Adenovirus-mediated Gene Transfer Research Articles

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma.

In the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation. In this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients. GM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit. Vaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

Induction of Immunological Antitumor Effects by the Combination of Adenovirus-Mediated Gene Transfer of B7-1 and Anti-Programmed Cell Death-1 Antibody in a Murine Squamous Cell Carcinoma Model.

The goal of this study was to evaluate the antitumor immune effects of B7-1 gene expression in addition to immune checkpoint inhibitor against squamous cell carcinoma. A murine SCC cell line, KLN205, was infected with adenoviral vector carrying B7-1 (AdB7). Infected cells were injected subcutaneously in the flanks of DBA/2 mice. Three weeks after implantation, anti-mouse PD-1 antibody (antiPD1) was intraperitonially administrated twice a week for a total of six times. CD80 was significantly overexpressed in the AdB7-infected tumors. IFN-gamma in the T cells in the spleen was significantly increased and tumor size was significantly reduced in the mice treated with both AdB7 and antiPD1. Targeted tumors treated with both AdB7 and antiPD1 exhibited significantly increased cell densities of total immune cells as well as Ki-67+ CD8+ T cells and decreased regulatory T cells. These results suggest that the B7-1 gene transfer may enhance the antitumor effect of anti-PD1 antibody against SCC.

Deficiency of skeletal muscle Agrin contributes to the pathogenesis of age-related sarcopenia in mice

Sarcopenia, a progressive and prevalent neuromuscular disorder, is characterized by age-related muscle wasting and weakening. Despite its widespread occurrence, the molecular underpinnings of this disease remain poorly understood. Herein, we report that levels of Agrin, an extracellular matrix (ECM) protein critical for neuromuscular formation, were decreased with age in the skeletal muscles of mice. The conditional loss of Agrin in myogenic progenitors and satellite cells (SCs) (Pax7 Cre:: Agrin flox/flox) causes premature muscle aging, manifesting a distinct sarcopenic phenotype in mice. Conversely, the elevation of a miniaturized form of Agrin in skeletal muscle through adenovirus-mediated gene transfer induces enhanced muscle capacity in aged mice. Mechanistic investigations suggest that Agrin-mediated improvement in muscle function occurs through the stimulation of Yap signaling and the concurrent upregulation of dystroglycan expression. Collectively, our findings underscore the pivotal role of Agrin in the aging process of skeletal muscles and propose Agrin as a potential therapeutic target for addressing sarcopenia.

Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase.

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Cysteine and glycine-rich protein 2 retards platelet-derived growth factor-BB-evoked phenotypic transition of airway smooth muscle cells by decreasing YAP/TAZ activity.

Cysteine and glycine-rich protein 2 (Csrp2) has emerged as a key factor in controlling the phenotypic modulation of smooth muscle cells. The phenotypic transition of airway smooth muscle cells (ASMCs) is a pivotal step in developing airway remodeling during the onset of asthma. However, whether Csrp2 mediates the phenotypic transition of ASMCs in airway remodeling during asthma onset is undetermined. This work aimed to address the link between Csrp2 and the phenotypic transition of ASMCs evoked by platelet-derived growth factor (PDGF)-BB in vitro. The overexpression or silencing of Csrp2 in ASMCs was achieved through adenovirus-mediated gene transfer. The expression of mRNA was measured by quantitative real-time-PCR. Protein levels were determined through Western blot analysis. Cell proliferation was detected by EdU assay and Calcein AM assays. Cell cycle distribution was assessed via fluorescence-activated cell sorting assay. Cell migration was evaluated using the scratch-wound assay. The transcriptional activity of Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) was measured using the luciferase reporter assay. A decline in Csrp2 level occurred in PDGF-BB-stimulated ASMCs. Increasing Csrp2 expression repressed the PDGF-BB-evoked proliferation and migration of ASMCs. Moreover, increasing Csrp2 expression impeded the phenotypic change of PDGF-BB-stimulated ASMCs from a contractile phenotype into a synthetic/proliferative phenotype. On the contrary, the opposite effects were observed in Csrp2-silenced ASMCs. The activity of YAP/TAZ was elevated in PDGF-BB-stimulated ASMCs, which was weakened by Csrp2 overexpression or enhanced by Csrp2 silencing. The YAP/TAZ activator could reverse Csrp2-overexpression-mediated suppression of the PDGF-BB-evoked phenotypic switching of ASMCs, while the YAP/TAZ suppressor could dimmish Csrp2-silencing-mediated enhancement on PDGF-BB-evoked phenotypic switching of ASMCs. In summary, Csrp2 serves as a determinant for the phenotypic switching of ASMCs. Increasing Csrp2 is able to impede PDGF-BB-evoked phenotypic change of ASMCs from a synthetic phenotype into a synthetic/proliferative phenotype through the effects on YAP/TAZ. This work implies that Csrp2 may be a key player in airway remodeling during the onset of asthma.

