Recruiting In Vitro Transcribed mRNA against Cancer Immunotherapy: A Contemporary Appraisal of the Current Landscape.

Over 100 innovative in vitro transcribed (IVT)-mRNAs are presently undergoing clinical trials, with a projected substantial impact on the pharmaceutical market in the near future. Τhe idea behind this is that after the successful cellular internalization of IVT-mRNAs, they are subsequently translated into proteins with therapeutic or prophylactic relevance. Simultaneously, cancer immunotherapy employs diverse strategies to mobilize the immune system in the battle against cancer. Therefore, in this review, the fundamental principles of IVT-mRNA to its recruitment in cancer immunotherapy, are discussed and analyzed. More specifically, this review paper focuses on the development of mRNA vaccines, the exploitation of neoantigens, as well as Chimeric Antigen Receptor (CAR) T-Cells, showcasing their clinical applications and the ongoing trials for the development of next-generation immunotherapeutics. Furthermore, this study investigates the synergistic potential of combining the CAR immunotherapy and the IVT-mRNAs by introducing our research group novel, patented delivery method that utilizes the Protein Transduction Domain (PTD) technology to transduce the IVT-mRNAs encoding the CAR of interest into the Natural Killer (NK)-92 cells, highlighting the potential for enhancing the CAR NK cell potency, efficiency, and bioenergetics. While IVT-mRNA technology brings exciting progress to cancer immunotherapy, several challenges and limitations must be acknowledged, such as safety, toxicity, and delivery issues. This comprehensive exploration of IVT-mRNA technology, in line with its applications in cancer therapeutics, offers valuable insights into the opportunities and challenges in the evolving landscape of cancer immunotherapy, setting the stage for future advancements in the field.

Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass, Dicentrarchus labrax.

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues-eye, oesophagus, gills and skin-as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes.

The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus-oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml-1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

Targeting mitochondrial bioenergetics by combination treatment with imatinib and dichloroacetate in human erythroleukemic K­562 and colorectal HCT­116 cancer cells.

Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCΟ2 and FRATAXIN (FXN) genes involved in the heme­dependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IM­sensitive K­562 chronic myeloid leukemia cells (K­562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K­562 and IM­chemoresistant K­562 chronic myeloid leukemia cells (K­562R), as well as human colorectal carcinoma cells HCT­116 (+/+p53) and (­/­p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2 µM) and/or DCA (4 mΜ) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde­3­phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor­1a, heme oxygenase­1, NF­κB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K­562 and considerable (>45%) in HCT­116 (+/+p53) cultures, but less in K­562R and HCT­116 (­/­p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flow­cytometry, in K­562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.

Protein Transduction Domain-Mediated Delivery of Recombinant Proteins and In Vitro Transcribed mRNAs for Protein Replacement Therapy of Human Severe Genetic Mitochondrial Disorders: The Case of Sco2 Deficiency.

Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness.

An Innovative PTD-IVT-mRNA Delivery Platform for CAR Immunotherapy of ErbB(+) Solid Tumor Neoplastic Cells.

Chimeric antigen receptor (CAR) immunotherapy includes the genetic modification of immune cells to carry such a receptor and, thus, recognize cancer cell surface antigens. Viral transfection is currently the preferred method, but it carries the risk of off-target mutagenicity. Other transfection platforms have thus been proposed, such the in vitro transcribed (IVT)-mRNAs. In this study, we exploited our innovative, patented delivery platform to produce protein transduction domain (PTD)-IVT-mRNAs for the expression of CAR on NK-92 cells. CAR T1E-engineered NK-92 cells, harboring the sequence of T1E single-chain fragment variant (scFv) to recognize the ErbB receptor, bearing either CD28 or 4-1BB as co-stimulatory signaling domains, were prepared and assessed for their effectiveness in two different ErbB(+) cancer cell lines. Our results showed that the PTD-IVT-mRNA of CAR was safely transduced and expressed into NK-92 cells. CAR T1E-engineered NK-92 cells provoked high levels of cell death (25-33%) as effector cells against both HSC-3 (oral squamous carcinoma) and MCF-7 (breast metastatic adenocarcinoma) human cells in the co-incubation assays. In conclusion, the application of our novel PTD-IVT-mRNA delivery platform to NK-92 cells gave promising results towards future CAR immunotherapy approaches.

Serum pharmacokinetics of clindamycin hydrochloride in normal dogs when administered at two dosage regimens.

