| 1. |
- Anwar, Mohiemen, et al.
(author)
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Ixovex-1, a novel oncolytic E1B-mutated adenovirus
- 2022
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In: Cancer Gene Therapy. - : Springer Nature. - 0929-1903 .- 1476-5500. ; 29:11, s. 1628-1635
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Journal article (peer-reviewed)abstract
- There is a great demand for improved oncolytic viruses that selectively replicate within cancer cells while sparing normal cells. Here, we describe a novel oncolytic adenovirus, Ixovex-1, that obtains a cancer-selective replication phenotype by modulating the level of expression of the different, alternatively spliced E1B mRNA isoforms. Ixovex-1 is a recombinant adenovirus that carries a single point mutation in the E1B-93R 3' splice acceptor site that results in overexpression of the E1B-156R splice isoform. In this paper, we studied the characteristics of this novel oncolytic adenovirus by validating its in vitro behaviour in a panel of normal cells and cancer cells. We additionally studied its anti-tumour efficacy in vivo. Ixovex-1 significantly inhibited tumour growth and prolonged survival of mice in an immune-deficient lung carcinoma tumour implantation model. In complementation experiments, overexpression of E1B-156R was shown to increase the oncolytic index of both Ad5wt and ONYX-015. In contrast to prior viruses of similar type, Ixovex-1 includes a functional E3B region for better in vivo efficacy. Throughout this study, the Ixovex-1 virus has been proven to be superior in competency compared to a virus with multiple deletions.
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| 2. |
- Darweesh, Mahmoud, et al.
(author)
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ZC3H11A loss of function enhances NF-κB signaling through defective IκBα protein expression
- 2022
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In: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 13
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Journal article (peer-reviewed)abstract
- ZC3H11A is a cellular protein associated with the transcription export (TREX) complex that is induced during heat-shock. Several nuclear-replicating viruses exploit the mRNA export mechanism of ZC3H11A protein for their efficient replication. Here we show that ZC3H11A protein plays a role in regulation of NF-kappa B signal transduction. Depletion of ZC3H11A resulted in enhanced NF-kappa B mediated signaling, with upregulation of numerous innate immune related mRNAs, including IL-6 and a large group of interferon-stimulated genes. IL-6 upregulation in the absence of the ZC3H11A protein correlated with an increased NF-kappa B transcription factor binding to the IL-6 promoter and decreased IL-6 mRNA decay. The enhanced NF-kappa B signaling pathway in ZC3H11A deficient cells correlated with a defect in I kappa B alpha inhibitory mRNA and protein accumulation. Upon ZC3H11A depletion The I kappa B alpha mRNA was retained in the cell nucleus resulting in failure to maintain normal levels of the cytoplasmic I kappa B alpha mRNA and protein that is essential for its inhibitory feedback loop on NF-kappa B activity. These findings indicate towards a previously unknown mechanism of ZC3H11A in regulating the NF-kappa B pathway at the level of IkB alpha mRNA export.
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| 3. |
- Deng, Siyuan, et al.
