Adenoviral Delivery Research Articles

Temporally and Spatially Controlled Age-Related Prostate Cancer Model in Mice.

The initiation and progression of prostate cancer (PCa) are associated with aging. In the history of age-related PCa research, mice have become a more popular animal model option than any other species due to their short lifespan and rapid reproduction. However, PCa in mice is usually induced at a relatively young age, while it spontaneously develops in humans at an older age. Thus, it is essential to develop a method by which the PCa initiation and progression timeline can be strictly controlled to mimic human physiological conditions. One milestone in this field was the identification of the prostate-specific transcription factor, Probasin (Pb), which allowed for the prostate-specific expression of genes knocked into the mice's genome. Another milestone is the establishment of the preclinical mouse model with Pten conditionally knocked out in the prostate tissue, which closely mimics the formation and growth of human PCa. Hereby, we present the prostate-specific temporally and spatially controlled Pten knockout PCa mouse model that can be induced using an adenovirus-based Cre-LoxP system. The Cre recombinase (Cre) is inserted into an adenovirus vector. Unlike Pb-Cre knock-in models (which are spatially but not temporally controlled), the expression of Cre is activated to knock out Pten from the mice's prostate epithelial cells once injected. The viral delivery procedures strictly control the location and time of Pten knockout. This novel approach provides a powerful age-related murine model for PCa, emphasizing the effect of aging on prostate carcinogenesis. Key features • In vivo delivery of Cre recombinase adenovirus (Ad-Cre-Luc) in Pten LoxP/LoxP (L/L) mice. • Generation of Cre-expressing Ad-Cre-Luc-mediated ablation of Pten in anterior prostate epithelial cells of adult Pten L/L mice at different ages. • The Ad-Cre-Luc-mediated ablation of Pten leads to hyperplasia that progresses through prostatic intraepithelial neoplasia (PIN) to adenocarcinoma. • PIN refers to the non-cancerous growth of epithelial cells in the prostate tissue-not cancer but a precursor of prostate cancer [1].

Adenoviral delivery of the CIITA transgene induces T-cell-mediated killing in glioblastoma organoids.

The immunosuppressive nature of the tumor microenvironment poses a significant challenge to effective immunotherapies against glioblastoma (GB). Boosting the immune response is critical for successful therapy. Here, we adopted a cancer gene therapy approach to induce T-cell-mediated killing of the tumor through increased activation of the immune system. Patient-based three-dimensional (3D) GB models were infected with a replication-deficient adenovirus (AdV) armed with the class II major histocompatibility complex (MHC-II) transactivator (CIITA) gene (Ad-CIITA). Successful induction of surface MHC-II was achieved in infected GB cell lines and primary human GB organoids. Infection with an AdV carrying a mutant form of CIITA with a single amino acid substitution resulted in cytoplasmic accumulation of CIITA without subsequent MHC-II expression. Co-culture of infected tumor cells with either peripheral blood mononuclear cells (PBMCs) or isolated T-cells led to dramatic breakdown of GB organoids. Intriguingly, both wild-type and mutant Ad-CIITA, but not unarmed AdV, triggered immune-mediated tumor cell death in the co-culture system, suggesting an at least partially MHC-II-independent process. We further show that the observed cancer cell killing requires the presence of either CD8+ or CD4+ T-cells and direct contact between GB and immune cells. We did not, however, detect evidence of activation of canonical T-cell-mediated cell death pathways. Although the precise mechanism remains to be determined, these findings highlight the potential of AdV-mediated CIITA delivery to enhance T-cell-mediated immunity against GB.

