The Coxsackievirus and Adenovirus Receptor Has a Short Half-Life in Epithelial Cells

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAREx8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAREx8 and determine the feasibility of receptor down regulation, the half-life of total and apical localized CAREx8 was determined and correlated with adenovirus transduction. Total and apical CAREx8 has a relatively short half-life of approximately 2 h. The half-life of apical CAREx8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application

Current Approaches for Glioma Gene Therapy and Virotherapy

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in the adult population and it carries a dismal prognosis. Inefficient drug delivery across the blood brain barrier (BBB), an immunosuppressive tumor microenvironment (TME) and development of drug resistance are key barriers to successful glioma treatment. Since gliomas occur through sequential acquisition of genetic alterations, gene therapy, which enables to modification of the genetic make-up of target cells, appears to be a promising approach to overcome the obstacles encountered by current therapeutic strategies. Gene therapy is a rapidly evolving field with the ultimate goal of achieving specific delivery of therapeutic molecules using either viral or non-viral delivery vehicles. Gene therapy can also be used to enhance immune responses to tumor antigens, reprogram the TME aiming at blocking glioma-mediated immunosuppression and normalize angiogenesis. Nano-particles-mediated gene therapy is currently being developed to overcome the BBB for glioma treatment. Another approach to enhance the anti-glioma efficacy is the implementation of viro-immunotherapy using oncolytic viruses, which are immunogenic. Oncolytic viruses kill tumor cells due to cancer cell-specific viral replication, and can also initiate an anti-tumor immunity. However, concerns still remain related to off target effects, and therapeutic and transduction efficiency. In this review, we describe the rationale and strategies as well as advantages and disadvantages of current gene therapy approaches against gliomas in clinical and preclinical studies. This includes different delivery systems comprising of viral, and non-viral delivery platforms along with suicide/prodrug, oncolytic, cytokine, and tumor suppressor-mediated gene therapy approaches. In addition, advances in glioma treatment through BBB-disruptive gene therapy and anti-EGFRvIII/VEGFR gene therapy are also discussed. Finally, we discuss the results of gene therapy-mediated human clinical trials for gliomas. In summary, we highlight the progress, prospects and remaining challenges of gene therapies aiming at broadening our understanding and highlighting the therapeutic arsenal for GBM.Fil: Banerjee, Kaushik. University of Michigan; Estados UnidosFil: Núñez Aguilera, Felipe Javier. Fundación Instituto Leloir; ArgentinaFil: Haase, Santiago. University of Michigan; Estados UnidosFil: McClellan, Brandon L.. University of Michigan; Estados UnidosFil: Faisal, Syed M.. University of Michigan; Estados UnidosFil: Carney, Stephen V.. University of Michigan; Estados UnidosFil: Yu, Jin. University of Michigan; Estados UnidosFil: Alghamri, Mahmoud S.. University of Michigan; Estados UnidosFil: Asad, Antonela Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Nicola Candia, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Varela, Maria Luisa. University of Michigan; Estados UnidosFil: Candolfi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Lowenstein, Pedro R.. University of Michigan; Estados UnidosFil: Castro, Maria G.. University of Michigan; Estados Unido

Immunotherapy for gliomas: shedding light on progress in preclinical and clinical development

