Fast Track Adaptation of Oncolytic Coxsackie B3 Virus to Resistant Colorectal Cancer Cells - a Method to Personalize Virotherapy.

BACKGROUND: The efficacy of oncolytic viruses (OV) in cancer treatment depends on their ability to successfully infect and destroy tumor cells. However, patients' tumors vary, and in the case of individual insensitivity to an OV, therapeutic efficacy is limited. Here, we present a protocol for rapid generation of tumor cell-specific adapted oncolytic coxsackievirus B3 (CVB3) with enhanced oncolytic potential and a satisfactory safety profile. This is achieved by combining directed viral evolution (DVE) with genetic modification of the viral genome and the use of a microRNA-dependent regulatory tool. METHODS: The oncolytic CVB3 variant PD-H was adapted to the refractory colorectal carcinoma cell line Colo320 through serial passaging. XTT assays and virus plaque assays were used to determine virus cytotoxicity and virus replication in vitro. Recombinant PD-H variants were generated through virus mutagenesis. Apoptosis was detected by Western blots, Caspase 3/7 assays, and DAPI staining. The therapeutic efficacy and safety of the adapted recombinant OV PD-SK-375TS were assessed in vivo using a subcutaneous Colo320 xenograft mouse model. RESULTS: PD-H was adapted to the colorectal cancer cell line Colo320 within 10 passages. Sequencing of passage 10 virus P-10 revealed a heterogenous virus population with five nucleotide mutations resulting in amino acid substitutions. The genotypically homogeneous OV PD-SK was generated by inserting the five detected mutations of P-10 into the genome of PD-H. PD-SK showed significantly stronger replication and cytotoxicity than PD-H in Colo320 cells, but not in other colorectal carcinoma cell lines. Increase of apoptosis induction was detected as key mechanisms of Colo320 cell-specific adaptation of PD-SK. For in vivo safety PD-SK was engineered with target sites of the miR-375 (miR-375TS) to exclude virus replication in normal tissues. PD-SK-375TS, unlike the PD-H-375TS not adapted homolog suppressed the growth of subcutaneous Colo320 tumors in nude mice without causing any side effects. CONCLUSION: Taken together, here we present an optimized protocol for the rapid generation of tumor cell-specific adapted oncolytic CVB3 based on the oncolytic CVB3 strain PD-H. The protocol is promising for the generation of personalized OV for tumor therapy and has the potential to be applied to other OV.

Combination of FOLFOXIRI Drugs with Oncolytic Coxsackie B3 Virus PD-H Synergistically Induces Oncolysis in the Refractory Colorectal Cancer Cell Line Colo320.

FOLFOXIRI chemotherapy is a first-line therapy for advanced or metastatic colorectal cancer (CRC), yet its therapeutic efficacy remains limited. Immunostimulatory therapies like oncolytic viruses can complement chemotherapies by fostering the infiltration of the tumor by immune cells and enhancing drug cytotoxicity. In this study, we explored the effect of combining the FOLFOXIRI chemotherapeutic agents with the oncolytic coxsackievirus B3 (CVB3) PD-H in the CRC cell line Colo320. Additionally, we examined the impact of the drugs on the expression of microRNAs (miRs), which could be used to increase the safety of oncolytic CVB3 containing corresponding miR target sites (miR-TS). The measurement of cytotoxic activity using the Chou-Talalay combination index approach revealed that PD-H synergistically enhanced the cytotoxic activity of oxaliplatin (OX), 5-fluorouracil (5-FU) and SN-38. PD-H replication was not affected by OX and SN-38 but inhibited by high concentrations of 5-FU. MiR expression levels were not or only slightly elevated by the drugs or with drug/PD-H combinations on Colo320 cells. Moreover, the drug treatment did not increase the mutation rate of the miR-TS inserted into the PD-H genome. The results demonstrate that the combination of FOLFOXIRI drugs and PD-H may be a promising approach to enhance the therapeutic effect of FOLFOXIRI therapy in CRC.

Xeno-Free 3D Bioprinted Liver Model for Hepatotoxicity Assessment.

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85-92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86-92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97-101%), akin to a Matrigel-based liver model (83-102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Biocatalytic Nucleobase Diversification of 4'-Thionucleosides and Application of Derived 5-Ethynyl-4'-thiouridine for RNA synthesis detection.

Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4'-thiouridine in a one-pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4'-thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5-iodo-4'-thiouridine on multi-milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5-ethynyl-4'-thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Generation of a Perfusable 3D Lung Cancer Model by Digital Light Processing.