Dissecting immunological mechanisms underlying influenza viral nucleoprotein-induced mucosal immunity against diverse viral strains

ABSTRACT The nucleoprotein (NP) of type A influenza virus (IAV) is highly conserved across all virus strains, making it an attractive candidate antigen for universal vaccines. While various studies have explored NP-induced mucosal immunity, here we interrogated the mechanistic differences between intramuscular (IM) and intranasal (IN) delivery of a recombinant adenovirus carrying NP fused with a bifunctional CD40 ligand. Despite being less effective than IM delivery in inducing systemic cellular immune responses and antibody-dependent cellular cytotoxicity (ADCC), IN immunization elicited superior antigen-specific recall humoral and cellular response in the nasal associated lymphoid tissue (NALT) of the upper respiratory tract, the initial site of immune recognition and elimination of inhaled pathogens. IN vaccination also induced significantly stronger pulmonary T cell responses in the lower respiratory tract than IM vaccination, in particular the CD8 T cells. Moreover, blocking lymphocyte circulation abrogated IM but not IN immunization induced protection, illustrating the critical role of local memory immune response upon viral infection. Notably, the CD40-targeted nasal delivery not only improved the magnitude but also the breadth of protection, including against lethal challenge with a newly isolated highly pathogenic avian H5N1 strain. These findings are informative for the design of universal mucosal vaccines, where the predominant mode of protection is independent of neutralizing antibodies.

Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles

Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.

Enhanced Humoral and Cellular Immune Responses Elicited by Adenoviral Delivery of SARS-CoV-2 Receptor-Binding Motif Fused to Human Fc.

Background/Objectives: The receptor binding motif (RBM) of the SARS-CoV-2 spike protein is critical for viral entry into host cells. Development of a vaccine targeting this region is a promising strategy for COVID-19 prevention. To enhance the immunogenicity of SARS-CoV-2 vaccines, we developed an adenoviral vector expressing the RBM from the SARS-CoV-2 spike protein that fused to the human Fc (hFc) domain. Methods: The recombinant RBM_hFc fusion protein was successfully cloned into the pacAd5CMV-N-pA (pAd5) vector and expressed in HEK293 cells as a ~40 kDa protein. A recombinant adenovirus encoding RBM_hFc was subsequently generated and confirmed by cytopathic effect assay. Results: Western blot analysis verified the expression of RBM_hFc in the adenovirus (AdV). ELISA assays, validated for IgG detection, demonstrated a twofold increase in IgG antibody levels (M-1.090 at 450 nm; SD-±0.326; and 95% CI-0.250 [0.839 to 1.340]) in sera from BALB/c mice immunized with Ad/RBM_hFc, compared to the negative control group. Result suggests a robust humoral immune response induced by the Ad/RBM_hFc vaccine. Moreover, ELISpot assays demonstrated a tenfold increase in IFN-γ -producing cells (M-440 spot-forming cells; SD-±124.976; and 95% CI-75.522 [364.478 to 515.522]) in mice immunized with AdV/RBM_hFc compared to the negative control group. Result proved that AdV/RBM_hFc-stimulated a robust cellular immune response in animal model. Conclusions: Our findings indicate that the RBM_hFc fusion protein enhances both humoral and cellular immune responses. These results suggest the potential of adenoviral vectors carrying RBM_hFc as vaccine candidates. However, comprehensive evaluation of the protective efficacy of these adenoviral vectors will necessitate rigorous experimental studies.

Smart Accumulating Dual-Targeting Lipid Envelopes Equipping Oncolytic Adenovirus for Enhancing Cancer Gene Therapeutic Efficacy.

