Herpes Simplex Virus Thymidine Kinase Research Articles

A Systematic Review of Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Potential Treatment for Glioblastoma.

Glioblastoma (GBM) is an extremely aggressive brain tumor that has few available treatment options and a dismal prognosis. Recent research has highlighted the potential of extracellular vesicles (MSC-EVs) produced from mesenchymal stem cells as a potential treatment approach for GBM. MSC-EVs, including exosomes, microvesicles, and apoptotic bodies, perform a significant function in cellular communication and have shown promise in mediating anti-tumor effects. This systematic literature review aims to consolidate current findings on the therapeutic potential of MSC-EVs in GBM treatment. A systematic search was conducted across major medical databases (PubMed, Web of Science, and Scopus) up to September 2024 to identify studies investigating the use of MSC-derived EVs in GBM therapy. Keywords included "extracellular vesicles", "mesenchymal stem cells", "targeted therapies", "outcomes", "adverse events", "glioblastoma", and "exosomes". Inclusion criteria were studies published in English involving GBM models both in vivo and in vitro and those reporting on therapeutic outcomes of MSC-EVs. Data were extracted and analyzed based on EV characteristics, mechanisms of action, and therapeutic efficacy. The review identified several key studies demonstrating the anti-tumor effects of MSC-EVs in GBM models. A total of three studies were included, focusing on studies conducted between 2021 and 2023. The review included three studies that collectively enrolled a total of 18 patients. These studies were distributed across two years, with two trials published in 2023 (66.7%) and one in 2021 (33.3%). The mean age of the participants ranged from 37 to 57 years. In terms of gender distribution, males were the predominant group in all studies. Prior to receiving MSC-EV therapy, all patients had undergone standard treatments for GBM, including surgery, chemotherapy (CT), and, in some cases, radiation therapy (RT). In all three studies, the targeted treatment involved the administration of herpes simplex virus thymidine kinase (HSVtk) gene therapy delivered to the tumor site, then 14 days of ganciclovir treatment. Outcomes across the studies indicated varying levels of efficacy for the MSC-EV-based therapy. The larger 2023 study reported fewer encouraging outcomes, with a median PFS of 11.0 months (95% CI: 8.3-13.7) and a median OS of 16.0 months (95% CI: 14.3-17.7). Adverse effects were reported in only one of the studies, the 2021 trial, where patients experienced mild-to-moderate side effects, including fever, headache, and cerebrospinal fluid leukocytosis. A total of 11 studies on preclinical trials, using in vitro and in vivo models, were included, covering publications from 2010 to 2024. The studies utilized MSCs as delivery systems for various therapeutic agents (interleukin 12, interleukin 7, doxorubicin, paclitaxel), reflecting the versatility of these cells in targeted cancer therapies. MSC-derived EVs represent a promising therapeutic approach for GBM, offering multiple mechanisms to inhibit tumor growth and enhance treatment efficacy. Their ability to deliver bioactive molecules and modulate the tumor microenvironment underscores their potential as a novel, cell-free therapeutic strategy. Future studies should optimize EV production and delivery methods and fully understand their long-term effects in clinical settings to harness their therapeutic potential in GBM treatment.

Optimization of a lentivirus-mediated gene therapy targeting HIV-1 RNA to eliminate HIV-1-infected cells

Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA trans-splicing at the HIV-1 D4 donor site. AUG1-deficient HSVtk therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 tat1. D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where trans-spliced HIV-1 tat/HSVtk mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in HSVtk ΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted in silico correlated with the propensity of opt 2 payloads to induce HIV-1 RNA trans-splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach.

Establishing an induced infertile chicken line for efficient germline transmission of exogenous PGCs

Primordial germ cells (PGCs) are the stem-cell population of adult animal gametes, which develop into sperm or eggs. It can be propagated in vitro and injected into the host chicken for genome editing to obtain germline chimeric chicken. However, it has the limitation that the host embryo contains endogenous PGCs, which raises complications, resultantly donor PGCs fail to compete, and transmission efficiency reduced. Therefore, to increase the transmission efficiency, we generated a novel sterile chicken with the inducible elimination of endogenous PGCs in the host. This is the first study that applied the herpes simplex virus thymidine kinase (HSV-TK) cell ablation system in avian. CRISPR/Cas9-mediated homology-directed repair was performed to localize the HSV-TK suicide gene to the last exon of the deleted in azoospermia-like (DAZL) gene, and ganciclovir (GCV) was added to induce the apoptosis in the germ cells of the host embryo. The sterilized host embryo introduced genome-edited PGCs to produce chimeric chicken carrying exogenous germ cells only. It was observed that the germline transmission efficiency was 100% achieved, and the obtained chicks were purely from donor breeds. The technologies established in the current study have important applications in germplasm conservation and gene editing in chicken.

