HDAC inhibitors activate lipid peroxidation and ferroptosis in gastric cancer.

Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death. In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level. Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model. We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.

Spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery.

Spray-drying of nucleic acid-based drugs designed for gene therapy or gene knockdown is associated with many advantages including storage stability and handling as well as the possibility of pulmonary application. The encapsulation of nucleic acids in nanoparticles prior to spray-drying is one strategy for obtaining efficient formulations. This, however, strongly relies on the definition of optimal nanoparticles, excipients and spray-drying conditions. Among polymeric nanoparticles, polyethylenimine (PEI)-based complexes with or without chemical modifications have been described previously as very efficient for gene or oligonucleotide delivery. The tyrosine-modification of linear or branched low molecular weight PEIs, or of polypropylenimine (PPI) dendrimers, has led to high complex stability, improved cell uptake and transfection efficacy as well as high biocompatibility. In this study, we identify optimal spray-drying conditions for PEI-based nanoparticles containing large plasmid DNA or small siRNAs, and further explore the spray-drying of nanoparticles containing chemically modified polymers. Poly(vinyl alcohol) (PVA), but not trehalose or lactose, is particularly well-suited as excipient, retaining or even enhancing transfection efficacies compared to fresh complexes. A big mesh size is critically important as well, while the variation of the spray-drying temperature plays a minor role. Upon spray-drying, microparticles in a âˆ¼ 3.3 - 8.5 µm size range (laser granulometry) are obtained, dependent on the polymers. Upon their release from the spray-dried material, the nanoparticles show increased sizes and markedly altered zeta potentials as compared to their fresh counterparts. This may contribute to their high efficacy that is seen also after prolonged storage of the spray-dried material. We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.

Efficient polymeric nanoparticles for RNAi in macrophage reveal complex effects on polarization markers upon knockdown of STAT3/STAT6.

Tumor associated macrophages (TAMs) are the most abundant immune cell type in the tissue microenvironment, affecting tumor progression, metastasis and therapeutic response. Different macrophage activation ("polarization") states can be distinguished: resting (M0; non-activated), pro-inflammatory/anti-tumorigenic (M1) and anti-inflammatory/pro-tumorigenic (M2). When exploring macrophages as targets in novel cancer immunotherapy approaches, TAM repolarization from the M2 into the M1 phenotype is an intriguing strategy to block their pro-tumoral and enhance their anti-tumoral properties. In the context of RNAi-based gene knockdown of M2 promoting genes, major bottlenecks include cellular siRNA delivery and correct intracellular processing. This is particularly true in case of macrophages as a cell type well-known to be notoriously hard-to-transfect. Among polymeric nanocarriers, the cationic polymer polyethylenimine (PEI) is widely explored for delivering nucleic acids. Further advanced nanocarriers are tyrosine-modified polymers based on PEI or polypropylenimine dendrimers (PPI) for highly efficient siRNA delivery in vitro and in vivo. In this paper, we explored a panel of PEI- or PPI-based nanoparticle systems for siRNA-mediated gene knockdown efficacy in macrophages and subsequent TAM repolarization. The tyrosine-modified linear 10 kDa PEI (LP10Y) or branched 5 kDa PEI (P5Y) as well as a tyrosine-modified PPI (PPI-Y) were found most efficient for gene knockdown in macrophage cell lines or primary macrophages, independent of their polarization. Knockdown of STAT6 or STAT3 led to repolarization of M2 macrophages, as indicated by alterations in various M2 and M1 marker levels. This highly specific approach also demonstrated non-redundant functions of STAT3 and STAT6. Importantly, macrophage re-polarization from M2 to M1 upon PPI-Y/siRNA-mediated STAT6 knockdown increased tumor cell phagocytosis in a co-culture model. In conclusion, we identify certain tyrosine-modified PEI- or PPI-based nanoparticles as particularly efficient for macrophage transfection, and the specific, siRNA-mediated STAT6 knockdown as a promising approach for macrophage repolarization and enhancement of their tumor cell suppressive role.

MicroRNA-30d-5p-A Potential New Therapeutic Target for Prevention of Ischemic Cardiomyopathy after Myocardial Infarction.