Systemic delivery of oncolytic adenovirus (oAd) for cancer gene therapy must overcome several limitations such as rapid clearance from the blood, nonspecific accumulation in the liver, and insufficient delivery to the tumor tissues. In the present report, a tumor microenvironment-triggered artificial lipid envelope composed of a pH-responsive sulfamethazine-based polymer (PUSSM)-conjugated phospholipid (DOPE-HZ-PUSSM) and another lipid decorated with epidermal growth factor receptor (EGFR) targeting peptide (GE11) (GE11-DOPE) was utilized to encapsulate replication-incompetent Ad (dAd) or oAd coexpressing short-hairpin RNA (shRNA) against Wnt5 (shWnt5) and decorin (dAd/LP-GE-PS or oAd/LP-GE-PS, respectively). In vitro studies demonstrated that dAd/LP-GE-PS transduced breast cancer cells in a pH-responsive and EGFR-specific manner, showing a higher level of transduction than naked Ad under a mildly acidic pH of 6.0 in EGFR-positive cell lines. In vivo biodistribution analyses revealed that systemic administration of oAd/LP-GE-PS leads to a significantly higher level of intratumoral virion accumulation compared to naked oAd, oAd encapsulated in a liposome without PUSSM or EGFR targeting peptide moiety (oAd/LP), or oAd encapsulated in a liposome with EGFR targeting peptide alone (oAd/LP-GE) in an EGFR overexpressing MDA-MB-468 breast tumor xenograft model, showing that both pH sensitivity and EGFR targeting ability were integral to effective systemic delivery of oAd. Further, systemic administration of all liposomal oAd formulations (oAd/LP, oAd/LP-GE, and oAd/LP-GE-PS) showed significantly attenuated hepatic accumulation of the virus compared to naked oAd. Collectively, our findings demonstrated that pH-sensitive and EGFR-targeted liposomal systemic delivery of oAd can be a promising strategy to address the conventional limitations of oAd to effectively treat EGFR-positive cancer in a safe manner.

Oncolytic adenovirus encoding decorin and CD40 ligand inhibits tumor growth and liver metastasis via immune activation in murine colorectal tumor model

Colorectal cancer (CRC) is the second common cause of cancer mortality worldwide, and it still lacks effective approaches for relapsed and metastatic CRC. Recently, oncolytic virus has been emerged as a promising immune therapeutic strategy. In this study, we develop a novel oncolytic adenovirus, rAd.mDCN.mCD40L, which drive oncolytic activity by telomerase reverse transcriptase promoter (TERTp). rAd.mDCN.mCD40L expressed both mouse genes of decorin (mDCN) and CD40 ligand (mCD40L), and produced effective cytotoxicity in both human and mouse CRC cells. Moreover, oncolytic adenovirus mediated mDCN over-expression inhibited Met expression in vitro. In CT26 subcutaneous tumor model, intratumorally delivery of oncolytic adenoviruses could inhibit tumor growth and liver metastasis, while mDCN and/or mCD40L armed oncolytic adenoviruses produced much more impressive responses. No obvious toxicity was detected in lung, liver and spleen. Moreover, mDCN and/or mCD40L armed oncolytic adenoviruses altered the immune state to activate anti-tumor responses, including increasing CD8+ T effector cells and CD4+ memory T cells, reducing MDSCs and Tregs in peripheral blood. Furthermore, mDCN and/or mCD40L armed oncolytic adenoviruses mediated mDCN and/or mCD40L expression in tumors, and up-regulated Th1 cytokines and reduced Th2 cytokines in tumors, which will be benefit for remodeling tumor microenvironment. Importantly, rAd.mDCN.mCD40L and rAd.mCD40L prevented tumor liver metastasis much more effectively than rAd.Null and rAd.mDCN. Therefore, rAd.mDCN.mCD40L and rAd.mCD40L are promising approaches for CRC therapy.

Abstract We121: Doxorubicin-Induced Cardiomyocyte Death is mediated by Lysosomal Membrane Permeabilization