MDB-07. A SUICIDE GENE THERAPY TARGETING SOX9-POSITIVE THERAPY-RESISTANT MEDULLOBLASTOMA CELLS

Abstract The transcription factor SOX9 is a stem cell factor and a glial fate marker in the developing brain. SOX9 levels correlate with worse prognosis and relapse in Group 3 medulloblastoma, a malignant pediatric brain tumor with poor survival. SOX9 activation promotes epidermal to mesenchymal transition and leptomeningeal spread. It is also upregulated by and facilitates cisplatin resistance in medulloblastoma cells. Direct SOX9 inhibition has proven difficult and few targeted substances have emerged from drug screening efforts. SOX9 acts as a selective and potent activator of a minimal collagen type II alpha 1 enhancer element. We here show that cells with SOX9 activity can be efficiently and specifically labeled when this SOX9 response element is coupled to a minimal promoter (S9RE-minP) driving GFP or luciferase. We further show that S9RE-minP construct can be used to drive herpes simplex virus thymidine kinase (TK) in a suicide gene construct that can convert ganciclovir to a cytotoxic drug in mitotic SOX9-positive cells. The gene therapy worked efficiently together with cisplatin to eradicate SOX9-positive medulloblastoma cells in vitro and could be safely combined and synergize with fractionated radiation in vivo. We suggest that this could be an attractive approach to target therapy-resistant cells in malignant pediatric brain tumors.

A lentivirus-vectored feline erythropoietin gene therapy strategy in tissue culture and rodent models for the potential treatment of chronic renal disease-associated anemia.

The aim of this study was to assess the efficacy and safety of a third-generation lentivirus-based vector encoding the feline erythropoietin (EPO) (feEPO) gene in vitro and in rodent models in vivo. This vector incorporates a genetic mechanism to facilitate the termination of the therapeutic effect in the event of supraphysiologic polycythemia, the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene." CFRK cells and replication-defective lentiviral vectors encoding feEPO were used for in vitro experiments. Eight Fischer rats were enrolled in the pilot in vivo study, 24 EPO-deficient mice were used in the initial mouse study, and 15 EPO-deficient mice were enrolled in the final mouse study. Efficacy of a third-generation lentivirus encoding feEPO was determined in vitro using western blot assays. Subsequently, in a series of rodent experiments, animals were administered the viral vector in progressively increasing inoculation doses with serial measurements of blood packed cell volume (PCV) over time. We documented production of feEPO protein in transduced CRFK cells with subsequent cessation of production when treated with the HSV-TK substrate ganciclovir. In vivo, we demonstrated variably persistent elevated PCV values in treated rats and mice with eventual return to baseline values over time. These results provide justification for a lentiviral gene therapy approach to the treatment of nonregenerative anemia associated with chronic renal disease in cats.

Abstract P32: Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease

Abstract Cell type-specific gene delivery which restricts gene transfer to therapy-relevant cells, hence reducing unwanted side effects caused by the ectopic expression of transgenes, remains one of the biggest challenges. To mitigate potential off-target effects, we developed a programmable cell type-specific gene therapy using an RNA trans-splicing-based approach for gene replacement therapy or suicide gene therapy that can selectively express therapeutic proteins or suicide signals based on the cell’s endogenous pre-mRNA expression profile. Using the liver as a medical target organ, we developed a hepatocellular carcinoma (HCC)-specific trans-splicing based Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy approach. We rationally designed suicide RNAs that can target multiple HCC-related pre-mRNA biomarkers thereby increasing the overall concentration of targetable pre-mRNA. For delivery, non-viral redosable dumbbell-shaped DNA vectors were designed, manufactured, and tested in tissue culture cells, in 3D patient-derived xenograft spheroids and in mouse model. For cell-type specific delivery, the conjugation of dumbbell vectors with GalNAc3 residues enabled targeted delivery into hepatoblastoma-derived human tissue culture cells. To deliver nucleic acids into the mitochondria of human cells, we studied the long non-coding β2.7 RNA of the human cytomegalovirus which was previously reported to colocalise with mitochondrial complex I. Using thermodynamic profiling, we identified four thermodynamically conserved structural subdomains within the β2.7 RNA that are responsible for its mitochondrial targeting activity. A vector comprising eight mitochondrial targeting domains exhibited a three times higher mitochondrial targeting activity as compared with the parental β2.7 RNA. The amalgamation of mitochondrial targeting RNA and trans-splicing allows cell type-specific transcription of mRNA which can be delivered into mitochondria for mitochondrial gene therapy. Trans-splicing based gene therapy represent a technology that is highly translational offering promising perspectives towards suicide gene therapy of any cancer types or incurable infections with integrating viruses and may enable cell type-specific mitochondrial gene therapy. Citation Format: Pei She Loh (Rachel), Sushmita Poddar, Kaustav Chatterjee, Chiu Jing Wen, Derrick Wee Zan Liang, Fritz Lai, Chen Qingfeng, Volker Patzel. Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P32.

