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Aims: CAR-T cell therapy has proven its importance in cancer treatment and currently there are five approved products on the market. However, these products are based on viral vectors, which presents a risk of insertional mutagenesis, it can also be immunogenic and its manufacture is extremely expensive. The development of a nonviral vector, suitable for an efficient and stable transfection of NK and T cells is the goal of this study. Methods: In this study we use a special episomal vector (extrachromosomal vectors) with i) scaffold/matrix attachment region (S/MAR) motifs that can actively mediate the episomal maintenance, ii) a plasmid replication enhancer, the replication-Initiation Region (IR) from the β-globin locus (pEP-IR) and iii) a CAR anti CD19 (pEP-IR-CAR). We electroporated activated and non-activated T cells and NK cells with different plasmid concentrations. We evaluated transfection efficiency and viability. Results and discussion: Our findings showed that unstimulated T cells are transfected more efficiently than activated T cells. Transfection of unstimulated T cells with 20 ug of supercoiled pEP-IR-GFP plasmid reached 22 to 63% of GFP and viability around 54% depending on the culture condition used after electroporation (FBS% and IL2). We then accessed the efficacy of pEP-IR-CAR and CAR expression, confirmed in 3,2% of the cells with 89% of viability. Cells expressing CAR on their surface were marked with anti-CAR antibody Biotin-SP-conjugated and selected by anti-Biotin microbeads, achieving an enrichment of 54,9% CAR+ cells. Conclusions: Our results showed that we established an optimized protocol resulting in an acceptable transfection efficiency and the transfected cells can be selected. So, the next step is to perform in vitro cytotoxicity and in vivo assays with the episomal CAR+ cells. Funding: Financially supported by FAPESP 2020/09206-4, FAPESP 2019/25309-0, CTC Center for Cell-based Therapy (FAPESP 2013/08135-2), and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773).

Introduction: Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. We hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy [Interleukin-27 (IL-27)] could be used to treat bone-metastatic prostate cancer and exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. Methods: To test this hypothesis, in vivo experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells that are able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), and/or a therapeutic gene (IL-27). Sonoporation was used to aid gene delivery. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Results: Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing was used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone quality by enhancing osteoblast differentiation and inhibiting osteoclast differentiation. Discussion: Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach will enable a significant reduction in chemotherapy-associated toxicity, enhance sensitivity to immunotherapy, and improve bone quality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kumar, Mulia and Figueiredo.)

Nonviral and nonintegrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable safety profile, significantly lower risk for insertional mutagenesis, and a lesser potential for innate immune reactions, in addition to their low production cost. Over the past few years, attempts have been made to generate highly functional nonviral vectors that display long-term maintenance within cells and promote more sustained gene expression relative to conventional plasmids. Extensive research into the parameters that stabilize the episomal DNA within dividing and nondividing cells has shed light into the genetic and epigenetic mechanisms that govern replication and transcription of episomal DNA within a mammalian nucleus in long-term cell culture. Episomal vectors based on scaffold/matrix attachment regions (S/MARs) do not integrate into the genomic DNA and address the serious problem of plasmid loss during mitosis by providing mitotic stability to established plasmids, which results in long-term transfection and transgene expression. The inclusion, in such vectors, of an origin of replication-initiation region-from the human genome has greatly enhanced their performance in primary cell culture. A number of vectors that function as episomes have arisen, which are either devoid or depleted of harmful CpG sequences and bacterial genes, and their effectiveness, as well as that of nonintegrating viral episomes, is enhanced when combined with S/MAR elements. As a result of these advances, an "S/MAR technology" has emerged for the production of efficient episomal vectors. Significant research continues in this field and innovations, in combination with promising systems based on nanoparticles and potentially combined with physical delivery methods, will enable the generation of optimized systems with scale-up and clinical application suitability utilizing episomal vectors.

G protein-coupled receptors (GPCRs) are implicated in the regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but can also engage downstream kinases and effectors through scaffolding interactions mediated by β-arrestin. Here, we investigated whether β-arrestin signaling regulates anxiety-like and fear-related behavior in mice in response to activation of the GPCR δ-opioid receptor (δOR or DOR). Administration of β-arrestin-biased δOR agonists to male C57BL/6 mice revealed β-arrestin 2-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the dorsal hippocampus and amygdala and β-arrestin 1-dependent activation of ERK1/2 in the nucleus accumbens. In mice, β-arrestin-biased agonist treatment was associated with reduced anxiety-like and fear-related behaviors, with some overlapping and isoform-specific input. In contrast, applying a G protein-biased δOR agonist decreased ERK1/2 activity in all three regions as well as the dorsal striatum and was associated with increased fear-related behavior without effects on baseline anxiety. Our results indicate a complex picture of δOR neuromodulation in which β-arrestin 1- and 2-dependent ERK signaling in specific brain subregions suppresses behaviors associated with anxiety and fear and opposes the effects of G protein-biased signaling. Overall, our findings highlight the importance of noncanonical β-arrestin-dependent GPCR signaling in the regulation of these interrelated emotions., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The mitogen-activated protein kinases (MAPKs) are a family of protein kinases that regulate crucial neuronal functions such as neuronal differentiation, proliferation, and apoptosis through phosphorylation of subsequent protein kinases. The three classical MAPK subfamilies, extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase have been linked to various neurological disorders often in conjunction with activation of a wide range of G protein-coupled receptors and receptor tyrosine kinases. Many studies investigating MAPK function in these disorders rely on histochemistry or immunoblotting that require brain isolation following euthanasia. Here, we evaluated to what degree different modes of anesthesia/euthanasia impact MAPK activity in adult male and female C57BL/6 mice. Mice were decapitated following ketamine/xylazine or isoflurane anesthesia, carbon dioxide asphyxiation, or without anesthesia. We selectively chose five brain regions (the prefrontal cortex, the dorsal hippocampus, the dorsal striatum, the nucleus accumbens, and the amygdala) that are heavily implicated in neuropsychiatric disorders. We found that relative to carbon dioxide asphyxiation, the other methods displayed significantly stronger ERK1/2 phosphorylation in select brain regions of male and female mice, with no pronounced sex difference. A similar, yet, less pronounced trend was observed for JNK activity, whereas the choice of euthanasia method did not differentially impact p38 phosphorylation. Our study results reveal how small differences in experimental design may impact whether one will be able to detect drug- or disease-related changes in MAPK activity. These findings are timely in a period where experimental rigor is emphasized to increase reproducibility of research.