Doxorubicin (DOX) is a potent antineoplastic drug with broad-spectrum capabilities. However, its clinical use is hindered by dose-dependent cardiotoxicity, often leading to heart failure. This study investigates the connection between DOX-induced cardiotoxicity and lysosomal dysfunction, focusing on lysosomal membrane permeabilization (LMP) as a central mechanism in cardiomyocyte death. LMP occurs when there is a disruption in the integrity of the lysosomal membrane, releasing lysosomal enzymes into the cytoplasm. We tested the hypothesis that DOX induces LMP, contributing to cardiomyocyte death. Utilizing a tfGalectin3 LMP reporter and Lysosensor staining, we verified LMP occurrence in DOX-treated H9C2 cardiomyoblast cells, as indicated by compromised lysosomal acidity and increased release of lysosomal protease Cathepsin D (CTSD). Western blot analysis showed elevated CTSD expression following DOX exposure, while DQRed BSA staining demonstrated diminished lysosomal proteolysis, signifying lysosomal dysfunction. Intriguingly, downregulating CTSD via siRNA aggravates cardiomyocyte death, whereas enhancing CTSD expression through adenoviral delivery exerts opposite effects, suggesting a protective role of CTSD in DOX cardiotoxicity. In vivo study using a tfGalectin3 LMP reporter mouse validated the ability of DOX to induce LMP in cardiac tissue. CTSD heterozygous knockout mice exhibited deteriorated heart function compared to the wild type, underscoring CTSD's protective role against DOX-induced cardiotoxicity. In summary, our findings highlight the critical impact of DOX on lysosomal stability and protease activity, leading to cardiomyocyte death and cardiac dysfunction. Further studies are warranted to explore potential interventions, such as imidazolines and apolipoproteins, to reinforce LMP stability and safeguard against DOX-induced cardiotoxicity.

Mesenchymal stromal cells protect combined oncolytic and helper-dependent adenoviruses from humoral immunity

Systemic delivery of oncolytic and immunomodulatory adenoviruses may be required for optimal effects on human malignancies. Mesenchymal stromal cells (MSCs) can serve as delivery systems for cancer therapeutics due to their ability to transport and shield these agents whilst homing to tumors. We now use MSCs to deliver a clinically validated binary oncolytic and helper dependent adenovirus combination (CAdVEC) to tumor cells. We show successful oncolysis and helper-dependent virus function in tumor cells even in the presence of plasma from adenovirus seropositive donors. In both two- and three-dimensional cultures, CAdVEC function is eliminated even at high dilutions of seropositive plasma but is well sustained when CAdVEC is delivered by MSCs. These results provide a robust in vitro model to measure oncolytic and helper dependent virus spread and demonstrate a beneficial role of using MSCs for systemic delivery of CAdVEC even in the presence of a neutralizing humoral response.

CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both invivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an invivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Glucocorticoid Resistance Increases PPARγ Activity and Drives Hepatic Lipid Accumulation via Increased Eicosanoid and Monoacylglyceride Synthesis

A diet high in fat induces adiposity and obesity, and chronic feeding can lead to multiple metabolic complications, including type II diabetes, non-alcoholic fatty liver disease, and cardiovascular disease. The glucocorticoid receptor (GR) regulates genes that control metabolism. The GR has two isoforms (GRα and GRβ) that arise from the GR gene ( NR3C1) due to the alternative splicing of exon 9. The GRα isoform is the classical receptor that binds to glucocorticoids. However, the GRβ isoform has a truncated C-terminus. It cannot bind glucocorticoid steroids, and as a result, this isoform induces a glucocorticoid-resistant state when its expression is increased. We hypothesized that increasing levels of GRβ from long-term excess glucocorticoid exposure or a high-fat diet induces adiposity by affecting lipid metabolism and lipogenesis, causing fat to accumulate in the adipose and liver tissues. We first overexpressed GRβ (GRβ-Ad) or control (Vec-Ad) using adenovirus delivery and fed the mice a normal chow diet. The GRβ-Ad mice had significantly higher lipid accumulation within five days. Western blotting analysis revealed a significant increase (p-value <0.05) in the GRβ-Ad livers for fatty acid synthase (FAS) expression, and there was a remodeling of the hepatic lipid content as measured by mass spectrometry lipidomic analysis. The data indicate an increased level of monoacylglyceride lipid species and some species of long-chain triacylglycerides and diacylglycerides. mRNA analysis revealed a significant increase (p-value <0.05) in Carbohydrate-responsive element-binding protein (ChREBP), PPARγ, and Cyp2j6, indicating an increase in de novo lipogenesis and eicosanoid synthesis. We conclude that glucocorticoid resistance due to increased levels of GRβ drives de novo lipogenesis and increases eicosanoid synthesis, possibly via synergism with PPARγ. A better understanding of the GR isoforms and glucocorticoid resistance could benefit people with obesity and other metabolic disorders to improve obesity-associated comorbidities. R01DK121797 (T.D.H.J.), R01DA058933 (T.D.H.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10μM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8μM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

