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.

Establishing a strategy for sequencing of T cell–redirecting therapies for relapsed/refractory multiple myeloma (RRMM) is a pressing clinical need. We longitudinally tracked the clinical and immunologic impact of bispecific T cell–engaging antibodies (BsAb) as bridging therapy (BT) to subsequent B-cell maturation antigen–directed chimeric antigen receptor T (CAR-T) cell therapies in 52 patients with RRMM. BsAbs were a potent and safe option for BT, achieving the highest overall response rate (100%) to BT compared with chemotherapy, anti-CD38, or anti-SLAMF7 antibody–based regimens (46%). We observed early CD4+CAR+ and delayed CD8+CAR+ T-cell expansion in patients receiving BsAbs as BT. In vitro cytotoxicity of CAR-T cells was comparable among BT options. Single-cell analyses revealed increased clonality in the CD4+ and CD8+ T-cell compartments in patients with previous exposure to BsAbs at leukapheresis and on day 30 after CAR-T cell infusion. This study demonstrates the feasibility and efficacy of BT with BsAbs for CAR-T cell therapy in RRMM. Significance: CAR-T cell therapy and BsAbs have revolutionized treatment of triple-class refractory multiple myeloma; however, optimal sequencing is unknown. We demonstrate that BT with BsAb before B-cell maturation antigen–directed CAR-T cell therapy is safe and effective, which might have implications for other hematologic malignancies as well. See related commentary by Bal and Costa, p. 10

Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in fish. IL-15L genes with intact open reading frames (ORFs) and evidence of transcription, and a recent past of purifying selection, were found for cattle, horse, sheep, pig and rabbit. In human and mouse the IL-15L ORF is incapacitated. Although deduced IL-15L proteins share only ~21 % overall amino acid identity with IL-15, they share many of the IL-15 residues important for binding to receptor chain IL-15Rα, and recombinant bovine IL-15L was shown to interact with IL-15Rα indeed. Comparison of sequence motifs indicates that capacity for binding IL-15Rα is an ancestral characteristic of the IL-2/15/15L family, in accordance with a recent study which showed that in fish both IL-2 and IL-15 can bind IL-15Rα. Evidence reveals that the species lineage leading to mammals started out with three similar cytokines IL-2, IL-15 and IL-15L, and that later in evolution (1) IL-2 and IL-2Rα receptor chain acquired a new and specific binding mode and (2) IL-15L was lost in several but not all groups of mammals. The present study forms an important step forward in understanding this potent family of cytokines, and may help to improve future strategies for their application in veterinarian and human medicine.

In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE. Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM). Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified. The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may result in improved outcomes.

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.

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.