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.

Abstract 7250: Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors

Abstract GEN2 is a non-replicating retroviral gene transfer vector encoding a sequence-optimized herpes simplex thymidine kinase (HSV-TK), which activates the antiherpetic pro-drug ganciclovir (GCV), resulting in chain termination during DNA replication, triggering cancer cell apoptosis and release of neoantigens (NA). GEN2 also encodes hGM-CSF, which may augment antitumor immunity by enhancing NA presentation to immune effector cells. GEN2 in combination with valganciclovir (an oral formulation of GCV) was evaluated in a dose-escalating Phase 1 clinical trial in 61 adult patients (pts) with an advanced solid tumor (NCT04313868), with serial assessment of PBMCs to detect any GEN2 integration and replication competent retrovirus (RCR). Methods: PBMCs were obtained in Cycle 1 (following 1st/3rd dose), prior to and 2 hours after Cycle 2, and thereafter at 6-month intervals for 15 years, or until death or withdrawal of consent. Digital droplet PCR (ddPCR) for psi and envelope gene sequences in extracted genomic DNA from PBMCs was used to detect vector genome integration and replication competent retrovirus (RCR), respectively. Results: All 82 samples obtained from the intravenous cohorts 1-12 were below LOD (Limit of Detection) for RCR. All results for integration were BLOQ (Below Limit of Quantitation), whereas 4 samples collected at two hours following the end of infusion were above LOD, likely resulting from non-integrated cDNA. GEN2 continues to be studied in clinical trials in adult patients with solid tumors. Per the 2020 FDA Guidance for Industry (Long Term Follow-Up After Administration of Human Gene Therapy Products), serial assessment of vector integration is mandated throughout a pt’s participation in a gene therapy clinical trial and for 15 years following the last dose. These guidance documents were originally conceived when gene therapy was developed for treatment of rare hereditary diseases in pediatric pts; however, adult pts enrolled in early-stage clinical trials for cancer typically do not have a prolonged life expectancy (BMC Cancer 2011 11:426). In the context of proper and warranted usage of pts’ blood for testing, attention should be drawn to the limited expectation for a 2nd malignancy within the lifespan of this pt population, and the absence of detectable RCR in cancer patients receiving retroviral vectors over decades. A recent review noted no evidence of RCR in 1,595 post-treatment PBMCs obtained from 60 clinical trials (Mol Ther 2023 31(3):801). Our results confirm that GEN2 also shows negligible genotoxic risk for gene therapy of cancer at doses tested to date. Citation Format: Alison L. Hannah, Noriyuki Kasahara, Li Liu, Allen Wang, Joe McNulty, Robert G. Johnson. Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7250.

Engineering of Rhesus Monkey Pluripotent Stem Cells for Noninvasive Survey and Remote Control after Brain Allotransplantation