Real-Time MRI Stereotaxis Aided Smartflow Convection Delivery of DNX-2401

INTRODUCTION: The Combination Adenovirus + Pembrolizumab to Trigger Immune Virus Effects (CAPTIVE) study is a phase II clinical trial testing the efficacy of a recombinant adenovirus DNX-2401 combined with the immune checkpoint inhibitor pembrolizumab. METHODS: Prospective case series. RESULTS: Precise catheter placement was achieved in all patients treated, and no adverse events were noted. Average radial error from target was 0.9 mm. The average procedural time was 3 hours and 16 minutes, and comparable to other CED techniques. In two patients, delivery of DNX-2401 was visualized as T1-hypointensity using an MRI taken within 30 minutes of procedure completion. These patients exhibited partial response based on RANO assessment. Infusion associated T1-hypointensity in tumor core was not seen in the other patients, who showed no evidence of therapeutic response. CONCLUSIONS: Our pilot study supports the safety of MRI guided SmartFlow convection delivery of the DNX-2401 adenovirus in recurrent glioblastoma patients. Visualization of drug delivery may serve as an imaging biomarker for therapeutic response.

Tumor-targeted delivery of copper-manganese biomineralized oncolytic adenovirus for colorectal cancer immunotherapy

Oncolytic viral therapy (OVT) is a novel anti-tumor immunotherapy approach, specifically replicating within tumor cells. Currently, oncolytic viruses are mainly administered by intratumoral injection. However, achieving good results for distant metastatic tumors is challenging. In this study, a multifunctional oncolytic adenovirus, OA@CuMnCs, was developed using bimetallic ions copper and manganese. These metal cations form a biomineralized coating on the virus's surface, reducing immune clearance. It is known that viruses upregulate the expression of PD-L1. Copper ions in OA@CuMnCs can decrease the PD-L1 expression of tumor cells, thereby promoting immune cell-related factor release. This process involves antigen presentation and the combination of immature dendritic cells, transforming them into mature dendritic cells. It changes "cold" tumors into "hot" tumors, further inducing immunogenic cell death. While oncolytic virus replication requires oxygen, manganese ions in OA@CuMnCs can react with endogenous hydrogen peroxide. This reaction produces oxygen, enhancing the virus's replication ability and the tumor lysis effect. Thus, this multifunctionally coated OA@CuMnCs demonstrates potent amplification in immunotherapy efficacy, and shows great potential for further clinical OVT. Statement of significanceOncolytic virus therapy (OVs) is a new anti-tumor immunotherapy method that can specifically replicate in tumor cells. Although the oncolytic virus can achieve a therapeutic effect on some non-metastatic tumors through direct intratumoral injection, there are still three major defects in the treatment of metastatic tumors: immune response, hypoxia effect, and administration route. Various studies have shown that the immune response in vivo can be overcome by modifying or wrapping the surface protein of the oncolytic virus. In this paper, a multifunctional coating of copper and manganese was prepared by combining the advantages of copper and manganese ions. The coating has a simple preparation method and mild conditions, and can effectively enhance tumor immunotherapy.