(1) Background and Objective: MicroRNAs (miRs) are biomarkers for assessing the extent of cardiac remodeling after myocardial infarction (MI) and important predictors of clinical outcome in heart failure. Overexpression of miR-30d-5p appears to have a cardioprotective effect. The aim of the present study was to demonstrate whether miR-30d-5p could be used as a potential therapeutic target to improve post-MI adverse remodeling. (2) Methods and Results: MiR profiling was performed by next-generation sequencing to assess different expression patterns in ischemic vs. healthy myocardium in a rat model of MI. MiR-30d-5p was significantly downregulated (p < 0.001) in ischemic myocardium and was selected as a promising target. A mimic of miR-30d-5p was administered in the treatment group, whereas the control group received non-functional, scrambled siRNA. To measure the effect of miR-30d-5p on infarct area size of the left ventricle, the rats were randomized and treated with miR-30d-5p or scrambled siRNA. Histological planimetry was performed 72 h and 6 weeks after induction of MI. Infarct area was significantly reduced at 72 h and at 6 weeks by using miR-30d-5p (72 h: 22.89 ± 7.66% vs. 35.96 ± 9.27%, p = 0.0136; 6 weeks: 6.93 ± 4.58% vs. 12.48 ± 7.09%, p = 0.0172). To gain insight into infarct healing, scratch assays were used to obtain information on cell migration in human umbilical vein endothelial cells (HUVECs). Gap closure was significantly faster in the mimic-treated cells 20 h post-scratching (12.4% more than the scrambled control after 20 h; p = 0.013). To analyze the anti-apoptotic quality of miR-30d-5p, the ratio between phosphorylated p53 and total p53 was evaluated in human cardiomyocytes using ELISA. Under the influence of the miR-30d-5p mimic, cardiomyocytes demonstrated a decreased pp53/total p53 ratio (0.66 ± 0.08 vs. 0.81 ± 0.17), showing a distinct tendency (p = 0.055) to decrease the apoptosis rate compared to the control group. (3) Conclusion: Using a mimic of miR-30d-5p underlines the cardioprotective effect of miR-30d-5p in MI and could reduce the risk for development of ischemic cardiomyopathy.

The effect of novel tyrosine-modified polyethyleneimines on human albumin structure - Thermodynamic and spectroscopic study.

The interaction of proteins with nanoparticle components are crucial for the evaluation of nanoparticle function, toxicity and biodistribution. Polyethyleneimines (PEIs) with defined tyrosine modifications are a class of novel polymers designed for improved siRNA delivery. Their interactions with biomacromolecules are still poorly described. This paper analyzes the interaction of different tyrosine-modified PEIs with human serum albumin as the most abundant serum protein. The ability of tyrosine modified, linear or branched PEIs to bind human serum albumin (HSA) was analyzed and further characterized. The interaction with hydrophobic parts of protein were studied using 1- nilinonaphthalene-8-sulfonic acid (ANS) and changes in the HSA secondary structure were evaluated using circular dichroism (CD). Complex formation and sizes were studied by transmission electron microscopy (TEM) and dynamic light scattering methods (DLS). We demonstrate that tyrosine modified PEIs are able to bind human serum albumin. Based on thermodynamic studies, van der Waals interaction, H-bonding and hydrophobic interactions are determined as main molecular forces involved in complex formation. Analysis of secondary structures revealed that the polymers decreased α-helix content, while increasing levels of randomly folded structures. Complex formation was confirmed by TEM and DLS. These findings are crucial for understanding polymer-protein interactions and the properties of nanoparticles.

Antibody-Drug Conjugates as a Targeted Therapeutic Approach Across Entities in Oncology.

BACKGROUND: Cancer is no longer treated on the basis of its histological lineage alone; more and more drugs are being developed that are directed toward specific molecular and immunological features. Monoclonal antibodies are one type of selectively acting therapeutic agent. As part of this development, antibody-drug conjugates ("ADCs") have been approved in recent years for the treatment of hematologic and solid malignancies. METHODS: This review is based on pertinent articles retrieved by a selective search in PubMed, as well as on papers presented at international congresses of specialist societies such as the European Society for Medical Oncology, the American Society of Clinical Oncology, and the American Association for Cancer Research, and information published on the websites of the European Medicines Agency, the Food and Drug Administration, and the German Joint Federal Committee. RESULTS: The efficacy of the nine ADCs currently approved in the European Union (as of 12/2022) is derived from technical improvements in the conjugation process, the introduction of novel linkers for the covalent binding of cytotoxic agents to the Fc portion of the antibody, and the development of new, potent cytotoxic agents. Compared to conventional cancer therapies, the approved ADCs improve treatment outcomes with respect to tumor remission, time to tumor progression and, in some cases, overall survival by specifically channeling cytotoxic agents into the malignant target cells and thereby limiting, at least to some extent, the exposure of healthy tissue to adverse effects. Various potential side effects still require attention, including venous occlusive disease, pneumonitis, ocular keratopathy, and skin rash. The development of effective ADCs requires the identification of tumor-selective targets to which ADCs can bind. CONCLUSION: ADCs are a novel category of drugs for the treatment of cancer. Their approval is mainly, but not exclusively, based on the favorable findings of randomized, controlled phase III trials. ADCs are already helping to improve the outcomes of treatment for cancer.

