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.

The noncoding RNA LINC00152 conveys contradicting effects in different glioblastoma cells.

Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor, characterized by its high genetic heterogeneity. In search of novel putative therapeutic RNA targets we investigated the role of the oncogenic long noncoding RNA LINC00152 (CYTOR, and STAiR18) in A172 glioblastoma cells. Here, we are the first to describe, that LINC00152 unexpectedly acts in a tumor suppressive manner in this cell line. SiRNA-based knockdown of LINC00152 enhanced malignant tumor behaviors including proliferation, cell cycle entry, migration, and invasion, contradicting previous studies using U87-MG and LN229 glioblastoma cells. Furthermore, LINC00152 knockdown had no influence on survival of A172 glioblastoma cells. In a genome wide transcription analysis of A172 and U87-MG glioblastoma cells, we identified 70 LINC00152 target genes involved in locomotion, cell migration, and motility in A172 cells, whereas in U87-MG cells only 40 target genes were detected. The LINC00152-regulated genes found in A172 differed from those identified in U87-MG glioblastoma cells, none of them being regulated in both cell lines. These findings underline the strong genetic heterogeneity of glioblastoma and point to a potential, yet unknown risk addressing LINC00152 lncRNA as a prospective therapeutic target in GBM.

ProstaTrend-A Multivariable Prognostic RNA Expression Score for Aggressive Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is the most prevalent solid cancer among men in Western Countries. The clinical behavior of localized PCa is highly variable. Some cancers are aggressive leading to death, while others can even be monitored safely. Hence, there is a high clinical need for precise biomarkers for identification of aggressive disease in addition to established clinical parameters. OBJECTIVE: To develop an RNA expression-based score for the prediction of PCa prognosis that facilitates clinical decision making. DESIGN, SETTING, AND PARTICIPANTS: We assessed 233 tissue specimens of PCa patients with long-term follow-up data from fresh-frozen radical prostatectomies (RPs), from formalin-fixed and paraffin-embedded RP specimens and biopsies by transcriptome-wide next-generation sequencing and customized expression microarrays. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We applied Cox proportional hazard models to the cohorts from different platforms and specimen types. Evidence from these models was combined by fixed-effect meta-analysis to identify genes predictive of the time to death of disease (DoD). Genes were combined by a weighted median approach into a prognostic score called ProstaTrend and transferred for the prediction of biochemical recurrence (BCR) after RP in an independent cohort of The Cancer Genome Atlas (TCGA). RESULTS AND LIMITATIONS: ProstaTrend comprising ∼1400 genes was significantly associated with DoD in the training cohort of PCa patients treated by RP (leave-one-out cross-validation, Cox regression: p=2e-09) and with BCR in the TCGA validation cohort (Cox regression: p=3e-06). The prognostic impact persisted after multivariable Cox regression analysis adjusting for Gleason grading group (GG) ≥3 and resection status (p=0.001; DoD, training cohort) and for GG≥3, pathological stage ≥T3, and resection state (p=0.037; BCR, validation cohort). CONCLUSIONS: ProstaTrend is a transcriptome-based score that predicts DoD and BCR in cohorts of PCa patients treated with RP. PATIENT SUMMARY: ProstaTrend provides molecular patient risk stratification after radical prostatectomy.

The Role of lncRNAs TAPIR-1 and -2 as Diagnostic Markers and Potential Therapeutic Targets in Prostate Cancer.

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa.

Master and servant: LINC00152 - a STAT3-induced long noncoding RNA regulates STAT3 in a positive feedback in human multiple myeloma.