Lung cancer still has one of the highest morbidity and mortality rates among all types of cancer. Its incidence continues to increase, especially in developing countries. Although the medical field has witnessed the development of targeted therapies, new treatment options need to be developed urgently. For the discovery of new drugs, human cancer models are required to study drug efficiency in a relevant setting. Here, we report the generation of a non-small cell lung cancer model with a perfusion system. The bioprinted model was produced by digital light processing (DLP). This technique has the advantage of including simulated human blood vessels, and its simple assembly and maintenance allow for easy testing of drug candidates. In a proof-of-concept study, we applied gemcitabine and determined the IC50 values in the 3D models and 2D monolayer cultures and compared the response of the model under static and dynamic cultivation by perfusion. As the drug must penetrate the hydrogel to reach the cells, the IC50 value was three orders of magnitude higher for bioprinted constructs than for 2D cell cultures. Compared to static cultivation, the viability of cells in the bioprinted 3D model was significantly increased by approximately 60% in the perfusion system. Dynamic cultivation also enhanced the cytotoxicity of the tested drug, and the drug-mediated apoptosis was increased with a fourfold higher fraction of cells with a signal for the apoptosis marker caspase-3 and a sixfold higher fraction of cells positive for PARP-1. Altogether, this easily reproducible cancer model can be used for initial testing of the cytotoxicity of new anticancer substances. For subsequent in-depth characterization of candidate drugs, further improvements will be necessary, such as the generation of a multi-cell type lung cancer model and the lining of vascular structures with endothelial cells.

Evaluating the Suitability of 3D Bioprinted Samples for Experimental Radiotherapy: A Pilot Study.

Radiotherapy is an important component in the treatment of lung cancer, one of the most common cancers worldwide, frequently resulting in death within only a few years of diagnosis. In order to evaluate new therapeutic approaches and compare their efficiency with regard to tumour control at a pre-clinical stage, it is important to develop standardized samples which can serve as inter-institutional outcome controls, independent of differences in local technical parameters or specific techniques. Recent developments in 3D bioprinting techniques could provide a sophisticated solution to this challenge. We have conducted a pilot project to evaluate the suitability of standardized samples generated from 3D printed human lung cancer cells in radiotherapy studies. The samples were irradiated at high dose rates using both broad beam and microbeam techniques. We found the 3D printed constructs to be sufficiently mechanically stable for use in microbeam studies with peak doses up to 400 Gy to test for cytotoxicity, DNA damage, and cancer cell death in vitro. The results of this study show how 3D structures generated from human lung cancer cells in an additive printing process can be used to study the effects of radiotherapy in a standardized manner.

Bioprinted Cancer Model of Neuroblastoma in a Renal Microenvironment as an Efficiently Applicable Drug Testing Platform.

Development of new anticancer drugs with currently available animal models is hampered by the fact that human cancer cells are embedded in an animal-derived environment. Neuroblastoma is the most common extracranial solid malignancy of childhood. Major obstacles include managing chemotherapy-resistant relapses and resistance to induction therapy, leading to early death in very-high-risk patients. Here, we present a three-dimensional (3D) model for neuroblastoma composed of IMR-32 cells with amplified genes of the myelocytomatosis viral related oncogene MYCN and the anaplastic lymphoma kinase (ALK) in a renal environment of exclusively human origin, made of human embryonic kidney 293 cells and primary human kidney fibroblasts. The model was produced with two pneumatic extrusion printheads using a commercially available bioprinter. Two drugs were exemplarily tested in this model: While the histone deacetylase inhibitor panobinostat selectively killed the cancer cells by apoptosis induction but did not affect renal cells in the therapeutically effective concentration range, the peptidyl nucleoside antibiotic blasticidin induced cell death in both cell types. Importantly, differences in sensitivity between two-dimensional (2D) and 3D cultures were cell-type specific, making the therapeutic window broader in the bioprinted model and demonstrating the value of studying anticancer drugs in human 3D models. Altogether, this cancer model allows testing cytotoxicity and tumor selectivity of new anticancer drugs, and the open scaffold design enables the free exchange of tumor and microenvironment by any cell type.

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations.

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.

Deciphering the Enigmatic Biological Functions of RNA Guanine-Quadruplex Motifs in Human Cells.