Individual hematotoxicity prediction of further chemotherapy cycles by dynamic mathematical models in patients with gastrointestinal tumors.

PURPOSE: Hematotoxicity is a common side-effect of cytotoxic gastrointestinal (GI) cancer therapies. An unsolved problem is to predict the individual risk therefore to decide on treatment adaptions. We applied an established biomathematical prediction model and primarily evaluated its predictive value in patients undergoing chemotherapy for GI cancers in curative intent. METHODS: In a prospective, observational multicenter study on patients with gastro-esophageal or pancreatic cancer (n = 28) receiving myelosuppressive adjuvant or neoadjuvant chemotherapy (FLO(T) or FOLFIRINOX), individual model parameters were learned based on patients' observed laboratory values during the first chemotherapy cycle and further external data resources. Grades of hematotoxicity of subsequent cycles were predicted by model simulation and compared with observed data. RESULTS: The most common high-grade hematological toxicity was neutropenia [19/28 patients (68%)]. For the FLO(T) regimen, individual grades of thrombocytopenia and leukopenia could be well predicted for cycles 2-4, as well as grades of neutropenia for cycle 2. Prediction accuracy for neutropenia in the third and fourth cycle differed by one toxicity grade on average. For the FOLFIRINOX-regimen, thrombocytopenia predictions showed a maximum deviation of one toxicity grade up to the end of therapy (8 cycles). Deviations of predictions were less than one degree on average up to cycle 4 for neutropenia, and up to cycle 6 for leukopenia. CONCLUSION: The biomathematical model showed excellent short-term and decent long-term prediction performance for all relevant hematological side effects associated with FLO(T)/FOLFIRINOX. Clinical utility of this precision-medicine approach needs to be further investigated in a larger cohort.

Pose, duplicate, then elaborate: Steps towards increased affinity for inhibitors targeting the specificity surface of the Pim-1 kinase.

In this study, fragment-sized hits binding to Pim-1 kinase with initially modest affinity were further optimized by combining computational, synthetic and crystallographic expertise, eventually resulting in potent ligands with affinities in the nanomolar range that address rarely-targeted regions of Pim-1 kinase. Starting from a set of crystallographically validated, chemically distinct fragments that bind to Pim-1 kinase but lack typical nucleotide mimetic structures, a library of extended fragments was built by exhaustive in silico reactions. After docking, minimization, clustering, visual inspection of the top-ranked compounds, and evaluation of ease of synthetic accessibility, either the original compound or a close derivative was synthesized and tested against Pim-1. For compounds showing the highest degree of Pim-1 inhibition the binding mode was determined crystallographically. Following a structure-guided approach, these were further optimized in a subsequent design cycle improving the compound's initial affinity by several orders of magnitude while synthesizing only a comparatively modest number of derivatives. The combination of computational and experimental approaches resulted in the development of a reasonably potent, novel molecular scaffold for inhibition of Pim-1 that targets specific surface regions, such as the interaction with R122 and P123 of the hinge region, which has been less frequently investigated in similar studies.

Experimental in vitro, ex vivo and in vivo models in prostate cancer research.