(author)
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Development of a New Hyaluronic Acid Based Redox-Responsive Nanohydrogel for the Encapsulation of Oncolytic Viruses for Cancer Immunotherapy
- 2021
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In: Nanomaterials. - : MDPI. - 2079-4991. ; 11:1
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Journal article (peer-reviewed)abstract
- Oncolytic viruses (OVs) are emerging as promising and potential anti-cancer therapeutic agents, not only able to kill cancer cells directly by selective intracellular viral replication, but also to promote an immune response against tumor. Unfortunately, the bioavailability under systemic administration of OVs is limited because of undesired inactivation caused by host immune system and neutralizing antibodies in the bloodstream. To address this issue, a novel hyaluronic acid based redox responsive nanohydrogel was developed in this study as delivery system for OVs, with the aim to protect the OVs following systemic administration. The nanohydrogel was formulated by water in oil (W/O) nanoemulsion method and cross-linked by disulfide bonds derived from the thiol groups of synthesized thiolated hyaluronic acid. One DNA OV Ad[I/PPT-E1A] and one RNA OV Rigvir(R) ECHO-7 were encapsulated into the developed nanohydrogel, respectively, in view of their potential of immunovirotherapy to treat cancers. The nanohydrogels showed particle size of approximately 300-400 nm and negative zeta potential of around -13 mV by dynamic light scattering (DLS). A uniform spherical shape of the nanohydrogel was observed under the scanning electron microscope (SEM) and transmission electron microscope (TEM), especially, the successfully loading of OV into nanohydrogel was revealed by TEM. The crosslinking between the hyaluronic acid chains was confirmed by the appearance of new peak assigned to disulfide bond in Raman spectrum. Furthermore, the redox responsive ability of the nanohydrogel was determined by incubating the nanohydrogel into phosphate buffered saline (PBS) pH 7.4 with 10 mu M or 10 mM glutathione at 37 degrees C which stimulate the normal physiological environment (extracellular) or reductive environment (intracellular or tumoral). The relative turbidity of the sample was real time monitored by DLS which indicated that the nanohydrogel could rapidly degrade within 10 h in the reductive environment due to the cleavage of disulfide bonds, while maintaining the stability in the normal physiological environment after 5 days. Additionally, in vitro cytotoxicity assays demonstrated a good oncolytic activity of OVs-loaded nanohydrogel against the specific cancer cell lines. Overall, the results indicated that the developed nanohydrogel is a delivery system appropriate for viral drugs, due to its hydrophilic and porous nature, and also thanks to its capacity to maintain the stability and activity of encapsulated viruses. Thus, nanohydrogel can be considered as a promising candidate carrier for systemic administration of oncolytic immunovirotherapy.
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| 4. |
- Depreter, Barbara, et al.
(author)
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TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment
- 2020
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In: Haematologica. - : FERRATA STORTI FOUNDATION. - 0390-6078 .- 1592-8721. ; 105:5, s. 1306-1316
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Journal article (peer-reviewed)abstract
- Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor ? chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a Emslike tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.
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| 5. |
- Georganaki, Maria, et al.
(author)
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Tumor endothelial cell up-regulation of IDO1 is an immunosuppressive feed-back mechanism that reduces the response to CD40-stimulating immunotherapy
- 2020
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In: Oncoimmunology. - : TAYLOR & FRANCIS INC. - 2162-4011 .- 2162-402X. ; 9:1
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Journal article (peer-reviewed)abstract
- CD40-stimulating immunotherapy can elicit potent anti-tumor responses by activating dendritic cells and enhancing T-cell priming. Tumor vessels orchestrate T-cell recruitment during immune response, but the effect of CD40-stimulating immunotherapy on tumor endothelial cells has not been evaluated. Here, we have investigated how tumor endothelial cells transcriptionally respond to CD40-stimulating immunotherapy by isolating tumor endothelial cells from agonistic CD40 mAb- or isotype-treated mice bearing B16-F10 melanoma, and performing RNA-sequencing. Gene set enrichment analysis revealed that agonistic CD40 mAb therapy increased interferon (IFN)-related responses in tumor endothelial cells, including up-regulation of the immunosuppressive enzyme Indoleamine 2, 3-Dioxygenase 1 (IDO1). IDO1 was predominantly expressed in endothelial cells within the tumor microenvironment, and its expression in tumor endothelium was positively correlated to T-cell infiltration and to increased intratumoral expression of IFN gamma. In vitro, endothelial cells up-regulated IDO1 in response to T-cell-derived IFN gamma, but not in response to CD40-stimulation. Combining agonistic CD40 mAb therapy with the IDO1 inhibitor epacadostat delayed tumor growth in B16-F10 melanoma, associated with increased activation of tumor-infiltrating T-cells. Hereby, we show that the tumor endothelial cells up-regulate IDO1 upon CD40-stimulating immunotherapy in response to increased IFN gamma-secretion by T-cells, revealing a novel immunosuppressive feedback mechanism whereby tumor vessels limit T-cell activation.
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| 6. |
- Iscaro, Alessandra, et al.