The aim of this cross-over study was to compare clindamycin pharmacokinetics in the serum of clinically normal dogs when administered orally at two dosage regimens (5.5 mg/kg, twice daily, and 11 mg/kg, once daily), separated by a 1 week wash-out period. Serum samples were obtained from six clinically normal laboratory beagles before, 3, 6, 9 and 12 h after the first and fifth dose of clindamycin at 5.5 mg/kg, twice daily, and before, 3, 6, 9, 12, 18 and 24 h after the first and third dose at 11 mg/kg, once daily. Serum clindamycin concentrations were determined by reverse-phase liquid chromatography coupled with mass spectrometry. Results were analysed using Student's paired t-test, at a 5% level of significance. Values of pharmacokinetic parameters that differed significantly between the two dosage regimens included the following: maximal concentration and area under the concentration-time curve were higher at 11 mg/kg, once daily, than at 5.5 mg/kg, twice daily; and, more importantly, the ratio of AUC(0-24) to the minimal inhibitory concentration (MIC) value of 0.5 µg/mL for a 24 h period (AUC(0-24)/MIC) was higher when clindamycin was administered at 11 than at 5.5 mg/kg, at least during the first day of drug administration. Therefore, a better pharmacokinetic profile may be expected when clindamycin is administered at 11 mg/kg, once daily, for the treatment of canine pyoderma caused by Staphylococcus pseudintermedius.

Development of a novel PTD-mediated IVT-mRNA delivery platform for potential protein replacement therapy of metabolic/genetic disorders.

The potential clinical applications of the powerful in vitro-transcribed (IVT)-mRNAs, to restore defective protein functions, strongly depend on their successful intracellular delivery and transient translation through the development of safe and efficient delivery platforms. In this study, an innovative (international patent-pending) methodology was developed, combining the IVT-mRNAs with the protein transduction domain (PTD) technology, as an efficient delivery platform. Based on the PTD technology, which enables the intracellular delivery of various cargoes intracellularly, successful conjugation of a PTD to the IVT-mRNAs was achieved and evaluated by band-shift assay and NMR spectroscopy. In addition, the PTD-IVT-mRNAs were applied and evaluated in two protein-disease models, including the mitochondrial disorder fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency (attributed to SCO2 gene mutations) and ß-thalassemia. The PTD-IVT-mRNA of SCO2 was successfully transduced and translated to the corresponding Sco2 protein inside the primary fibroblasts of a SCO2/COX-deficient patient, whereas the PTD-IVT-mRNA of ß-globin was transduced and translated in bone marrow cells, derived from three ß-thalassemic patients. The transducibility and the structural stability of the PDT-IVT-mRNAs, in both cases, were confirmed at the RNA and protein levels. We propose that our novel delivery platform could be clinically applicable as a protein therapy for metabolic/genetic disorders.

Mechanisms involved in the induced differentiation of leukemia cells.

Despite the remarkable progress achieved in the treatment of leukemias over the last several years, many problems (multidrug resistance [MDR], cellular heterogeneity, heterogeneous molecular abnormalities, karyotypic instability, and lack of selective action of antineoplastic agents) still remain. The recent progress in tumor molecular biology has revealed that leukemias are likely to arise from disruption of differentiation of early hematopoietic progenitors that fail to give birth to cell lineage restricted phenotypes. Evidence supporting such mechanisms has been derived from studying bone marrow leukemiogenesis and analyzing differentiation of leukemic cell lines in culture that serve as models of erythroleukemic (murine erythroleukemia [MEL] and human leukemia [K562] cells) and myeloid (human promyelocytic leukemia [HL-60] cells) cell maturation. This paper reviews the current concepts of differentiation, the chemical/pharmacological inducing agents developed thus far, and the mechanisms involved in initiation of leukemic cell differentiation. Emphasis was given on commitment and the cell lineage transcriptional factors as key regulators of terminal differentiation as well as on membrane-mediated events and signaling pathways involved in hematopoietic cell differentiation. The developmental program of MEL cells was presented in considerable depth. It is quite remarkable that the erythrocytic maturation of these cells is orchestrated into specific subprograms and gene expression patterns, suggesting that leukemic cell differentiation represents a highly coordinated set of events that lead to irreversible growth arrest and expression of cell lineage restricted phenotypes. In MEL and other leukemic cells, differentiation appears to be accompanied by differentiation-dependent apoptosis (DDA), an event that can be exploited chemotherapeutically. The mechanisms by which the chemical inducers promote differentiation of leukemic cells have been discussed.

Mechanisms of action of differentiation inducers: detection of inducer binding protein(s) in murine erythroleukemia cells.

We have shown previously that murine erythroleukemia (MEL) and human neuroectodermal RD/TE-671 cells are induced to differentiate by ureido derivatives of pyridine (UDPs) and may contain inducer binding protein(s). In the present study, we prepared radiolabeled [3H]UDP [2-(3-ethylureido)-6-[3H]-acetylaminopyridine] as ligand and investigated whether it interacts selectively with novel binding proteins. MEL and RD/TE-671 cells, incubated with the inducer [3H]UDP and subsequently fractionated, yielded a radiolabeled postmitochondrial soluble fraction containing the [3H]UDP-protein complex. We purified the UDP binding protein by using UDP-sepharose affinity chromatography, gel filtration, and SDS-PAGE electrophoresis and analyzed its structure. The data presented here indicate for the first time that the inducer UDP interacts with a 38,333 +/- 30 Da binding protein(s) (p38), of unknown function, in both cell lines. Microsequencing and sequence alignment search revealed that the p38 protein(s) contains at least two homologous domains, one being part of ABC-type transporters and another found in the Wingless-type (Wnt) proteins. Kinetic analysis revealed that the p38 forms a relatively stable protein complex with [3H]UDP that accumulates within the cytosol and nucleus of MEL cells during the precommitment period. This complex, however, decays later on after commitment to erythroid maturation has been initiated. De novo protein and mRNA synthesis is needed for the UDP-p38 complex to form, as shown by the use of metabolic inhibitors. Purified p38 was used to develop an anti-p38 polyclonal serum, and Western blot analysis revealed that the level of p38 was quite similar in both UDP-inducible and -resistant MEL subclones that we developed. Although only a portion of the primary structure of the p38 is known from microsequencing, the mechanism by which the UDP-p38 complex contributes to induction of differentiation in both UDP-responsive mouse MEL and human RD/TE-671 cells is discussed.