Androgen deprivation therapy has a central role in the treatment of advanced prostate cancer, often causing initial tumour remission before increasing independence from signal transduction mechanisms of the androgen receptor and then eventual disease progression. Novel treatment approaches are urgently needed, but only a fraction of promising drug candidates from the laboratory will eventually reach clinical approval, highlighting the demand for critical assessment of current preclinical models. Such models include standard, genetically modified and patient-derived cell lines, spheroid and organoid culture models, scaffold and hydrogel cultures, tissue slices, tumour xenograft models, patient-derived xenograft and circulating tumour cell eXplant models as well as transgenic and knockout mouse models. These models need to account for inter-patient and intra-patient heterogeneity, the acquisition of primary or secondary resistance, the interaction of tumour cells with their microenvironment, which make crucial contributions to tumour progression and resistance, as well as the effects of the 3D tissue network on drug penetration, bioavailability and efficacy.

Analysis of polymeric nanoparticle properties for siRNA/DNA delivery in a tumor xenograft tissue slice air-liquid interface model.

BACKGROUND: Classical two-dimensional (2D) cell culture as a drug or nanoparticle test system only poorly recapitulates in vivo conditions. Animal studies are costly, ethically controversial, and preclude large-scale testing. METHODS AND RESULTS: We established a three-dimensional (3D) tissue slice air-liquid interface (ALI) culture model for nanoparticle testing. We developed an optimized procedure for the reproducible generation of large sets of tissue slices from tumor xenografts that retain their tissue architecture. When used for the analysis of nanoparticles based on chemically modified polyethylenimines (PEIs) to deliver siRNA or DNA, differences in transfection efficacy and cytotoxicity between nanoparticles were observed more clearly than in 2D cell culture. While nanoparticle efficacies between cell culture and the tissue slice model overall correlated, the tissue slice model also identified particularly suitable candidates whose efficacy was underestimated in 2D cell culture and had already been shown in previous in vivo studies. CONCLUSION: The ex vivo 3D tissue slice ALI culture model is a powerful system that allows the effective evaluation of biological nanoparticle efficacy and biocompatibility in an intact tissue environment. It is comparably inexpensive, time-saving, and follows the 3R principle, while allowing the identification of critical nanoparticle properties and optimal candidates for in vivo applications.

LPA receptor 1 (LPAR1) is a novel interaction partner of Filamin A that promotes Filamin A phosphorylation, MRTF-A transcriptional activity and oncogene-induced senescence.

Myocardin-related transcription factors A and B (MRTFs) are coactivators of Serum Response Factor (SRF), which controls fundamental biological processes such as cell growth, migration, and differentiation. MRTF and SRF transcriptional activity play an important role in hepatocellular carcinoma (HCC) growth, which represents the second leading cause of cancer-related mortality in humans worldwide. We, therefore, searched for druggable targets in HCC that regulate MRTF/SRF transcriptional activity and can be exploited therapeutically for HCC therapy. We identified the G protein-coupled lysophosphatidic acid receptor 1 (LPAR1) as a novel interaction partner of MRTF-A and Filamin A (FLNA) using fluorescence resonance energy transfer-(FRET) and proximity ligation assay (PLA) in vitro in HCC cells and in vivo in organoids. We found that LPAR1 promotes FLNA phosphorylation at S2152 which enhances the complex formation of FLNA and MRTF-A, actin polymerization, and MRTF transcriptional activity. Pharmacological blockade or depletion of LPAR1 prevents FLNA phosphorylation and complex formation with MRTF-A, resulting in reduced MRTF/SRF target gene expression and oncogene-induced senescence. Thus, inhibition of the LPAR1-FLNA-MRTF-A interaction represents a promising strategy for HCC therapy.

Therapeutic siRNA: State-of-the-Art and Future Perspectives.

The highly specific induction of RNA interference-mediated gene knockdown, based on the direct application of small interfering RNAs (siRNAs), opens novel avenues towards innovative therapies. Two decades after the discovery of the RNA interference mechanism, the first siRNA drugs received approval for clinical use by the US Food and Drug Administration and the European Medicines Agency between 2018 and 2022. These are mainly based on an siRNA conjugation with a targeting moiety for liver hepatocytes, N-acetylgalactosamine, and cover the treatment of acute hepatic porphyria, transthyretin-mediated amyloidosis, hypercholesterolemia, and primary hyperoxaluria type 1. Still, the development of siRNA therapeutics faces several challenges and issues, including the definition of optimal siRNAs in terms of target, sequence, and chemical modifications, siRNA delivery to its intended site of action, and the absence of unspecific off-target effects. Further siRNA drugs are in clinical studies, based on different delivery systems and covering a wide range of different pathologies including metabolic diseases, hematology, infectious diseases, oncology, ocular diseases, and others. This article reviews the knowledge on siRNA design and chemical modification, as well as issues related to siRNA delivery that may be addressed using different delivery systems. Details on the mode of action and clinical status of the various siRNA therapeutics are provided, before giving an outlook on issues regarding the future of siRNA drugs and on their potential as one emerging standard modality in pharmacotherapy. Notably, this may also cover otherwise un-druggable diseases, the definition of non-coding RNAs as targets, and novel concepts of personalized and combination treatment regimens.