BACKGROUND: The survival of INA-6 human multiple myeloma cells is strictly dependent upon the Interleukin-6-activated transcription factor STAT3. Although transcriptional analyses have revealed many genes regulated by STAT3, to date no protein-coding STAT3 target gene is known to mediate survival in INA-6 cells. Therefore, the aim here was to identify and analyze non-protein-coding STAT3 target genes. In addition to the oncogenic microRNA-21, we previously described five long noncoding RNAs (lncRNAs) induced by STAT3, named STAiRs. Here, we focus on STAT3-induced RNA 18 (STAiR18), an mRNA-like, long ncRNA that is duplicated in the human lineage. One STAiR18 locus is annotated as the already well described LINC00152/CYTOR, however, the other harbors the MIR4435-2HG gene and is, up to now, barely described. METHODS: CAPTURE-RNA-sequencing was used to analyze STAiR18 transcript architecture. To identify the STAiR18 and STAT3 phenotype, siRNA-based knockdowns were performed and microarrays were applied to identify their target genes. RNA-binding partners of STAiR18 were determined by Chromatin-Isolation-by-RNA-Purification (ChIRP) and subsequent sequencing. STAT3 expression in dependence of STAiR18 was investigated by immunoblots, chromatin- and RNA-immunoprecipitations. RESULTS: As identified by CAPTURE-RNA sequencing, a complex splice pattern originates from both STAiR18 loci, generating different transcripts. Knockdown of the most abundant STAiR18 isoforms dramatically decreased INA-6 cell vitality, suggesting a functional role in myeloma cells. Additionally, STAiR18 and STAT3 knockdowns yielded overlapping changes of transcription patterns in INA-6 cells, suggesting a close functional interplay between the two factors. Moreover, Chromatin isolation by RNA purification (ChIRP), followed by genome-wide RNA sequencing showed that STAiR18 associates specifically with the STAT3 primary transcript. Furthermore, the knockdown of STAiR18 reduced STAT3 levels on both the RNA and protein levels, suggesting a positive feedback between both molecules. Furthermore, STAiR18 knockdown changes the histone methylation status of the STAT3 locus, which explains the positive feedback and indicates that STAiR18 is an epigenetic modulator. CONCLUSION: Hence, STAiR18 is an important regulator of myeloma cell survival and is strongly associated with the oncogenic function of STAT3. The close functional interplay between STAT3 and STAiR18 suggests a novel principle of regulatory interactions between long ncRNAs and signaling pathways.

Quantification of Multiple Bacteria in Calcified Structural Valvular Heart Disease.

Genome studies of heart valve tissue (HVT) in patients with structural valvular heart disease (sVHD) and acute infective endocarditis (aIE) showed polymicrobial infections. Subject of this study is the quantification of bacterial DNA in HVT of sVHD in comparison to aIE. It will be examined whether the bacterial DNA concentration can be used as surrogate marker to differentiate chronic and acute infections. DNA was isolated from HVT of 100 patients with sVHD and 23 microbiologically positively tested patients with aIE. Selected pathogens (Cutibacterium acnes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium difficile, and Klebsiella pneumoniae) were quantified using TaqMan-qPCR. Polymicrobial infiltration of HVT was investigated by immunohistologic methods. Of 100 sVHD patients, 94 tested positive for bacteria by 16S-rDNA and 72 by TaqMan-qPCR. In 29% of the sVHD cohort and in 70% of the aIE cohort, a coinfection with more than 2 bacteria was observed as indication of a polymicrobial infection. The most common pathogens in the sVHD patients were C. acnes (59%; 5-4074 pg/mL), E. faecalis (16%, 174-2781 pg/mL), and S. aureus (15%, 8-105 pg/mL). The DNA concentration of E. faecalis (P = 0.0285) and S. aureus (P = 0.0149) is significantly lower in the sVHD cohort than in the aIE cohort. sVHD is associated with bacterial infection and infiltration of the HVT in a majority of cases. TaqMan-qPCR is a valid instrument for the specific detection of bacteria in HVT and allows discrimination between sVHD and aIE for E. faecalis and S. aureus.

Bacterial infiltration in structural heart valve disease.