Gliomas are infiltrating brain tumors associated with high morbidity and mortality. Current standard of care includes radiation, chemotherapy and surgical resection. Today, survival rates for malignant glioma patients remain dismal and unchanged for decades. The glioma microenvironment is highly immunosuppressive and consequently this has motivated the development of immunotherapies for counteracting this condition, enabling the immune cells within the tumor microenvironment to react against this tumor.Areas covered: The authors discuss immunotherapeutic strategies for glioma in phase-I/II clinical trials and illuminate their mechanisms of action, limitations and key challenges. They also examine promising approaches under preclinical development.Expert opinion: In the last decade there has been an expansion in immune-mediated anti-cancer therapies. In the glioma field, sophisticated strategies have been successfully implemented in preclinical models. Unfortunately, clinical trials have not yet yielded consistent results for glioma patients. This could be attributed to our limited understanding of the complex immune cell infiltration and its interaction with the tumor cells, the selected time for treatment, the combination with other therapies and the route of administration of the agent. Applying these modalities to treat malignant glioma is challenging, but many new alternatives are emerging to by-pass these hurdles.Fil: Garcia Fabiani, Maria Belen. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ventosa, Maria. University of Michigan; Estados UnidosFil: Comba, Andrea. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Candolfi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Nicola Candia, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Alghamri, Mahmoud S.. University of Michigan; Estados UnidosFil: Kadiyala, Padma. University of Michigan; Estados UnidosFil: Carney, Stephen. University of Michigan; Estados UnidosFil: Faisal, Syed M.. University of Michigan; Estados UnidosFil: Schwendeman, Anna. University of Michigan; Estados UnidosFil: Moon, James J.. University of Michigan; Estados UnidosFil: Scheetz, Lindsay. University of Michigan; Estados UnidosFil: Lahann, Joerg. University of Michigan; Estados UnidosFil: Mauser, Ava. University of Michigan; Estados UnidosFil: Lowenstein, Pedro R.. University of Michigan; Estados UnidosFil: Castro, Maria Gabriela. University of Michigan; Estados Unido

Genetic Alterations in Gliomas Remodel the Tumor Immune Microenvironment and Impact Immune-Mediated Therapies

High grade gliomas are malignant brain tumors that arise in the central nervous system, in patients of all ages. Currently, the standard of care, entailing surgery and chemo radiation, exhibits a survival rate of 14-17 months. Thus, there is an urgent need to develop new therapeutic strategies for these malignant brain tumors. Currently, immunotherapies represent an appealing approach to treat malignant gliomas, as the pre-clinical data has been encouraging. However, the translation of the discoveries from the bench to the bedside has not been as successful as with other types of cancer, and no long-lasting clinical benefits have been observed for glioma patients treated with immune-mediated therapies so far. This review aims to discuss our current knowledge about gliomas, their molecular particularities and the impact on the tumor immune microenvironment. Also, we discuss several murine models used to study these therapies pre-clinically and how the model selection can impact the outcomes of the approaches to be tested. Finally, we present different immunotherapy strategies being employed in clinical trials for glioma and the newest developments intended to harness the immune system against these incurable brain tumors.Fil: Garcia Fabiani, Maria Belen. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Haase, Santiago. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Comba, Andrea. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carney, Stephen. University of Michigan; Estados UnidosFil: McClellan, Brandon. University of Michigan; Estados UnidosFil: Banerjee, Kaushik. University of Michigan; Estados UnidosFil: Alghamri, Mahmoud S.. University of Michigan; Estados UnidosFil: Syed, Faisal. University of Michigan; Estados UnidosFil: Kadiyala, Padma. University of Michigan; Estados UnidosFil: Nuñez, Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Candolfi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Asad, Antonela Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: González, Nazareno. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Aikins, Marisa E.. University of Michigan; Estados UnidosFil: Schwendeman, Anna. University of Michigan; Estados UnidosFil: Moon, James J.. University of Michigan; Estados UnidosFil: Lowenstein, Pedro R.. University of Michigan; Estados UnidosFil: Castro, Maria G.. University of Michigan; Estados Unido