Transcriptional cofactor dyxin mediates hypertrophic response in the heart during angiotensin II-induced hypertension.

Dyxin is a LIM-domain containing transcriptional regulator protein shown to play a role in a hypertrophic response in the heart. Here, the effect of adenoviral dyxin overexpression was studied on cardiac function and gene expression in the normal heart and in angiotensin II (Ang II)-induced hypertension in rats. The adenovirus-mediated intramyocardial gene transfer of dyxin (1.5x109 infectious units/animal) was performed into the left ventricle (LV) of Sprague-Dawley rats with and without the Ang II (33 μg/kg/h) infusion, administered via osmotic minipumps for 1 and 2 weeks. Echocardiography was used to assess the structural and functional changes. Dyxin expression and localization in the heart was analyzed with quantitative RT-PCR and immunohistochemistry, respectively. In the normal rat heart, the adenoviral overexpression of dyxin did not alter LV function in normal hearts as assessed by echocardiography. Dyxin was found to be localized in the cardiomyocytes as shown by the immunohistochemical staining. In Ang II-induced hypertrophy, echocardiographic data revealed a significant increase in the posterior wall diameter both in systole (21%, P<0.05) and diastole (21%, P<0.01) as well as in the diameter of the interventricular septum in systole (19%, P<0.05) in the dyxin-injected group compared with the LacZ-injected animals after two weeks of Ang II infusion. Interestingly, a significant decrease in the levels of both atrial natriuretic peptide (ANP) mRNA (55%, P<0.01) and B-type natriuretic peptide (BNP) mRNA (68%, P<0.05) was observed in the dyxin-injected group compared with the LacZ control group after one week of Ang II infusion. These results indicate that dyxin overexpression was deteriorative against pressure overload by inducing structural changes in the LV in rats. Interestingly, simultaneous adenoviral overexpression of dyxin suppressed the Ang II-induced changes of ANP and BNP genes suggesting that dyxin might have a role as a regulator of the cardiac hypertrophic gene program.

Abstract P1155: P62 Overexpression Rescues Defective Autophagy Flux In Cardiomyocytes Deficient In P21 Activated Kinase 1

P21-Activated Kinase 1 (PAK1) is a serine-threonine kinase that plays a critical role in cardiomyocyte survival under numerous stressful conditions. However, the underlying mechanisms for PAK1’s cardioprotective effects remain unclear. Autophagy is a lysosome-dependent degradation pathway for eliminating and recycling long-lived proteins and unwanted cellular components, which is essential for cellular homeostasis. In this study, we investigated whether and how PAK1 regulates autophagy in cardiomyocytes. PAK1 was either silenced by siRNA knockdown or overexpressed by adenovirus-mediated gene transfer in H9c2 cardiac myoblast cells. Autophagy flux was determined by measuring LC3-II protein levels or by a fluorescent autophagy reporter with and without the lysosomal protease inhibitors pepstatin A (pepA) and Aloxistatin (E64d). PAK1 downregulation reduced LC3-II levels in the total cell lysates, which were not affected by pepA and E64d, suggesting that knockdown of PAK1 inhibited autophagy flux. Inversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 was sufficient to accelerate autophagy flux. Interestingly, the expression of p62, a ubiquitin-binding autophagic adaptor, was concurrently upregulated by PAK1 overexpression or downregulated by PAK1 knockdown, suggesting that p62 may mediates PAK1-dependent autophagy. Indeed, overexpression of p62 restored autophagy flux in the absence of PAK1 (Figure 1). Together, these results suggest that PAK1 is both sufficient and necessary for maintaining normal autophagy function in cardiomyocytes, which is likely mediated by p62.