Advanced technologies of targeted differentiation and genetic engineering in pluripotent stem cells (PSCs) offer production of purposefully designed cells for transplantation. Here, we engineer rhesus monkey PSCs (rhPSCs) to produce safe and functional neural progenitors and neurons, which could be noninvasively surveyed and controlled after brain transplantation. RhPSCs expressing hM3Dq-mCherry exhibited normal karyotype and had the ability to equally self-renew and differentiate into functional neurons as that of the mCherry expressing control. After allotransplantation into monkey prefrontal cortex, the hM3Dq-mCherry expressing rhPSC-derived cortical progenitors survived and matured gradually, which could be long-term surveyed by [18F]-Fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) and electroencephalogram (EEG) following clozapine-N-oxide (CNO) administration. Remote activation of transplanted neurons caused increased [18F]-Fluorodeoxyglucose uptake started at 3 months and reached a plateau 9-12 months post-transplantation. EEG analysis revealed a decrease in the high delta spectrum power while an increase in the beta power after CNO delivery. Notably, no seizure-like spikes were observed even after repeated CNO exposure. Magnetic resonance (MR) imaging found no overgrowth in all allografts. Expression of herpes simplex virus thymidine kinase (HSVtk), a Ganciclovir (GCV)-induced suicide gene in rhPSCs further assured the safety without sacrificing the functional outcome of brain grafts. Our study offers a feasible strategy for long-term noninvasive survey and remote control of brain-grafted neurons.

Development of an optimized, non-stem cell line for intranasal delivery of therapeutic cargo to the central nervous system.

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain.

Quantitative Structure–Activity Relationship in the Series of 5-Ethyluridine, N2-Guanine, and 6-Oxopurine Derivatives with Pronounced Anti-Herpetic Activity

A quantitative analysis of the relationship between the structure and inhibitory activity against the herpes simplex virus thymidine kinase (HSV-TK) was performed for the series of 5-ethyluridine, N2-guanine, and 6-oxopurines derivatives with pronounced anti-herpetic activity (IC50 = 0.09 ÷ 160,000 μmol/L) using the GUSAR 2019 software. On the basis of the MNA and QNA descriptors and whole-molecule descriptors using the self-consistent regression, 12 statistically significant consensus models for predicting numerical pIC50 values were constructed. These models demonstrated high predictive accuracy for the training and test sets. Molecular fragments of HSV-1 and HSV-2 TK inhibitors that enhance or diminish the anti-herpetic activity are considered. Virtual screening of the ChEMBL database using the developed QSAR models revealed 42 new effective HSV-1 and HSV-2 TK inhibitors. These compounds are promising for further research. The obtained data open up new opportunities for developing novel effective inhibitors of TK.

CTIM-28. FIRST-IN-HUMAN TRANSGENE DETECTION 17 MONTHS POST-ADENOVIRAL THERAPY

Abstract Gene therapy was developed as a promising approach to treat high-grade glioma (HGG). One of the main strategies is suicide gene therapy. This method introduces the expression of an enzyme that converts a non-toxic prodrug to an active toxic compound, thereby causing the death of transduced cells. Specifically, herpes simplex virus thymidine kinase (HSV1-TK) converts the pro-drug valacyclovir into phosphorylated valacyclovir. When cells initiate DNA replication, phosphorylated valacyclovir is incorporated into de novo DNA strands, causing DNA replication to arrest, followed by apoptosis. This approach is lethal only for replicating cells, (i.e., tumor cells), without harming non-dividing brain cells. An infected cell, expressing HSV1-TK, requires two further simultaneous events to induce cell death: (1) to be exposed to valacyclovir and (2) enter into cell division. Without either of these events, the cell will remain alive and will still express HSV1-TK. Previous work from our lab in murine models demonstrated the long-term expression of transgenes β-galactosidase and HSV1-TK up to 12 months after viral therapy administration (Zermansky et al 2001; Barcia et al 2007). We recently completed and published our Phase I clinical trial (NCT01811992), where dual vector viral therapy, Ad-TK/Ad-Flt3L, and valacyclovir was administered to patients suffering from HGG (Umemura et al, 2023). In paraffin embedded tumor samples from recurrent tumors to our surprise we detected HSV1-TK immunoreactivity up to 17 months after initial treatment. HSV1-TK immunoreactive cells were found in five out of eight patients’ samples. We analyzed HSV1-TK expression to further characterize HSV1-TK positive cells using combination immunohistochemistry, allowing us to identify them as tumor cells, neurons, or astrocytes. The presence of HSV-TK positive cells in the recurrent tumors strongly supports the administration of further cycles of pro drug (valacyclovir), following Ad-TK administration.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese-hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MAR), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR-1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+ hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells. This article is protected by copyright. All rights reserved.

EndoC-βH5 cells are storable and ready-to-use human pancreatic beta cells with physiological insulin secretion

ObjectivesReadily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-βH5 cells, the latest in the EndoC-βH cell family. MethodsEndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed transcriptome, immunological and extensive functional assays. ResultsReady to use EndoC-βH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-βH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-βH5 cells elicit specific A2-alloreactive CD8 T cell activation. ConclusionsEndoC-βH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.