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.Fil: Alghamri, Mahmoud S.. University Of Michigan Medical School; Estados UnidosFil: Banerjee, Kaushik. University Of Michigan Medical School; Estados UnidosFil: Mujeeb, Anzar A.. University Of Michigan Medical School; Estados UnidosFil: Mauser, Ava. University of Michigan; Estados UnidosFil: Taher, Ayman. University Of Michigan Medical School; Estados UnidosFil: Thalla, Rohit. University Of Michigan Medical School; Estados UnidosFil: McClellan, Brandon L.. University Of Michigan Medical School; Estados UnidosFil: Varela, Maria L.. University Of Michigan Medical School; Estados UnidosFil: Stamatovic, Svetlana M.. University Of Michigan Medical School; Estados UnidosFil: Martinez Revollar, Gabriela. University Of Michigan Medical School; Estados UnidosFil: Andjelkovic, Anuska V.. University Of Michigan Medical School; Estados UnidosFil: Gregory, Jason V.. University of Michigan; Estados UnidosFil: Kadiyala, Padma. University Of Michigan Medical School; Estados UnidosFil: Calinescu, Alexandra. University Of Michigan Medical School; Estados UnidosFil: Jiménez, Jennifer A.. University of Michigan; Estados UnidosFil: Apfelbaum, April A.. University of Michigan; Estados UnidosFil: Lawlor, Elizabeth R.. University of Washington; Estados UnidosFil: Carney, Stephen. University of Michigan; Estados UnidosFil: Comba, Andrea. University Of Michigan Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Faisal, Syed Mohd. University Of Michigan Medical School; Estados UnidosFil: Barissi, Marcus. University Of Michigan Medical School; Estados UnidosFil: Edwards, Marta B.. University Of Michigan Medical School; Estados UnidosFil: Appelman, Henry. University Of Michigan Medical School; Estados UnidosFil: Sun, Yilun. Case Western Reserve University; Estados UnidosFil: Gan, Jingyao. University of Michigan; Estados UnidosFil: Ackermann, Rose. University of Michigan; Estados UnidosFil: Schwendeman, Anna. University of Michigan; Estados UnidosFil: Candolfi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Olin, Michael R.. University of Minnesota; Estados UnidosFil: Lahann, Joerg. University of Michigan; Estados UnidosFil: Lowenstein, Pedro R.. University of Michigan; Estados UnidosFil: Castro, Maria G.. University of Michigan; Estados Unido

Enhanced Angiotensin II-induced Cardiac and Aortic Remodeling in ACE2 Knockout Mice

Angiotensin-converting enzyme 2 (ACE2) is present in the heart and thought to exert protective functions. We conducted studies in ACE2 deficient mice to determine whether enzyme loss would exacerbate the cardiac and vascular pathological responses to chronic subcutaneous (sc) angiotensin II (Ang II) infusion. Eight-week-old male ACE2 knockout (KO) and wild type (WT) mice were infused with Ang II (1000 ng/kg per min, 4 weeks) using mini-osmotic pumps. Blood pressure (radiotelemetry), cardiac function (echocardiography, echo), cardiac/aortic structure (histology, collagen, and oxidative stress), and vascular inflammation were examined. Before Ang II infusion, ACE2 KO mice showed unaltered cardiac function and blood pressure. After 4 weeks of Ang II infusion, the mean arterial pressure (MAP) increased from 96 ± 2 to 136 ± 17 mm Hg (∼40%) in WT and from 104 ± 5 to 141 ± 13 mm Hg (∼ 35%) in ACE2 KO. While there were no differences in MAP between groups, the ACE2 KO responded differently to the hypertensive stimulus. Echo analysis revealed severe myocardial dysfunction in Ang II-infused ACE2 KO (Ang ACE2 KO). Ejection fraction was lower (39% versus 50%) as was fractional shortening (27% versus 38%) in ACE2 KO versus WT, respectively. Cardiac dysfunction was associated with hypertrophic cardiomyopathy shown by increased left-ventricular wall thickness, average cardiomyocyte cross-sectional area, and heart weight/body weight ratio. Collagen staining in the myocardium and aorta revealed increased collagen in Ang ACE2 KO, suggestive of remodeling. Results also showed enhanced oxidative stress in the myocardium and aorta of Ang ACE2 KO. There was a 3-fold elevation in macrophage inflammatory protein 1α (MIP 1α) in the aorta of ACE2 KO. Studies in the ACE2 KO model reveal the importance of ACE2 in the maladaptive cardiac and aortic responses to Ang II stimulation, seen as enhanced remodeling using physiological, structural, and biochemical markers. Results document a cardio- and vascular-protective role of ACE2 under pathological conditions