Expression and Function of MAO A in Cardiac Cells by Means of Adenovirus-Mediated Gene Transfer.

Gene-transfer methods are useful to study the structural or functional roles of recombinant proteins in vitro. In particular, adenovirus-mediated gene transduction results in strong efficiency and high level of expression in primary cells such as cardiomyocytes, which are difficult to transfect with classical methods. Here, we describe a protocol that enables efficient expression of MAO A in both primary cells and cell lines. Following expression of recombinant MAO A, substrate-induced activation of the enzyme can be assessed by measuring production of reactive oxygen species and downstream signal transduction pathways in cells. This model allows to decipher the biological function of MAO A on metabolism, mitochondrial fitness, cell death/survival, and proliferation.

337.11: Adenovirus-Mediated Adiponectin Gene Transfer in a Rat Model of Orthotopic Left Lung Transplantation

337.11: Adenovirus-Mediated Adiponectin Gene Transfer in a Rat Model of Orthotopic Left Lung Transplantation

Using adenovirus-mediated gene transfer to study the effect of myeloperoxidase on plasma lipid levels, HDL structure and functionality in mice expressing human apoA-I forms

Apolipoprotein A-I (apoA-I), the main protein component of High-Density Lipoprotein (HDL), is modified in plasma and the arterial wall by various enzymes. Myeloperoxidase (MPO), a leukocyte-derived peroxidase, is highly expressed during inflammation and associates with HDL reducing its functionality and contributing to atherosclerosis. In the present study we sought to explore further the effect of MPO on HDL structure and functionality in vivo using adenovirus-mediated gene transfer of human MPO combined with human apoA-I forms containing substitutions at MPO-sensitive sites or wild type apoA-I. We found that overexpression of MPO in mice significantly increased plasma apoA-I and HDL levels without affecting the expression of genes involved in HDL biogenesis or catabolism in the liver. Overexpression of MPO in the liver reduced the expression of pro-inflammatory genes and increased or did not affect the expression of anti-inflammatory genes suggesting that MPO had no toxic effects in this organ. In the plasma of mice overexpressing MPO, no significant alterations in HDL size or electrophoretic mobility was observed with the exception of mice expressing apoA-I (M148A) which showed enriched pre-β relative to α HDL particles, suggesting that the apoA-I (M148A) mutation may interfere with HDL remodelling. Overexpression of MPO was associated with reduced anti-oxidant capacity of HDL particles in all mice. Interestingly, HDL particles bearing apoA-I (Y192A) showed enhanced ABCA1-dependent cholesterol efflux from macrophages which was not affected by MPO and these mice had reduced levels of LDL-c. These findings provide new insights on the role of specific amino acid residues of apoA-I in HDL structure and function following modification by MPO. This knowledge may facilitate the development of novel therapies based on improved HDL forms for patients with chronic diseases that are characterized by dysfunctional HDL.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Enforced dual-specificity tyrosine-regulated kinase 2 expression by adenovirus-mediated gene transfer inhibits tumor growth and metastasis of colorectal cancer.

Colorectal cancer is one of the most common gastrointestinal tumors with good outcomes; however, with distant metastasis, the outcomes are poor. Novel treatment methods are urgently needed. Our in vitro studies indicate that dual‐specificity tyrosine‐regulated kinase 2 (DYRK2) functions as a tumor suppressor in colorectal cancer by regulating cell survival, proliferation, and apoptosis induction. In addition, DYRK2 expression is decreased in tumor tissues compared to nontumor tissues in colorectal cancer, indicating a correlation with clinical prognosis. In this context, we devised a novel therapeutic strategy to overexpress DYRK2 in tumors by adenovirus‐mediated gene transfer. The present study shows that overexpression of DYRK2 in colon cancer cell lines by adenovirus inhibits cell proliferation and induces apoptosis in vitro. Furthermore, in mouse subcutaneous xenograft and liver metastasis models, enforced expression of DYRK2 by direct or intravenous injection of adenovirus to the tumor significantly inhibits tumor growth. Taken together, these findings show that adenovirus‐based overexpression of DYRK2 could be a novel gene therapy for liver metastasis of colorectal cancer.

Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells

Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.

Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

Zebra finch is a representative animal model for studying the molecular basis of human disorders of vocal development and communication. Accordingly, various functional studies of zebra finch have knocked down or introduced foreign genes in vivo; however, their germline transmission efficiency is remarkably low. The primordial germ cell (PGC)-mediated method is preferred for avian transgenic studies; however, use of this method is restricted in zebra finch due to the lack of an efficient gene transfer method for the germline. To target primary germ cells that are difficult to transfect and manipulate, an adenovirus-mediated gene transfer system with high efficiency in a wide range of cell types may be useful. Here, we isolated and characterized two types of primary germline-competent stem cells, PGCs and spermatogonial stem cells (SSCs), from embryonic and adult reproductive tissues of zebra finch and demonstrated that genes were most efficiently transferred into these cells using an adenovirus-mediated system. This system was successfully used to generate gene-edited PGCs in vitro. These results are expected to improve transgenic zebra finch production.

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by “negative” and “positive” PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

An Efficient Method for Adenovirus Production.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by "negative" and "positive" PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Advanced genetic engineering to achieve in vivo targeting of adenovirus utilizing camelid single domain antibody

For the developing field of gene therapy the successful address of the basic requirement effective gene delivery has remained a critical barrier. In this regard, the “Holy Grail” vector envisioned by the field's pioneers embodied the ability to achieve efficient and specific in vivo gene delivery. Functional linkage of antibody selectivity with viral vector efficiency represented a logical strategy but has been elusive. Here we have addressed this key issue by developing the technical means to pair antibody-based targeting with adenoviral-mediated gene transfer. Our novel method allows efficient and specific gene delivery. Importantly, our studies validated the achievement of this key vectorology mandate in the context of in vivo gene delivery. Vectors capable of effective in vivo delivery embody the potential to dramatically expand the range of successful gene therapy cures.

ONCOLYSIS MEDIATED BY INFB AND ARF GENE TRANSFER ACTIVATES KEY COMPONENTS OF THE IMMUNE SYSTEM

<h3>Background</h3> The importance of the immune system for controlling neoplasms was highlighted with the success of checkpoint blockade therapy. New cancer therapeutics seek to break the immune tolerance inherent in tumor progression. To this end, some virus-mediate gene therapy approaches have been shown to induce oncolysis accompanied by emission of immunogenic cell death (ICD) factors, thus attracting and activating cells of the immune system. <h3>Objective</h3> To use combined adenovirus-mediated gene transfer of p14ARF and human interferon-β (IFNβ) to human melanoma cells to induce oncolysis, ICD and subsequent activation of immune cells. <h3>Methods</h3> Cell death was measured by PI and/or AnnexinV staining. Detection of calreticulin was performed by cytometry, while ATP and IFNβ were detected by ELISA. DC and lymphocytes from healthy donors were cultivated ex vivo. <h3>Results</h3> IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. However, detection of critical markers of ICD) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. In situ gene therapy for s.c. SK-MEL-147 tumors in Nod-Scid mice controlled tumor growth and increased animal survival in response to IFNβ alone or in combination with p14ARF. However, the immunocompromised animal model cannot demonstrate the expected immune activation. Thus, we co-cultured SK-MEL-147 transduced with our combination of adenoviruses with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors. These Mo-DCs showed increased levels of activation markers HLA-DR, CD80, and CD86. Next, we used these activated Mo-DCs to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. T cells primed in this way were co-cultured with fresh non-transduced SK-MEL-147, resulting in tumor cell killing. Currently we are expanding these studies to include three-dimensional cultures of melanoma cell lines and also the use of human melanoma samples in patient derived organoid (PDO) models. We expect to reveal the influence of oncolysis on tumor infiltrating cells, including DCs, macrophages, NK and T cells. <h3>Conclusion</h3> Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

Overexpression of Activating Transcription Factor 3 Alleviates Cardiac Microvascular Ischemia/Reperfusion Injury in Rats.