Guanine-rich sequences in nucleic acids can form noncanonical structures known as guanine quadruplexes (G-quadruplexes), which constitute a not yet fully elucidated layer of regulatory function for central cellular processes. RNA G-quadruplexes have been shown to be involved in the modulation of translation, the regulation of (alternative) splicing, and the subcellular transport of mRNAs, among other processes. However, in living cells, an equilibrium between the formation of G-quadruplex structures and their unwinding by RNA helicases is likely. The extent to which G-rich sequences adopt G-quadruplex structures in living eukaryotic cells is currently a matter of debate. Multiple lines of evidence confirm the intracellular formation of G-quadruplex structures, such as their detection by immunochemical approaches, fluorogenic probes, and in vivo nuclear magnetic resonance. However, intracellular chemical probing suggests most if not all are in an unfolded state. It is therefore tempting to speculate that some G-quadruplex structures are only temporarily formed when they are required to contribute to the fine-tuning of the processes mentioned above. Future research should focus on the analysis of G-quadruplex formation under physiological conditions, which will allow the re-evaluation of the biological function of G-quadruplex motifs in regulatory processes in their natural environment and at physiological expression levels. This will help in the elucidation of their significance in the regulation of central processes in molecular biology and the exploitation of their potential as therapeutic targets.

RNAi-mediated knockdown of the Rhau helicase preferentially depletes proteins with a Guanine-quadruplex motif in the 5'-UTR of their mRNA.

Guanine-quadruplex (G-quadruplex) structures in mRNAs have been shown to modulate gene expression. However, the overall biological relevance of this process is under debate, as cellular helicases unwind G-quadruplex structures. The helicase Rhau (encoded by the DHX36 gene) was reported to be the major source of RNA G-quadruplex resolving activity in lysates of human cells. In the current study, we depleted Rhau by RNAi-mediated silencing and analyzed the effect on proteins whose mRNAs harbor a G-quadruplex motif in their 5'-UTRs. A targeted investigation of the proto-oncogenes Bcl-2 and NRAS, which are well-known examples for the translational repression of G-quadruplex structures, did not reveal effects caused by Rhau silencing. We therefore carried out a global analysis of changes in protein levels by label-free quantification using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Following Rhau knockdown, of all the identified proteins, only 1.9% were significantly downregulated to at least 70%. According to a bioinformatic analysis with the QGRS mapper, 33% of the downregulated proteins were predicted to harbor a G-quadruplex motif in the 5'-UTR of their respective mRNAs, compared to only 11% in the complete dataset. This indicates that in an unexpectedly small set of genes, in which G-quadruplex motifs are unusually common in the 5'-UTR of their mRNAs, Rhau helicase is responsible for the regulation of their expression.

3D organ models-Revolution in pharmacological research?

3D organ models have gained increasing attention as novel preclinical test systems and alternatives to animal testing. Over the years, many excellent in vitro tissue models have been developed. In parallel, microfluidic organ-on-a-chip tissue cultures have gained increasing interest for their ability to house several organ models on a single device and interlink these within a human-like environment. In contrast to these advancements, the development of human disease models is still in its infancy. Although major advances have recently been made, efforts still need to be intensified. Human disease models have proven valuable for their ability to closely mimic disease patterns in vitro, permitting the study of pathophysiological features and new treatment options. Although animal studies remain the gold standard for preclinical testing, they have major drawbacks such as high cost and ongoing controversy over their predictive value for several human conditions. Moreover, there is growing political and social pressure to develop alternatives to animal models, clearly promoting the search for valid, cost-efficient and easy-to-handle systems lacking interspecies-related differences. In this review, we discuss the current state of the art regarding 3D organ as well as the opportunities, limitations and future implications of their use.

Alternatively spliced variants of the 5'-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif.

The 5'-UTR of the actin-related protein 2/3 complex subunit 2 (ARPC2) mRNA exists in two variants. Using a bicistronic reporter construct, the present study demonstrates that the longer variant of the 5'-UTR harbours an internal ribosome entry site (IRES) which is lacking in the shorter one. Multiple control assays confirmed that only this variant promotes cap-independent translation. Furthermore, it includes a guanine-rich region that is capable of forming a guanine-quadruplex (G-quadruplex) structure which was found to contribute to the IRES activity. To investigate the cellular function of the IRES element, we determined the expression level of ARPC2 at various cell densities. At high cell density, the relative ARPC2 protein level increases, supporting the presumed function of IRES elements in driving the expression of certain genes under stressful conditions that compromise cap-dependent translation. Based on chemical probing experiments and computer-based predictions, we propose a structural model of the IRES element, which includes the G-quadruplex motif exposed from the central stem-loop element. Taken together, our study describes the functional relevance of two alternative 5'-UTR splice variants of the ARPC2 mRNA, one of which contains an IRES element with a G-quadruplex as a central motif, promoting translation under stressful cellular conditions.

RNA interference-based functional knockdown of the voltage-gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus vectors.