Enhanced Angiotensin II-induced Cardiac and Aortic Remodeling in ACE2 Knockout Mice

Angiotensin-converting enzyme 2 (ACE2) is present in the heart and thought to exert protective functions. We conducted studies in ACE2 deficient mice to determine whether enzyme loss would exacerbate the cardiac and vascular pathological responses to chronic subcutaneous (sc) angiotensin II (Ang II) infusion. Eight-week-old male ACE2 knockout (KO) and wild type (WT) mice were infused with Ang II (1000 ng/kg per min, 4 weeks) using mini-osmotic pumps. Blood pressure (radiotelemetry), cardiac function (echocardiography, echo), cardiac/aortic structure (histology, collagen, and oxidative stress), and vascular inflammation were examined. Before Ang II infusion, ACE2 KO mice showed unaltered cardiac function and blood pressure. After 4 weeks of Ang II infusion, the mean arterial pressure (MAP) increased from 96 ± 2 to 136 ± 17 mm Hg (∼40%) in WT and from 104 ± 5 to 141 ± 13 mm Hg (∼ 35%) in ACE2 KO. While there were no differences in MAP between groups, the ACE2 KO responded differently to the hypertensive stimulus. Echo analysis revealed severe myocardial dysfunction in Ang II-infused ACE2 KO (Ang ACE2 KO). Ejection fraction was lower (39% versus 50%) as was fractional shortening (27% versus 38%) in ACE2 KO versus WT, respectively. Cardiac dysfunction was associated with hypertrophic cardiomyopathy shown by increased left-ventricular wall thickness, average cardiomyocyte cross-sectional area, and heart weight/body weight ratio. Collagen staining in the myocardium and aorta revealed increased collagen in Ang ACE2 KO, suggestive of remodeling. Results also showed enhanced oxidative stress in the myocardium and aorta of Ang ACE2 KO. There was a 3-fold elevation in macrophage inflammatory protein 1α (MIP 1α) in the aorta of ACE2 KO. Studies in the ACE2 KO model reveal the importance of ACE2 in the maladaptive cardiac and aortic responses to Ang II stimulation, seen as enhanced remodeling using physiological, structural, and biochemical markers. Results document a cardio- and vascular-protective role of ACE2 under pathological conditions

Neutrophils adhered to the apical surface of polarized-MDCK cells augment AdV entry without decreasing the TER.

<p>A) MDCK-CAR^Ex8 cells were either mock- or DOX-induced. A neutrophil adhesion assay was performed with increasing numbers of neutrophils, as indicated. Immediately post-neutrophil adhesion, MDCK-CAR^Ex8 epithelia were infected with AdV5-β-gal for 1 h from the apical surface. 24 h later, viral entry was determined by qPCR analysis. Fold change in viral genomes, relative to AdV5-βGal entry in the absence of DOX and neutrophils, is shown. AdV entry from the apical surface was quantitated by qPCR analysis of polarized B) MDCK-CAR^Ex8 C) MDCK-mCherry and D) MDCK-CAR^Ex7 cells that were uninduced (circles), uninduced with adhered neutrophils (squares), or induced with DOX for 24 h prior to neutrophil adhesion (triangles). E) AdV5-β-gal entry from the apical surface of MDCK-CAR^Ex8 epithelia in the presence or absence of neutrophils and AdV5 FK or AdV3 FK. F) TER of mock- or Dox-induced MDCK-CAR^Ex8 epithelia was measured in the presence or absence of neutrophils. Error bars represent standard error of the mean (SEM) from three independent experiments. No significant difference was detected by one-way ANOVA. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.001 by one-way ANOVA and Bonferroni post hoc test.</p