Mucosal IFNλ1 mRNA-based immunomodulation effectively reduces SARS-CoV-2 induced mortality in mice.

RNA vaccines elicit protective immunity against SARS-CoV-2, but the use of mRNA as an antiviral immunotherapeutic is unexplored. Here, we investigate the activity of lipidoid nanoparticle (LNP)-formulated mRNA encoding human IFNλ1 (ETH47), which is a critical driver of innate immunity at mucosal surfaces protecting from viral infections. IFNλ1 mRNA administration promotes dose-dependent protein translation, induction of interferon-stimulated genes without relevant signs of unspecific immune stimulation, and dose-dependent inhibition of SARS-CoV-2 replication in vitro. Pulmonary administration of IFNλ1 mRNA in mice results in a potent reduction of virus load, virus-induced body weight loss and significantly increased survival. These data support the development of inhaled administration of IFNλ1 mRNA as a potential prophylactic option for individuals exposed to SARS-CoV-2 or at risk suffering from COVID-19. Based on the broad antiviral activity of IFNλ1 regardless of virus or variant, this approach might also be utilized for other respiratory viral infections or pandemic preparedness.

Therapeutic Applications of Aptamers.

Affinity reagents, or target-binding molecules, are quite versatile and are major workhorses in molecular biology and medicine. Antibodies are the most famous and frequently used type and they have been used for a wide range of applications, including laboratory techniques, diagnostics, and therapeutics. However, antibodies are not the only available affinity reagents and they do have significant drawbacks, including laborious and costly production. Aptamers are one potential alternative that have a variety of unique advantages. They are single stranded DNA or RNA molecules that can be selected for binding to many targets including proteins, carbohydrates, and small molecules-for which antibodies typically have low affinity. There are also a variety of cost-effective methods for producing and modifying nucleic acids in vitro without cells, whereas antibodies typically require cells or even whole animals. While there are also significant drawbacks to using aptamers in therapeutic applications, including low in vivo stability, aptamers have had success in clinical trials for treating a variety of diseases and two aptamer-based drugs have gained FDA approval. Aptamer development is still ongoing, which could lead to additional applications of aptamer therapeutics, including antitoxins, and combinatorial approaches with nanoparticles and other nucleic acid therapeutics that could improve efficacy.

CNS Delivery of Nucleic Acid Therapeutics: Beyond the Blood-Brain Barrier and Towards Specific Cellular Targeting.

Nucleic acid-based therapeutic molecules including small interfering RNA (siRNA), microRNA(miRNA), antisense oligonucleotides (ASOs), messenger RNA (mRNA), and DNA-based gene therapy have tremendous potential for treating diseases in the central nervous system (CNS). However, achieving clinically meaningful delivery to the brain and particularly to target cells and sub-cellular compartments is typically very challenging. Mediating cell-specific delivery in the CNS would be a crucial advance that mitigates off-target effects and toxicities. In this review, we describe these challenges and provide contemporary evidence of advances in cellular and sub-cellular delivery using a variety of delivery mechanisms and alternative routes of administration, including the nose-to-brain approach. Strategies to achieve subcellular localization, endosomal escape, cytosolic bioavailability, and nuclear transfer are also discussed. Ultimately, there are still many challenges to translating these experimental strategies into effective and clinically viable approaches for treating patients.

The year in cardiovascular medicine 2021: dyslipidaemia.

The past year was an exciting time for clinical lipidology when we learnt more about existing therapies as well as therapies targeting novel pathways discovered through genetic studies. LDL cholesterol remained the main target and a variety of drugs to lower LDL cholesterol through different mechanisms were explored. Emerging evidence on the atherogenity of triglyceride-rich lipoproteins led to renewed interest in lowering them with new treatments. Lp(a) was back in focus with evidence on causality and new targeted therapeutics which dramatically lower Lp(a) levels. We will be able to personalise lipid lowering therapy further with this enriched armamentarium once we have the results of the cardiovascular outcome studies with some of these new agents.

Extracellular Vesicles for Therapeutic Nucleic Acid Delivery: Loading Strategies and Challenges.