CAN-2409 plus prodrug with standard of care immune checkpoint inhibitor for patients with stage III/IV NSCLC.

TPS9162 Background: Immune checkpoint inhibitors (ICI) are a standard of care (SOC) treatment that have improved outcomes for patients (pts) with non-small cell lung cancer (NSCLC). However, most pts progress, signaling a need for combination approaches to further improve outcomes. CAN-2409 is a replication-defective adenoviral construct encoding the herpes simplex virus thymidine kinase (HSV-tk) gene that is injected intratumorally followed by 2 weeks of prodrug (valacyclovir or acyclovir). HSV-tk converts the prodrug in the tumor microenvironment into a toxic metabolite, resulting in immunogenic cell death, while the adenoviral proteins provide a strong pro-inflammatory signal. Together, this leads to in situ vaccination against the patient’s own tumor. As a result, CD8+ cytotoxic T cells are educated on how to recognize and kill tumor cells in both the injected tumor and the uninjected metastases. A completed neoadjuvant phase I trial in resectable NSCLC demonstrated safety and immune stimulation (Predina et al 2021), warranting further investigation of CAN-2409 + prodrug as a therapy that can lead to improved long-term outcomes for pts with NSCLC who have a suboptimal response to ICI. Methods: LuTK02 (NCT04495153) is an open-label, multi-arm, phase II clinical trial to evaluate CAN-2409 + prodrug added to SOC ICI (anti-PD-(L)1) therapy in pts with stage III/IV NSCLC presenting with inadequate response (as defined below) to ICI. Cohort 1 includes pts with nonresponding but stable disease ≥18 weeks after starting ICI; cohort 2 includes pts with progressive disease ≥18 weeks after starting ICI. Key eligibility criteria include RECIST-evaluable disease; an injectable lesion (via bronchoscopy or US/IR guidance); ability to continue ICI for the treatment period; no change of ICI or prior interruptions of > 4 weeks within 6 months of enrollment; and no focal therapy at > 3 sites of disease within 12 months prior to enrollment. Key exclusion criteria include a history of ICI immune-related adverse events and known alterations in EGFR, ALK, or ROS1. Eligible patients receive 2 injections of CAN-2409 approximately 6 weeks apart followed by prodrug for 14 days. After treatment of ~40 evaluable pts in cohort 2 with two injections of CAN-2409, the protocol has been amended to include a new cohort (n~20) of pts treated with three injections of CAN-2409 approximately 4 weeks apart, each followed by prodrug for 14 days, to further define the optimal therapeutic regimen. Primary endpoints are overall response and/or disease control rate per RECIST 1.1 criteria and safety of CAN-2409 + prodrug. Secondary endpoints include duration of response, PFS, OS, and biomarker studies. Exploratory endpoints include evaluation of effects in non-injected lesions, changes in non-target (per RECIST) lesions, tumor growth trajectory, and viral shedding. Clinical trial information: NCT04495153 .

Suicide gene therapy using allogeneic adipose tissue-derived mesenchymal stem cell gene delivery vehicles in recurrent glioblastoma multiforme: a first-in-human, dose-escalation, phase I clinical trial

BackgroundGlioblastoma multiforme (GBM) is associated with remarkably poor prognosis, and its treatment is challenging. This investigation aimed to evaluate the safety of suicide gene therapy using allogeneic adipose tissue-derived mesenchymal stem cells (ADSCs) carrying herpes simplex virus-thymidine kinase (HSV-TK) gene for the first time in patients with recurrent GBM.MethodsThis study was a first-in-human, open-label, single-arm, phase I clinical trial with a classic 3 + 3 dose escalation design. Patients who did not undergo surgery for their recurrence were included and received this gene therapy protocol. Patients received the intratumoral stereotactic injection of ADSCs according to the assigned dose followed by prodrug administration for 14 days. The first dosing cohort (n = 3) received 2.5 × 105 ADSCs; the second dosing cohort (n = 3) received 5 × 105 ADSCs; the third dosing cohort (n = 6) received 10 × 105 ADSCs. The primary outcome measure was the safety profile of the intervention.ResultsA total of 12 patients with recurrent GBM were recruited. The median follow-up was 16 (IQR, 14-18.5) months. This gene therapy protocol was safe and well tolerated. During the study period, eleven (91.7%) patients showed tumor progression, and nine (75.0%) died. The median overall survival (OS) was 16.0 months (95% CI 14.3–17.7) and the median progression-free survival (PFS) was 11.0 months (95% CI 8.3–13.7). A total of 8 and 4 patients showed partial response and stable disease, respectively. Moreover, significant changes were observed in volumetric analysis, peripheral blood cell counts, and cytokine profile.ConclusionsThe present clinical trial, for the first time, showed that suicide gene therapy using allogeneic ADSCs carrying the HSV-TK gene is safe in patients with recurrent GBM. Future phase II/III clinical trials with multiple arms are warranted to validate our findings and further investigate the efficacy of this protocol compared with standard therapy alone.Trial registration: Iranian Registry of Clinical Trials (IRCT), IRCT20200502047277N2. Registered 8 October 2020, https://www.irct.ir/.