Therapeutic miR-506-3p Replacement in Pancreatic Carcinoma Leads to Multiple Effects including Autophagy, Apoptosis, Senescence, and Mitochondrial Alterations In Vitro and In Vivo.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality. Considering its very poor prognosis, novel treatment options are urgently needed. MicroRNAs (miRNAs) are involved in the regulation of various physiological and pathological processes. In tumors, aberrant downregulation of given miRNAs may result in pathological overexpression of oncogenes, rendering miRNA replacement as a promising therapeutic strategy. In different tumor entities, miRNA-506-3p (miR506-3p) has been ambivalently described as tumor suppressing or oncogenic. In PDAC, miR-506 is mainly considered as a tumor-suppressing miRNA. In this study, we extensively analyze the cellular and molecular effects of miRNA-506-3p replacement in different PDAC cell lines. Beyond profound antiproliferation and induction of cell death and autophagy, we describe new cellular miR506-3p effects, i.e., induction of senescence and reactive oxygen species (ROS), as well as alterations in mitochondrial potential and structure, and identify multiple underlying molecular effects. In a preclinical therapy study, PDAC xenograft-bearing mice were treated with nanoparticle-formulated miRNA-506 mimics. Profound tumor inhibition upon systemic miRNA-506 administration was associated with multiple cellular and molecular effects. This demonstrates miRNA replacement as a potential therapeutic option for PDAC patients. Due to its broad mechanisms of action on multiple relevant target genes, miR506-3p is identified as a particularly powerful tumor-inhibitory miRNA.

Characterization of Drug Release from Mesoporous SiO2-Based Membranes with Variable Pore Structure and Geometry.

Transdermal drug delivery systems (TDDSs) play important roles in therapy due to distinct advantages over other forms and types of drug application. While common TDDS patches mainly consist of polymeric matrices so far, inorganic carriers show numerous advantages such as high mechanical stability, possible re-use and re-loading of drugs, and a broad chemical compatibility with therapeutically relevant compounds and chemical enhancers. Mesoporous glasses can be prepared in different monolithic shapes, and offer a particularly wide range of possible pore volumes, pore diameters, and specific surface areas. Further, they show high loading capacities and favorable physical, technical, and biological properties. Here, we explored for the first time monolithic SiO2-based carriers as sustained release systems of therapeutic drugs. In an ideally stirred vessel as model system, we systematically analyzed the influence of pore diameter, pore volume, and the dimensions of glass monoliths on the loading and sustained release of different drugs, including anastrozole, xylazine, imiquimod, levetiracetam, and flunixin. Through multilinear regression, we calculated the influence of different parameters on drug loading and diffusion coefficients. The systematic variation of the mesoporous glass properties revealed pore volumes and drug loading concentrations, but not pore diameter or pore surface area as important parameters of drug loading and release kinetics. Other relevant effectors include the occurrence of lateral diffusion within the carrier and drug-specific properties such as adsorption. The structure-property relationships derived from our data will allow further fine-tuning of the systems according to their desired properties as TDDS, thus guiding towards optimal systems for their use in transdermal drug applications.

Targeted Transposition of Minicircle DNA Using Single-Chain Antibody Conjugated Cyclodextrin-Modified Poly (Propylene Imine) Nanocarriers.

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise ß-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

Impact of medication on blood transcriptome reveals off-target regulations of beta-blockers.