(author)
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Targeting circulating monocytes with CCL2-loaded liposomes armed with an oncolytic adenovirus
- 2022
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In: Nanomedicine. - : Elsevier. - 1549-9634 .- 1549-9642. ; 40
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Journal article (peer-reviewed)abstract
- Oncolytic viruses (OVs) selectively replicate in and destroy cancer cells resulting in anti-tumor immunity. However, clinical use remains a challenge because of virus clearance upon intravenous delivery. OV packaging using a nanomedicine approach could overcome this. Here we encapsulate an oncolytic adenovirus (Ad[I/PPT-E1A]) into CCL2-coated liposomes in order to exploit recruitment of CCR2-expressing circulating monocytes into tumors. We demonstrate successful encapsulation of Ad[I/PPT-E1A] into CCL2-coated liposomes that were preferentially taken up by CCR2-expressing monocytes. No complex-related toxicities were observed following incubation with prostate tumor cells and the encapsulation did not affect virus oncolytic activity in vitro. Furthermore, intravenous administration of our nanomedicine resulted in a significant reduction in tumor size and pulmonary metastasis in prostate cancer-bearing mice whereby a 1000-fold less virus was needed compared to Ad[I/PPT-E1A] alone. Taken together our data provide an opportunity to target OVs via circulation to inaccessible tumors using liposome-assisted drug delivery. (c) 2021 Elsevier Inc. All rights reserved.
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| 7. |
- Jin, Chuan, 1986-, et al.
(author)
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CAR T cells expressing a bacterial virulence factor trigger potent bystander antitumour responses in solid cancers
- 2022
-
In: Nature Biomedical Engineering. - : Springer Nature. - 2157-846X. ; 6:7, s. 830-841
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Journal article (peer-reviewed)abstract
- Chimeric antigen receptor T cells (CAR T cells) are effective against haematologic malignancies. However, in solid tumours, their potency is hampered by local immunosuppression and by the heterogeneous expression of the antigen that the CAR targets. Here we show that CAR T cells expressing a pluripotent pro-inflammatory neutrophil-activating protein (NAP) from Helicobacter pylori trigger endogenous bystander T-cell responses against solid cancers. In mice with subcutaneous murine pancreatic ductal adenocarcinomas, neuroblastomas or colon carcinomas, CAR(NAP) T cells led to slower tumour growth and higher survival rates than conventional mouse CAR T cells, regardless of target antigen, tumour type and host haplotype. In tumours with heterogeneous antigen expression, NAP secretion induced the formation of an immunologically 'hot' microenvironment that supported dendritic cell maturation and bystander responses, as indicated by epitope spreading and infiltration of cytotoxic CD8(+) T cells targeting tumour-associated antigens other than the CAR-targeted antigen. CAR T cells armed with NAP neither increased off-tumour toxicity nor hampered the efficacy of CAR T cells, and hence may have advantageous translational potential. T cells expressing a pluripotent pro-inflammatory neutrophil-activating protein from Helicobacter pylori trigger endogenous bystander T-cell responses against solid cancers.
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| 8. |
- Jin, Chuan, 1986-, et al.