OBJECTIVES: The pathology of structural valvular heart disease (sVHD) ranges from basic diseases of rheumatologic origin to chronic degenerative remodeling processes after acute bacterial infections. Molecular genetic methods allow detection of the complete microbial spectrum in heart valve tissues independent of microbiological cultivation. In particular, whole-metagenome analysis is a sensitive and highly specific analytical method that allows a deeper insight into the pathogenicity of the diseases. In the present study we assessed the pathogen spectrum in heart valve tissue from 25 sVHD patients using molecular and microbiological methods. METHODS: Twenty-five sVHD patients were selected randomly from an observational cohort study (March 2016 to January 2017). The explanted native heart valves were examined using microbiological methods and immunohistological structural analysis. In addition, the bacterial metagenome of the heart valve tissue was determined using next-generation sequencing. RESULTS: The use of sonication as a pretreatment of valve tissue from 4 sVHD patients permitted successful detection of Clostridium difficile, Enterococcus faecalis, Staphylococcus saccharolyticus, and Staphylococcus haemolyticus using microbial cultivation. Histological staining revealed intramural localization. Metagenome analysis identified a higher rate of bacterial infiltration in 52% of cases. The pathogen spectrum included both gram-positive and gram-negative bacteria. CONCLUSIONS: Microbiological and molecular biological studies are necessary to detect the spectrum of bacteria in a calcified heart valve. Metagenome analysis is a valid method to gain new insight into the polymicrobial pathophysiology of sVHD. Our results suggest that an undetected proportion of sVHD might be triggered by chronic inflammation or influenced by secondary bacterial infiltration.

Gastric mucosal devitalization improves blood pressure, renin and cardiovascular lipid deposition in a rat model of obesity.

Background and study aims In lieu of the drawbacks of metabolic surgery, a method of mimicking resection of the gastric mucosa could be of value to those with obesity-related cardiovascular disease (CVD). Our study aims to investigate the effect of gastric mucosal devitalization (GMD) on blood pressure (BP) and cardiovascular lipid deposition in a rat model of obesity. Methods GMD of 70 % of the stomach was achieved by argon plasma coagulation. GMD was compared to sleeve gastrectomy (SG) and sham (SH) in a high-fat-diet-induced rat model of obesity (48 rats). At 8 weeks, we measured noninvasive BP, renin, vessel relaxation and ghrelin receptor regulation in the aorta. In addition, we quantified cardiac lipid deposition and lipid droplet deposition in cardiac muscle and aorta. Results GMD and SG were observed to have similar reductions in body weight, visceral adiposity, and serum lipid profile compared to SH rats. GMD resulted in a significant reduction in arterial BP compared to SH. Furthermore, there were significant reductions in plasma renin activity and percentage of phenylnephrine constriction to acetylcholine at the aortic ring in GMD rats compared to SH, providing insights into the mechanisms behind the reduced BP. Interestingly, the reduced BP occurred despite a reduction in endothelial ghrelin recteptor activation. Cardiac lipid content was significantly reduced in GMD rats. Lipid deposition, as illustrated by Nile Red stain, was reduced in cardiac muscle and the aorta. Conclusion GMD resulted in a significant improvement in BP, renin and cardiovascular lipid deposition. GMD deserves further attention as a method of treating obesity-related CVD.

p53 activates the PANK1/miRNA-107 gene leading to downregulation of CDK6 and p130 cell cycle proteins.

The tumor suppressor p53 is a central regulator of cell-cycle arrest and apoptosis by acting as a transcription factor to regulate numerous genes. We identified all human p53-regulated mRNAs by microarray analyses and searched for protein-coding genes which contain intronic miRNAs. Among others, this analysis yielded the panthothenate kinase 1 (PANK1) gene and its intronic miRNA-107. We showed that miRNA-107 and PANK1 are coregulated by p53 in different cell systems. The PANK1 protein, which catalyzes the rate-limiting step of coenzyme A biosynthesis, is also upregulated by p53. We observed that p53 directly activates PANK1 and miRNA-107 transcription through a binding site in the PANK1 promoter. Furthermore, p53 is recruited to the PANK1 promoter after DNA damage. In order to get more insight into miRNA-107 function we investigated its potential target genes. Cell-cycle regulators are significantly enriched among predicted miRNA-107 targets. We found miRNA-107-dependent regulation of two important regulators of G(1)/S progression, CDK6 and the RB-related 2 gene RBL2 (p130). CDK6 and p130 proteins are downregulated upon miRNA-107 expression. Our results uncover a novel miRNA-dependent signaling pathway which leads to downregulation of cell cycle proteins in the absence of transcriptional repression.

