PET) and bioluminescence imaging (BLI) for these cells in culture and in vivo in subcutaneous and intracranial glioma models. In addition, PET and BLI were used to further investigate and follow the fate of implanted C17.2-LITG cells in an intracranial glioma model. We show that both imaging modalities are sensitive in detecting reporter gene expressing NPCs; however, PET, by the use of 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG), detects NPCs only at sites of disrupted blood-brain barrier. Furthermore, both imaging modalities can be used to detect stem cell fate and migration and indicate excessive proliferation and aberrant migration. In conclusion, multimodal imaging can be used for longitudinal noninvasive monitoring of grafted NPCs in rodents.

High-Capacity Adenovirus Vector-Mediated Anti-Glioma Gene Therapy in the Presence of Systemic Antiadenovirus Immunity

Gene therapy is proposed as a novel therapeutic strategy for treating glioblastoma multiforme (GBM), a devastating brain cancer. In the clinic, antivector immune responses pose formidable challenges. Herein we demonstrate that high-capacity adenovirus vectors (HC-Ads) carrying the conditional cytotoxic gene herpes simplex virus type 1-thymidine kinase (TK) induce tumor regression and long-term survival in an intracranial glioma model, even in the presence of systemic antiadenovirus immunity, as could be encountered in patients. First-generation Ad-TK failed to elicit tumor regression in this model. These results pave the way for implementing HC-Ad-TK-mediated gene therapy as a powerful adjuvant for treating GBM.