Differentiation-dependent repression of c-myc, B22, COX II and COX IV genes in murine erythroleukemia (MEL) cells.

Murine erythroleukemia (MEL) cells provide a valuable model system for uncovering the cellular and molecular mechanisms of differentiation of proerythroid cells in culture. In order to characterize genes and gene expression patterns unique for erythropoiesis, we: (i) cloned and sequenced a 226bp cDNA encoding portion of the 3'-end B22 subunit of mitochondrial NADH-ubiquinone oxidoreductase (complex I); (ii) assessed the steady state level of RNA transcripts encoded by B22, cytochrome c oxidase (COX II, COX IV) and c-myc genes in MEL cells undergoing terminal differentiation induced by dimethylsulfoxide (DMSO) and/or 2-(3-ethylureido)-6-methylpyridine; and (iii) investigated whether the gene expression patterns of B22, COX IV and c-myc genes seen in differentiating cells are affected by N(6)-methyladenosine, an inhibitor of commitment and RNA methylation. These studies have indicated: (a) c-myc, COX II and COX IV genes exhibited biphasic expression pattern; a transient accumulation of c-myc, COX II and COX IV mRNAs was followed by a decline after 36hr incubation with DMSO and/or 2-(3-ethylureido)-6-methylpyridine, (b) B22 gene expression declined progressively in differentiated cells, (c) blockade of differentiation of MEL cells with N(6)-methyladenosine failed to prevent the transient accumulation of c-myc, COX II and COX IV mRNAs, but abrogated the irreversible expression of all four genes. These findings indicated that B22, c-myc, COX II and COX IV genes are gradually repressed in terminally differentiating MEL cells presumably via different patterns of expression (gradual vs. biphasic). Overall, these results showed that erythroid maturation of MEL cells is accompanied by transcriptional inactivation (or repression) of at least three genes encoding mitochondrial enzyme subunits involved in cell respiration.

The developmental program of murine erythroleukemia cells.

Hematopoietic stem cells (HSCs) or early progenitors respond to external stimuli in bone marrow and differentiate into cell-restricted lineages of blood cells of limited life span. In leukemias, however, early hematopoietic progenitors self-renew themselves, fail to respond to differentiation signals, and do not undergo programmed cell death (apoptosis). The basic mechanisms of differentiation and apoptosis of leukemia cells have been the long-term objective of our work. By exploiting widely studied murine and human leukemic cell systems as models of hematopoietic cell differentiation, we explored the mechanisms by which pharmaceutical agents initiate differentiation in leukemic systems. In this article, we present the developmental program of MEL cells with emphasis given on the role of commitment to terminal maturation. Commitment is initiated via inducer-receptor-mediated processes and leads to discrete patterns of expression of several genes that contribute to growth arrest at the G1 phase, expression of differentiated phenotype, and differentiation-dependent apoptosis (DDA). Overall, MEL erythroid cell differentiation represents a developmental program with a highly coordinated set of processes that is "triggered" by an inducer and functions via a network of genes and proteins interacting with each other harmonically to give birth to lineage-restricted phenotype.

Concentrations of clindamycin hydrochloride in homogenates of normal dog skin when administered at two oral dosage regimens.

BACKGROUND: Clindamycin is frequently used for the treatment of bacterial pyoderma. OBJECTIVE: To compare the pharmacokinetics of clindamycin in whole skin homogenates of normal dogs when administered orally at two dosage regimens (5.5 mg/kg BW, twice daily and 11 mg/kg BW, once daily). ANIMALS AND METHODS: Skin biopsies were obtained from six laboratory beagles before, 3, 6 and 12 h after the first and the fifth dose of clindamycin at the former regimen, as well as before, 3, 6, 12 and 24 h after the first and third dose at the latter regimen. Tissue was homogenized and clindamycin concentrations were measured by reverse-phase high-performance liquid chromatography coupled with mass spectrometry. Results were analyzed using Student's t-test at a level of significance of 0.05. RESULTS: Maximal concentration and area under the concentration-time curve, but not their relevant dose-normalized values, were higher at the dosage regimen of 11 mg/kg BW, once daily than at 5.5 mg/kg BW twice daily. CONCLUSIONS: The pharmacokinetic profile of clindamycin is at least equal, if not better, when this antimicrobial is administered at 11 mg/kg BW, once daily.