Exploring the DNA2-PNA heterotriplex formation in targeting the Bcl-2 gene promoter: A structural insight by physico-chemical and microsecond-scale MD investigation

Peptide Nucleic Acids (PNAs) represent a promising tool for gene modulation in anticancer treatment. The uncharged peptidyl backbone and the resistance to chemical and enzymatic degradation make PNAs highly advantageous to form stable hybrid complexes with complementary DNA and RNA strands, providing higher stability than the corresponding natural analogues. Our and other groups’ research has successfully shown that tailored PNA sequences can effectively downregulate the expression of human oncogenes using antigene, antisense, or anti-miRNA approaches. Specifically, we identified a seven bases-long PNA sequence, complementary to the longer loop of the main G-quadruplex structure formed by the bcl2midG4 promoter sequence, capable of downregulating the expression of the antiapoptotic Bcl-2 protein and enhancing the anticancer activity of an oncolytic adenovirus. Here, we extended the length of the PNA probe with the aim of including the double-stranded Bcl-2 promoter among the targets of the PNA probe. Our investigation primarily focused on the structural aspects of the resulting DNA2-PNA heterotriplex that were determined by employing conventional and accelerated microsecond-scale molecular dynamics simulations and chemical-physical analysis. Additionally, we conducted preliminary biological experiments using cytotoxicity assays on human A549 and MDA-MB-436 adenocarcinoma cell lines, employing the oncolytic adenovirus delivery strategy.

Zinc finger protein ZNF638 regulates triglyceride metabolism via ANGPTL8 in an estrogen dependent manner

Background and aimTriglyceride (TG) levels are closely related to obesity, fatty liver and cardiovascular diseases, while the regulatory factors and mechanism for triglyceride homeostasis are still largely unknown. Zinc Finger Protein 638 (ZNF638) is a newly discovered member of zinc finger protein family for adipocyte function in vitro. The aim of the present work was to investigate the role of ZNF638 in regulating triglyceride metabolism in mice. MethodsWe generated ZNF638 adipose tissue specific knockout mice (ZNF638 FKO) by cross-breeding ZNF638 flox to Adiponectin-Cre mice and achieved adipose tissue ZNF638 overexpression via adenoviral mediated ZNF638 delivery in inguinal adipose tissue (iWAT) to examined the role and mechanisms of ZNF638 in fat biology and whole-body TG homeostasis. ResultsAlthough ZNF638 FKO mice showed similar body weights, body composition, glucose metabolism and serum parameters compared to wild-type mice under chow diet, serum TG levels in ZNF638 FKO mice were increased dramatically after refeeding compared to wild-type mice, accompanied with decreased endothelial lipoprotein lipase (LPL) activity and increased lipid absorption of the small intestine. Conversely, ZNF638 overexpression in iWAT reduced serum TG levels while enhanced LPL activity after refeeding in female C57BL/6J mice and obese ob/ob mice. Specifically, only female mice exhibited altered TG metabolism upon ZNF638 expression changes in fat. Mechanistically, RNA-sequencing analysis revealed that the TG regulator angiopoietin-like protein 8 (Angptl8) was highly expressed in iWAT of female ZNF638 FKO mice. Neutralizing circulating ANGPTL8 in female ZNF638 FKO mice abolished refeeding-induced TG elevation. Furthermore, we demonstrated that ZNF638 functions as a transcriptional repressor by recruiting HDAC1 for histone deacetylation and broad lipid metabolic gene suppression, including Angptl8 transcription inhibition. Moreover, we showed that the sexual dimorphism is possibly due to estrogen dependent regulation on ZNF638-ANGPTL8 axis. ConclusionWe revealed a role of ZNF638 in the regulation of triglyceride metabolism by affecting Angptl8 transcriptional level in adipose tissue with sexual dimorphism.

Intratumoral delivery of a Tim-3 antibody-encoding oncolytic adenovirus engages an effective antitumor immune response in liver cancer

Intratumoral delivery of a Tim-3 antibody-encoding oncolytic adenovirus engages an effective antitumor immune response in liver cancer

H3K36 methyltransferase NSD1 protects against osteoarthritis through regulating chondrocyte differentiation and cartilage homeostasis.