Activating transcription factor 3 (ATF3) has been confirmed to be responsive to oxidative stress and to negatively regulate the activity of Toll-like receptor 4 (TLR4). However, the effect of ATF3 on cardiac microvascular ischemia/reperfusion (I/R) injury remains unknown. The GEO2R online tool was employed to obtain differentially expressed genes GSE4105 and GSE122020, in two rat I/R injury microarray datasets. We established a rat myocardial I/R model in vivo, and also generated an in vitro hypoxia/reoxygenation (H/R) model of cardiomyoblast H9c2 cells. Overexpression of ATF3 was achieved by adenoviral-mediated gene transfer (Ad-ATF3). Rats were randomly divided into four groups: sham, I/R, I/R + Ad-Lacz (as a control), and I/R + Ad-ATF3. ELISA, CCK-8, DCFH-DA probe, qRT-PCR and Western blotting were used to determine the expression of ATF3, oxidative indices, cellular injury and TLR4/NF-κB pathway-associated proteins. Transmission electron microscopy, immunohistochemistry and immunofluorescence were used to detect the leukocyte infiltration and the alteration of microvascular morphology and function in vivo. Echocardiographic and hemodynamic data were also obtained. Bioinformatics analysis revealed that ATF3 was upregulated in I/R myocardia in two independent rat myocardial I/R models. Cardiac microvascular I/R injury included leukocyte infiltration, microvascular integrity disruption, and microvascular perfusion defect, which eventually resulted in the deterioration of hemodynamic parameters and heart function. Ad-ATF3 significantly restored microvascular function, increased cardiac microvascular perfusion, and improved hemodynamic parameters and heart function. Mechanistically, Ad-ATF3 ameliorated oxidative stress, inhibited TLR4/NF-κB pathway activation and down-regulated the expression of downstream proinflammatory cytokines in I/R myocardium in vivo and in H/R H9c2 cells in vitro. ATF3 overexpression protects against cardiac microvascular I/R injury in part by inhibiting the TLR4/NF-κB pathway and oxidative stress.

Nogo-C Inhibits Peripheral Nerve Regeneration by Regulating Schwann Cell Apoptosis and Dedifferentiation.

While Nogo protein demonstrably inhibits nerve regeneration in the central nervous system (CNS), its effect on Schwann cells in peripheral nerve repair and regeneration following sciatic nerve injury remains unknown. In this research, We assessed the post-injury expression of Nogo-C in an experimental mouse model of sciatic nerve-crush injury. Nogo-C knockout (Nogo-C–/–) mouse was generated to observe the effect of Nogo-C on sciatic nerve regeneration, Schwann cell apoptosis, and myelin disintegration after nerve injury, and the effects of Nogo-C on apoptosis and dedifferentiation of Schwann cells were observed in vitro. We found that the expression of Nogo-C protein at the distal end of the injured sciatic nerve increased in wild type (WT) mice. Compared with the injured WT mice, the proportion of neuronal apoptosis was significantly diminished and the myelin clearance rate was significantly elevated in injured Nogo-C–/– mice; the number of nerve fibers regenerated and the degree of myelination were significantly elevated in Nogo-C–/– mice on Day 14 after injury. In addition, the recovery of motor function was significantly accelerated in the injured Nogo-C–/– mice. The overexpression of Nogo-C in primary Schwann cells using adenovirus-mediated gene transfer promoted Schwann cells apoptosis. Nogo-C significantly reduced the ratio of c-Jun/krox-20 expression, indicating its inhibition of Schwann cell dedifferentiation. Above all, we hold the view that the expression of Nogo-C increases following peripheral nerve injury to promote Schwann cell apoptosis and inhibit Schwann cell dedifferentiation, thereby inhibiting peripheral nerve regeneration.