BACKGROUND: For many drugs, mechanisms of action with regard to desired effects and/or unwanted side effects are only incompletely understood. To investigate possible pleiotropic effects and respective molecular mechanisms, we describe here a catalogue of commonly used drugs and their impact on the blood transcriptome. METHODS AND RESULTS: From a population-based cohort in Germany (LIFE-Adult), we collected genome-wide gene-expression data in whole blood using in Illumina HT12v4 micro-arrays (n = 3,378; 19,974 gene expression probes per individual). Expression profiles were correlated with the intake of active substances as assessed by participants' medication. This resulted in a catalogue of fourteen substances that were identified as associated with differential gene expression for a total of 534 genes. As an independent replication cohort, an observational study of patients with suspected or confirmed stable coronary artery disease (CAD) or myocardial infarction (LIFE-Heart, n = 3,008, 19,966 gene expression probes per individual) was employed. Notably, we were able to replicate differential gene expression for three active substances affecting 80 genes in peripheral blood mononuclear cells (carvedilol: 25; prednisolone: 17; timolol: 38). Additionally, using gene ontology enrichment analysis, we demonstrated for timolol a significant enrichment in 23 pathways, 19 of them including either GPER1 or PDE4B. In the case of carvedilol, we showed that, beside genes with well-established association with hypertension (GPER1, PDE4B and TNFAIP3), the drug also affects genes that are only indirectly linked to hypertension due to their effects on artery walls or their role in lipid biosynthesis. CONCLUSIONS: Our developed catalogue of blood gene expressions profiles affected by medication can be used to support both, drug repurposing and the identification of possible off-target effects.

Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2.

In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. 'host shutoff factor') as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.

Amphiphilic Anionic Oligomer-Stabilized Calcium Phosphate Nanoparticles with Prospects in siRNA Delivery via Convection-Enhanced Delivery.

Convection-enhanced delivery (CED) has been introduced as a concept in cancer treatment to generate high local concentrations of anticancer therapeutics and overcome the limited diffusional distribution, e.g., in the brain. RNA interference provides interesting therapeutic options to fight cancer cells but requires nanoparticulate (NP) carriers with a size below 100 nm as well as a low zeta potential for CED application. In this study, we investigated calcium phosphate NPs (CaP-NPs) as siRNA carriers for CED application. Since CaP-NPs tend to aggregate, we introduced a new terpolymer (o14PEGMA(1:1:2.5) NH3) for stabilization of CaP-NPs intended for delivery of siRNA to brain cancer cells. This small terpolymer provides PEG chains for steric stabilization, and a fat alcohol to improve interfacial activity, as well as maleic anhydrides that allow for both labeling and high affinity to Ca(II) in the hydrolyzed state. In a systematic approach, we varied the Ca/P ratio as well as the terpolymer concentration and successfully stabilized NPs with the desired properties. Labeling of the terpolymer with the fluorescent dye Cy5 revealed the terpolymer's high affinity to CaP. Importantly, we also determined a high efficiency of siRNA binding to the NPs that caused very effective survivin siRNA silencing in F98 rat brain cancer cells. Cytotoxicity investigations with a standard cell line resulted in minor and transient effects; no adverse effects were observed in organotypic brain slice cultures. However, more specific cytotoxicity investigations are required. This study provides a systematic and mechanistic analysis characterizing the effects of the first oligomer of a new class of stabilizers for siRNA-loaded CaP-NPs.

STAT3 Enhances Sensitivity of Glioblastoma to Drug-Induced Autophagy-Dependent Cell Death.

Glioblastoma (GBM) is a devastating disease and the most common primary brain malignancy of adults with a median survival barely exceeding one year. Recent findings suggest that the antipsychotic drug pimozide triggers an autophagy-dependent, lysosomal type of cell death in GBM cells with possible implications for GBM therapy. One oncoprotein that is often overactivated in these tumors and associated with a particularly dismal prognosis is Signal Transducer and Activator of Transcription 3 (STAT3). Here, we used isogenic human and murine GBM knockout cell lines, advanced fluorescence microscopy, transcriptomic analysis and FACS-based assessment of cell viability to show that STAT3 has an underappreciated, context-dependent role in drug-induced cell death. Specifically, we demonstrate that depletion of STAT3 significantly enhances cell survival after treatment with Pimozide, suggesting that STAT3 confers a particular vulnerability to GBM. Furthermore, we show that active STAT3 has no major influence on the early steps of the autophagy pathway, but exacerbates drug-induced lysosomal membrane permeabilization (LMP) and release of cathepsins into the cytosol. Collectively, our findings support the concept of exploiting the pro-death functions of autophagy and LMP for GBM therapy and to further determine whether STAT3 can be employed as a treatment predictor for highly apoptosis-resistant, but autophagy-proficient cancers.