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

Generation of a 3D Liver Model Comprising Human Extracellular Matrix in an Alginate/Gelatin-Based Bioink by Extrusion Bioprinting for Infection and Transduction Studies.

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

The Coxsackievirus and Adenovirus Receptor: Glycosylation and the Extracellular D2 Domain Are Not Required for Coxsackievirus B3 Infection.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily (IgSF) and functions as a receptor for coxsackie B viruses (CVBs). The extracellular portion of CAR comprises two glycosylated immunoglobulin-like domains, D1 and D2. CAR-D1 binds to the virus and is essential for virus infection; however, it is not known whether D2 is also important for infection, and the role of glycosylation has not been explored. To understand the function of these structural components in CAR-mediated CVB3 infection, we generated a panel of human (h) CAR deletion and substitution mutants and analyzed their functionality as CVB receptors, examining both virus binding and replication. Lack of glycosylation of the CAR-D1 or -D2 domains did not adversely affect CVB3 binding or infection, indicating that the glycosylation of CAR is not required for its receptor functions. Deletion of the D2 domain reduced CVB3 binding, with a proportionate reduction in the efficiency of virus infection. Replacement of D2 with the homologous D2 domain from chicken CAR, or with the heterologous type C2 immunoglobulin-like domain from IgSF11, another IgSF member, fully restored receptor function; however, replacement of CAR-D2 with domains from CD155 or CD80 restored function only in part. These data indicate that glycosylation of the extracellular domain of hCAR plays no role in CVB3 receptor function and that CAR-D2 is not specifically required. The D2 domain may function largely as a spacer permitting virus access to D1; however, the data may also suggest that D2 affects virus binding by influencing the conformation of D1. IMPORTANCE: An important step in virus infection is the initial interaction of the virus with its cellular receptor. Although the role in infection of the extracellular CAR-D1, cytoplasmic, and transmembrane domains have been analyzed extensively, nothing is known about the function of CAR-D2 and the extracellular glycosylation of CAR. Our data indicate that glycosylation of the extracellular CAR domain has only minor importance for the function of CAR as CVB3 receptor and that the D2 domain is not essential per se but contributes to receptor function by promoting the exposure of the D1 domain on the cell surface. These results contribute to our understanding of the coxsackievirus-receptor interactions.

Identification and characterization of RNA guanine-quadruplex binding proteins.

Guanine quadruplex (G-quadruplex) motifs in the 5' untranslated region (5'-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation.

Combination of RNA interference and virus receptor trap exerts additive antiviral activity in coxsackievirus B3-induced myocarditis in mice.

BACKGROUND: Coxsackievirus B3 (CVB3) is a major heart pathogen against which no therapy exists to date. The potential of a combination treatment consisting of a proteinaceous virus receptor trap and an RNA interference-based component to prevent CVB3-induced myocarditis was investigated. METHODS AND RESULTS: A soluble variant of the extracellular domain of the coxsackievirus-adenovirus receptor (sCAR-Fc) was expressed from an adenoviral vector and 2 short hairpin RNAs (shRdRp2.4) directed against CVB3 were delivered by an adeno-associated virus (AAV) vector. Cell culture experiments revealed additive antiviral activity of the combined application. In a CVB3-induced mouse myocarditis model, both components applied individually significantly reduced inflammation and viral load in the heart. The combination exerted an additive antiviral effect and reduced heart pathology. Hemodynamic measurement revealed that infection with CVB3 resulted in impaired heart function, as illustrated by a drastically reduced cardiac output and impaired contractility and relaxation. Treatment with either sCAR-Fc or shRdRp2.4 significantly improved these parameters. Importantly, the combination of both components led to a further significant improvement of heart function. CONCLUSIONS: Combination of sCAR-Fc and shRdRp2.4 exerted additive effects and was significantly more effective than either of the single treatments in inhibiting CVB3-induced myocarditis and preventing cardiac dysfunction.

Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.

Development of a highly stable, active small interfering RNA with broad activity against SARS-CoV viruses.

Treatment options for COVID-19 remain limited. Here, we report the optimization of an siRNA targeting the highly conserved leader region of SARS-CoV-2. The siRNA was rendered nuclease resistant by the introduction of modified nucleotides without loss of activity. Importantly, the siRNA also retained its inhibitory activity against the emerged omicron sublineage variant BA.2, which occurred after the siRNA was designed and is resistant to other antiviral agents such as antibodies. In addition, we show that a second highly active siRNA designed against the viral 5'-UTR can be applied as a rescue molecule, to minimize the spread of escape mutations. We therefore consider our siRNA-based molecules to be promising broadly active candidates for the treatment of current and future SARS-CoV-2 variants.