Abstract CT196: A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer

Abstract Introduction: Men with locally recurrent prostate cancer, after definitive radiotherapy, have few therapeutic options. Oncolytic adenovirus-mediated cytotoxic gene therapy is an investigational cancer therapy. Delivery of the suicide gene to the tumor is by direct intratumoral or systemic injection of a viral vector containing the suicide gene. Our approach incorporates yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK), to confer sensitivity to 5-fluorocytosine (5-FC) and Valganciclovir (vGCV), respectively. The pro-drugs are converted into active drugs that inhibit DNA damage repair. Here we report the safety of oncolytic adenovirus-mediated suicide gene therapy that incorporates an interleukin-12 (IL12) gene for treatment of recurrent prostate cancer. Methods: In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL12) was injected into tumors of 15 subjects with recurrent prostate cancer (T1c-T2) at escalating doses (1 × 1010, 3 × 1010, 1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-FC and vGCV for 7 days. The study endpoint was toxicity through day 30. Experimental endpoints included measurements of serum IL12, interferon gamma (IFNγ), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells (PBMC) and proliferation markers were analyzed by flow cytometry. Results and Conclusions: Fifteen patients received Ad5-yCD/mutTKSR39rep-hIL-12 and oral 5-FC and vGCV. Approximately 92% of the 115 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39rep-hIL-12 DNA was detected in the blood of only two patients. Elevated serum IL12, IFNγ, and CXCL10 levels were detected in 57%, 93%, and 79% of subjects, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39rep-hIL-12 administration in cohort 5. The study did not detect a significant difference in the PSA doubling time (PSADT) between pre and post treatment by paired Wilcoxon rank test (p=0.17). There was no correlation between adenoviral dose and PSADT in each cohort separately or pooled (cohorts1-3 and cohorts 4-5). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39 rep-hIL-12) was well tolerated when administered locally to prostate tumors. Citation Format: Shyam Nyati, Hans Stricker, Kenneth L. Barton, Piln Li, Mohamed Elshaikh, Haythem Ali, Stephen L. Brown, Clara Hwang, James Peabody, Svend O. Freytag, Benjamin Movsas, Farzan Siddiqui. A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT196.

Therapeutic effects of anti-GM2 CAR-T cells expressing IL-7 and CCL19 for GM2-positive solid cancer in xenograft model.

While chimeric antigen receptor (CAR)-T cell therapy has demonstrated excellent efficacy in hematopoietic malignancies, its clinical application in solid cancers has yet to be achieved. One of the reasons for such hurdle is a lack of suitable CAR targets in solid cancers. GM2 is one of the gangliosides, a group of glycosphingolipids with sialic acid in the glycan, and overexpressed in various types of solid cancers. In this study, by using interleukin (IL)-7 and chemokine (C-C motif) ligand 19 (CCL19)-producing human CAR-T system which we previously developed, a possibility of GM2 as a solid tumor target for CAR-T cell therapy was explored in a mouse model with human small-cell lung cancer. Treatment with anti-GM2 IL-7/CCL19-producing CAR-T cells induced complete tumor regression along with an abundant T cell infiltration into the solid tumor tissue and long-term memory responses, without any detectable adverse events. In addition, as measures to control cytokine-release syndrome and neurotoxicity which could occur in association with clinical use of CAR-T cells, we incorporated Herpes simplex virus-thymidine kinase (HSV-TK), a suicide system to trigger apoptosis by administration of ganciclovir (GCV). HSV-TK-expressing anti-GM2 IL-7/CCL19-producing human CAR-T cells were efficiently eliminated by GCV administration in vivo. Our study revealed the promising therapeutic efficacy of anti-GM2 IL-7/CCL19-producing human CAR-T cells with an enhanced safety for clinical application in the treatment of patients with GM2-positive solid cancers.