Expression of toxin-related human mono-ADP-ribosyltransferase 3 in human testes.

AIM: To investigate wether the corresponding protein of mono-ADP-ribosyltransferase 3 (ART3) mRNA is expressed in human testes and, if so, whether the expression is cell type-specific. METHODS: ART3 mRNA was determined in human testes and sperm by reverse transcription-polymerase chain reaction (RT-PCR). The glycosyl-phosphatidylinositol linkage of ART3 was shown by treating ART3-transfected HEK-293-T cells with phospholipase C. Fluorescent activated cell sorter (FACS)-analyses were used to detect ART3 on mature spermatozoa and immunohistological studies to detect the protein in testes. RESULTS: ART3 protein was shown to be present in testes. It was found on spermatocytes only. It was absent from spermatogonia, spermatids and spermatozoa. The absence of ART3 from spermatozoa was confirmed by FACS-analysis. ART3 protein was detected neither within a seminoma nor on Leydig cells. CONCLUSION: Here we show for the first time that ART3 protein is expressed in testes in particular on spermatocytes, indicating that ART3 exerts a specific function only required at a particular stage of spermatogenesis.

Analysis of mono-ADP-ribosyltransferase 4 gene expression in human monocytes: splicing pattern and potential regulatory elements.

Mono-ADP-ribosyltransferase (ART) 4 belongs to a family of ectoenzymes that catalyze the transfer of ADP-ribose from NAD+ to a target protein. ART4 could be detected on HEL cells and erythrocytes by FACS analysis while it was absent from activated monocytes, despite the presence of ART4 mRNA in these cells. The predicted glycosylphosphatidylinositol (GPI) linkage of ART4 could be verified by showing that treatment of erythrocytes, HEL cells and ART4-transfected HEK-293-T cells with phosphatidylinositol-specific phospholipase C results in a decrease in ART4 expression. Furthermore, an ART4 construct carrying an Ala285Val mutation that is critical for the formation of a GPI anchor failed to be expressed in transfected C-33A cells. Analysis of the gene structure revealed that the first of the three exons was at least 236 bp longer than previously published and that splicing occurred in the coding region of the mRNA from HEL cells and monocytes. When carrying out 5' inverse RACE-PCR we confirmed the existence of 5 ATGs in the 5' untranslated region (5'UTR). By deletion and site-directed mutagenesis of the ATGs, we showed that the first two ATGs impair translation and that both the 3rd and 5th ATG can be used for translation initiation after expression in C-33A cells. On analysis of the 3'UTR, which contains 2 adenylate/uridylate-rich elements (AREs), we detected one variant in monocytes that would be devoid of a GPI-anchor signal and thus could represent a secreted form of ART4. Thus, alternative splicing and the use of regulatory elements in the 5'UTR and 3'UTR represent means to control ART4 expression.

Mono-ADP-ribosyltransferases in human monocytes: regulation by lipopolysaccharide.

ADP-ribosyltransferase activity was shown to be present on the surface of human monocytes. Incubating the cells in the presence of BSA leads to an increase in enzyme activity. The acceptor amino acid mainly responsible for the ADP-ribose bond was identified as a cysteine residue. An increase in ADP-ribosyltransferase activity was observed when cells were treated for 16 h with bacterial lipopolysaccharide (LPS). Possible candidates for catalysing the reaction are mono-ADP-ribosyltransferases (ARTs). When measuring expression of the mRNA of ART1, 3, 4 and 5, only ART3 mRNA was detected in unstimulated monocytes. Upon stimulation for 16 h with LPS, lipoteichoic acid or peptidoglycan, ART4 mRNA was found to be expressed. No ART4 signal appeared after a 4 h exposure of the cells to LPS. Cell-surface proteins were labelled when incubating monocytes with [(32)P]NAD(+). Their molecular masses were 29, 33, 43, 45, 60 and 82 kDa. In response to LPS an additional protein of 31 kDa was found to be labelled. The bound label was resistant to treatment with NH(2)OH but sensitive to HgCl(2), characteristic of a cysteine-linked ADP-ribosylation.