Extracellular vesicles (EVs) are membrane vesicles released into the extracellular milieu by cells of various origins. They contain different biological cargoes, protecting them from degradation by environmental factors. There is an opinion that EVs have a number of advantages over synthetic carriers, creating new opportunities for drug delivery. In this review, we discuss the ability of EVs to function as carriers for therapeutic nucleic acids (tNAs), challenges associated with the use of such carriers in vivo, and various strategies for tNA loading into EVs.

mRNA Treatment Rescues Niemann-Pick Disease Type C1 in Patient Fibroblasts.

Messenger RNA (mRNA) holds great potential as a disease-modifying treatment for a wide array of monogenic disorders. Niemann-Pick disease type C1 (NP-C1) is an ultrarare monogenic disease that arises due to loss-of-function mutations in the NPC1 gene, resulting in the entrapment of unesterified cholesterol in the lysosomes of affected cells and a subsequent reduction in their capacity for cholesterol esterification. This causes severe damage to various organs including the brain, liver, and spleen. In this work, we describe the use of NPC1-encoded mRNA to rescue the protein insufficiency and pathogenic phenotype caused by biallelic NPC1 mutations in cultured fibroblasts derived from an NP-C1 patient. We first evaluated engineering strategies for the generation of potent mRNAs capable of eliciting high protein expression across multiple cell types. We observed that "GC3" codon optimization, coupled with N1-methylpseudouridine base modification, yielded an mRNA that was approximately 1000-fold more potent than wild-type, unmodified mRNA in a luciferase reporter assay and consistently superior to other mRNA variants. Our data suggest that the improved expression associated with this design strategy was due in large part to the increased secondary structure of the designed mRNAs. Both codon optimization and base modification appear to contribute to increased secondary structure. Applying these principles to the engineering of NPC1-encoded mRNA, we observed a normalization in NPC1 protein levels after mRNA treatment, as well as a rescue of the mutant phenotype. Specifically, mRNA treatment restored the cholesterol esterification capacity of patient cells to wild-type levels and induced a significant reduction in both unesterified cholesterol levels (>57% reduction compared to Lipofectamine-treated control in a cholesterol esterification assay) and lysosome size (157 µm2 reduction compared to Lipofectamine-treated control). These findings show that engineered mRNA can correct the deficit caused by NPC1 mutations. More broadly, they also serve to further validate the potential of this technology to correct diseases associated with loss-of-function mutations in genes coding for large, complex, intracellular proteins.

In Vitro Validation of the Therapeutic Potential of Dendrimer-Based Nanoformulations against Tumor Stem Cells.

Tumor cells with stem cell properties are considered to play major roles in promoting the development and malignant behavior of aggressive cancers. Therapeutic strategies that efficiently eradicate such tumor stem cells are of highest clinical need. Herein, we performed the validation of the polycationic phosphorus dendrimer-based approach for small interfering RNAs delivery in in vitro stem-like cells as models. As a therapeutic target, we chose Lyn, a member of the Src family kinases as an example of a prominent enzyme class widely discussed as a potent anti-cancer intervention point. Our selection is guided by our discovery that Lyn mRNA expression level in glioma, a class of brain tumors, possesses significant negative clinical predictive value, promoting its potential as a therapeutic target for future molecular-targeted treatments. We then showed that anti-Lyn siRNA, delivered into Lyn-expressing glioma cell model reduces the cell viability, a fact that was not observed in a cell model that lacks Lyn-expression. Furthermore, we have found that the dendrimer itself influences various parameters of the cells such as the expression of surface markers PD-L1, TIM-3 and CD47, targets for immune recognition and other biological processes suggested to be regulating glioblastoma cell invasion. Our findings prove the potential of dendrimer-based platforms for therapeutic applications, which might help to eradicate the population of cancer cells with augmented chemotherapy resistance. Moreover, the results further promote our functional stem cell technology as suitable component in early stage drug development.

Combined use of chemically modified nucleobases and nanostructured DNA for enhanced immunostimulatory activity of CpG oligodeoxynucleotide.