Osteoarthritis (OA) is one of the most common joint diseases, there are no effective disease-modifying drugs, and the pathological mechanisms of OA need further investigation. Here, we show that H3K36 methylations were decreased in senescent chondrocytes and age-related osteoarthritic cartilage. Prrx1-Cre inducible H3.3K36M transgenic mice showed articular cartilage destruction and osteophyte formation. Conditional knockout Nsd1Prrx1-Cre mice, but not Nsd2Prrx1-Cre or Setd2Prrx1-Cre mice, replicated the phenotype of K36M/+; Prrx1-Cre mice. Immunostaining results showed decreased anabolic and increased catabolic activities in Nsd1Prrx1-Cre mice, along with decreased chondrogenic differentiation. Transcriptome and ChIP-seq data revealed that Osr2 was a key factor affected by Nsd1. Intra-articular delivery of Osr2 adenovirus effectively improved the homeostasis of articular cartilage in Nsd1Prrx1-Cre mice. In human osteoarthritic cartilages, both mRNA and protein levels of NSD1 and OSR2 were decreased. Our results indicate that NSD1-induced H3K36 methylations and OSR2 expression play important roles in articular cartilage homeostasis and OA. Targeting H3K36 methylation and OSR2 would be a novel strategy for OA treatment.

Engineering a Novel Modular Adenoviral mRNA Delivery Platform Based on Tag/Catcher Bioconjugation.

mRNA vaccines have attracted widespread research attention with clear advantages in terms of molecular flexibility, rapid development, and potential for personalization. However, current mRNA vaccine platforms have not been optimized for induction of CD4/CD8 T cell responses. In addition, the mucosal administration of mRNA based on lipid nanoparticle technology faces challenges in clinical translation. In contrast, adenovirus-based vaccines induce strong T cell responses and have been approved for intranasal delivery. To leverage the inherent strengths of both the mRNA and adenovirus platforms, we developed a novel modular adenoviral mRNA delivery platform based on Tag/Catcher bioconjugation. Specifically, we engineered adenoviral vectors integrating Tag/Catcher proteins at specific locales on the Ad capsid proteins, allowing us to anchor mRNA to the surface of engineered Ad viruses. In proof-of-concept studies, the Ad-mRNA platform successfully mediated mRNA delivery and could be optimized via the highly flexible modular design of both the Ad-mRNA and protein bioconjugation systems.

Magnetic Resonance Imaging–Aided SmartFlow Convection Delivery of DNX-2401: A Pilot, Prospective Case Series

The Combination Adenovirus+ Pembrolizumab to Trigger Immune Virus Effects (CAPTIVE) study is a phase II clinical trial testing the efficacy of a recombinant adenovirus DNX-2401 combined with the immune checkpoint inhibitor pembrolizumab. Here, we report the first patients in this study who underwent viral delivery through real-time magnetic resonance imaging (MRI) stereotaxis-guided SmartFlow convection delivery of DNX-2401. Patients who underwent real-time MRI-guided DNX-2401 delivery through the SmartFlow convection catheter were prospectively followed. Precise catheter placement was achieved in all patients treated, and no adverse events were noted. Average radial error from target was 0.9 mm. Average procedural time was 3 hours 16 minutes and was comparable to other convection-enhanced delivery techniques. In 2 patients, delivery of DNX-2401 was visualized as >1 cm maximal diameter of T1 hypointensity infusate on MRI obtained immediately after completion of viral infusion. These patients exhibited partial response based on Response Assessment in Neuro-Oncology assessment. The remaining patient showed <1 cm maximal diameter of infusate on immediate postinfusion MRI and showed disease progression on subsequent MRI. Our pilot case series supports compatibility of the SmartFlow system with oncolytic adenovirus delivery and provides the basis for future validation studies.