(author)
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Intratumoral administration of pro-inflammatory allogeneic dendritic cells improved the anti-turnor response of systemic anti-CTLA-4 treatment via unleashing a T cell-dependent response
- 2022
-
In: Oncoimmunology. - : Informa UK Limited. - 2162-4011 .- 2162-402X. ; 11:1
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Journal article (peer-reviewed)abstract
- Immune checkpoint inhibitors (ICIs) have revolutionized the oncology field. However, a significant number of patients do not respond, at least partly due to the lack of preexisting anti-tumor T-cell immunity. Therefore, it is emergent to add an immune-priming step to improve efficacy. Here, we report a combined approach consisting of intratumoral administration of pro-inflammatory allogeneic dendritic cells (AlloDCs) and systemic treatment with alpha CTLA-4 that can drastically improve the anti-tumor efficacy compared to alpha CTLA-4 monotherapy. When evaluated in mice with large established CT-26 tumors, monotherapy with alpha CTLA-4 neither delayed tumor progression nor improved mice survival. However, combination treatment of AlloDCs and alpha CTLA-4 drastically improved the effectiveness, with 70% of mice being cured. This effect was T cell-dependent, and all survived mice rejected a subsequent tumor re-challenge. Further investigation revealed an immune-inflamed tumor microenvironment (TME) in the combination treatment group characterized by enhanced infiltration of activated antigen-presenting endogenous DCs and CD8(+) T cells with a tissue-resident memory (T-RM) phenotype (CD49a(+)CD103(+)). This correlated with elevated levels of tumor-specific CD39(+)CD103(+)CD8(+) T cells in the tumor and "tumor-matching" NKG2D(+)CD39(+)CX3CR1(+)CD8(+) T cells in peripheral blood. Moreover, splenocytes from mice in the combination treatment group secreted significantly higher IFN-gamma upon stimulation with the peptide from the endogenous CT-26 retroviral gp70 (model neoantigen), confirming the induction of a tumor-specific CD8(+) T-cell response. Taken together, these data indicate a strong anti-tumor synergy between AlloDCs and alpha CTLA-4 that warrant further clinical investigation with the corresponding human AlloDC product (ilixadencel) for patients receiving alpha CTLA-4 therapy.
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| 9. |
- Konstantinidis, Evangelos, 1990-
(author)
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CRISPR/Cas9-based therapies and the role of astrocytes in Alzheimer’s disease and Parkinson’s disease
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative disorders. Whereas the AD brain features plaques of amyloid-beta (Aβ) and neurofibrillary tangles of tau, the PD brain is characterized by Lewy bodies and Lewy neurites containing α-synuclein (αSyn). Rare familial disease forms have illustrated a central involvement of these proteins in the respective pathogeneses. Mutations in the genes for the presenilins (PSEN1, PSEN2) result in AD by an increased generation of the more aggregation prone Aβ42 peptide, whereas mutations in the αSyn gene (SNCA) cause PD by affecting aggregation of αSyn.This thesis has investigated the gene editing tool CRISPR/Cas9 as a potential treatment strategy against AD and PD. When targeting PSEN1 M146L in patient fibroblasts, the increased Aβ42/Aβ40 ratio was partially restored and the treatment typically normalized the mutation-induced conformation of presenilin 1. Moreover, the treatment did not cause any major off-target effects across the genome. For SNCA, both the wild-type form and the A53T mutant were targeted. Lentivirus-mediated delivery of CRISPR/Cas9 to patient fibroblasts and HEK293T cells led to a targeting efficiency of up to 87%. However, treatment of A53T mutant patient fibroblasts only resulted in low and inconsistent targeting efficiencies.During the course of AD, progressive cellular dysfunction and degeneration cause widespread neuronal death. Apart from neurons, also glial cells are affected by the disease process. Astrocytes, the most abundant glial cell type, play a key role in maintaining brain homeostasis. However, in a neurodegenerative environment, astrocytes enter a reactive and inflammatory state that can potentially harm nearby neurons.To further investigate the role of astrocytes in AD, we generated a co-culture system of human induced pluripotent stem cell-derived neurons and astrocytes. We observed a differential effect of direct and remote astrocytic control on neuronal viability and functionality. Physical astrocytic contact combined with the presence of Aβ resulted in increased phagocytosis and clearance of dead cells as well as a reduced neuronal activity. However, indirect contact via conditioned media from control astrocytes improved the viability of neurons, whereas addition of Aβ led to hyperactivity. Analyses of long-term astrocytic cultures revealed a persistent reactive state accompanied by a limited Aβ degradation capacity and severe cellular stress.Overall, this thesis has explored novel gene therapeutic strategies for AD and PD as well as contributed with knowledge regarding the role of astrocytes in AD progression.
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| 10. |
- Konstantinidis, Evangelos, et al.
(author)
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CRISPR-Cas9 treatment partially restores amyloid-β 42/40 in human fibroblasts with the Alzheimer's disease PSEN1 M146L mutation
- 2022
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In: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 28, s. 450-461
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Journal article (peer-reviewed)abstract
- Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-β (Aβ) peptide that deposits as plaques in the Alzheimer’s disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aβ42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aβ42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.
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