Abstract 6389: Differential role of CD8 cells and CD4 cells in anti-tumor immunity triggered by tumor cell apoptosis using HSVtk/GCV system

Abstract Immune cells can recognize tumor antigens and elicit an adaptive immune response, potentially resulting in tumor regression. Drug-induced dead tumor cells have been reported to enhance anti-tumor immunity, but results are conflicting, possibly due to the immunosuppressive effects of the cytotoxic drugs. To investigate whether apoptotic tumor cells trigger anti-tumor immunity independent of anti-cancer treatment, local immune responses were evaluated after direct induction of tumor cell apoptosis using a herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system. The inflammatory cytokines IFNγ and IL-12β and anti-inflammatory factor PD-1 and TGFβ1 were upregulated at the tumor site after apoptosis induction. HSV-tk/GCV-induced tumor cell apoptosis resulted in suppression of tumor growth in vivo and promoted the infiltration of CD8 and CD4 T lymphocytes in tumor. To explore the role of T cells after induction of tumor cell death, GCV was administered together with anti-CD8 or anti-CD4 depleting antibodies to syngeneic HSV-tk transfected tumor-bearing mice. CD8 T cell depletion abrogated the anti-tumor efficacy of GCV, indicating that tumor regression was mainly dependent on CD8 T cells. This anti-tumor immunity was enhanced by anti-PD-1 antibody injection. On the contrary, CD4 T cell depletion further inhibited tumor growth than GCV alone, suggesting the potential role of CD4 T cells in suppressive tumor immunity. To elucidate this immunological mechanism, tumor tissues were evaluated after tumor cell apoptosis and CD4 T cell depletion. Foxp3, a regulatory T cell marker, was decreased after CD4 T cell depletion. In addition, Arg1, a marker of immunosuppressive myeloid cells, was significantly downregulated, which was expressed on tumor-associated macrophages (TAMs) and monocytic myeloid-derived suppressor cells (mMDSCs). Collectively, these findings indicate that tumor cell apoptosis accelerates CD8 T cell-dependent anti-tumor immunity and CD4 T cell-mediated Arg1 production from TAMs and mMDSCs. Citation Format: Sho Umegaki, Hidekazu Shirota, Tomoyuki Iwasaki, Yuki Kasahara, Chikashi Ishioka. Differential role of CD8 cells and CD4 cells in anti-tumor immunity triggered by tumor cell apoptosis using HSVtk/GCV system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6389.

Molecular docking and dynamics supported investigation of antiviral activity of Lichen metabolites of Roccella montagnei: an in silico and in vitro study

Lichens are symbiotic organisms that have been traditionally used for treating different kinds of ailments. As there are only a few reports on the antiviral activity of lichens, we thought of evaluating the anti-Herpes simplex virus-1 (HSV-1) activity of methanolic extract of Roccella montagnei and their isolated compounds. Fractionation of crude methanolic extract of Roccella montagnei by column chromatography isolated two pure compounds. Antiviral activity was assessed using a CPE inhibition assay at non-cytotoxic concentrations on Vero cells. Molecular docking and dynamics studies were carried out against Herpes simplex type-1 thymidine kinase to understand the binding interactions of the isolated compounds with reference to acyclovir. Isolated compounds were characterized as methyl orsellinate and montagnetol by spectral methods. Methanolic extract of Roccella montagnei exhibited an EC50 value of 56.51 µg/ml, while the compounds methyl orsellinate and montagnetol offered EC50 values of 13.50 µg/ml and 37.52 µg/ml, respectively, against HSV-1 viral infection on Vero cell lines. The selectively index (SI) of montagnetol (10.93) was found to be higher when compared to that of methyl orsellinate (5.55), indicating its better anti-HSV-1 activity. The docking and dynamics studies showed montagnetol was stable throughout the 100 ns, having better interactions and docking scores with HSV-1 thymidine kinase than methyl orsellinate, as well as the standard. To understand the mechanism of montagnetol’s anti-HSV-1 activity, more research is required, and this could lead to the discovery of new and effective antiviral agents. Communicated by Ramaswamy H. Sarma