Oligodeoxynucleotide (ODN) containing a cytosine-phosphate-guanine (CpG) motif, or CpG ODN, is considered suitable for treating immune diseases, including allergies. Although the phosphorothioate modification is used to enhance the stability and immunostimulatory activity of CpG ODNs, it is associated with the risk of adverse effects. Construction of nanostructured DNA assemblies, such as tripod- and hexapod-like structured DNAs, tripodna and hexapodna, respectively, were also found to increase this activity. The chemical modification of nucleobases could be another approach for enhancing CpG ODN activity. Here, we examined whether chemically modified nucleobase substitutions can enhance CpG ODN activity by measuring tumor necrosis factor α (TNF-α) release after addition to murine macrophage-like RAW264.7 cells. First, the guanine at the 18th position of phosphodiester CpG 1668 was substituted with several chemically modified guanines, and then the various guanines were substituted. Among all tested substitutions, 15,18-thdG, in which two guanines outside the CpG motif were substituted with the 2-aminothieno[3,4-d]pyrimidine guanine mimic (thdG), was the most effective. Compared to 32P-CpG 1668, 32P-15,18-thdG was taken up more efficiently by the RAW264.7 cells. Then, 15,18-thdG was incorporated into tripodna and hexapodna. 15,18-thdG/tri- or hexapodna induced higher TNF-α release from the RAW264.7 cells than PO CpG 1668/tri- or hexapodna, respectively. These results indicate that the thdG substitution is a useful effective strategy for enhancing the immunostimulatory activity of CpG DNAs in both single stranded and DNA nanostructure forms.

miR-1293, a Candidate for miRNA-Based Cancer Therapeutics, Simultaneously Targets BRD4 and the DNA Repair Pathway.

BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, plays a role in the organization of super-enhancers and transcriptional activation of oncogenes in cancer and is recognized as a promising target for cancer therapy. microRNAs (miRNAs), endogenous small noncoding RNAs, cause mRNA degradation or inhibit protein translation of their target genes by binding to complementary sequences. miRNA mimics simultaneously targeting several tumor-promoting genes and BRD4 may be useful as therapeutic agents of tumor-suppressive miRNAs (TS-miRs) for cancer therapy. To investigate TS-miRs for the development of miRNA-based cancer therapeutics, we performed function-based screening in 10 cancer cell lines with a library containing 2,565 human miRNA mimics. Consequently, miR-1293, miR-876-3p, and miR-6571-5p were identified as TS-miRs targeting BRD4 in this screening. Notably, miR-1293 also suppressed DNA repair pathways by directly suppressing the DNA repair genes APEX1 (apurinic-apyrimidinic endonuclease 1), RPA1 (replication protein A1), and POLD4 (DNA polymerase delta 4, accessory subunit). Concurrent suppression of BRD4 and these DNA repair genes synergistically inhibited tumor cell growth in vitro. Furthermore, administration of miR-1293 suppressed in vivo tumor growth in a xenograft mouse model. These results suggest that miR-1293 is a candidate for the development of miRNA-based cancer therapeutics.

From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases.

Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.

Progress in the molecular pathogenesis and nucleic acid therapeutics for Parkinson's disease in the precision medicine era.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders that manifest various motor and nonmotor symptoms. Although currently available therapies can alleviate some of the symptoms, the disease continues to progress, leading eventually to severe motor and cognitive decline and reduced life expectancy. The past two decades have witnessed rapid progress in our understanding of the molecular and genetic pathogenesis of the disease, paving the way for the development of new therapeutic approaches to arrest or delay the neurodegenerative process. As a result of these advances, biomarker-driven subtyping is making it possible to stratify PD patients into more homogeneous subgroups that may better respond to potential genetic-molecular pathway targeted disease-modifying therapies. Therapeutic nucleic acid oligomers can bind to target gene sequences with very high specificity in a base-pairing manner and precisely modulate downstream molecular events. Recently, nucleic acid therapeutics have proven effective in the treatment of a number of severe neurological and neuromuscular disorders, drawing increasing attention to the possibility of developing novel molecular therapies for PD. In this review, we update the molecular pathogenesis of PD and discuss progress in the use of antisense oligonucleotides, small interfering RNAs, short hairpin RNAs, aptamers, and microRNA-based therapeutics to target critical elements in the pathogenesis of PD that could have the potential to modify disease progression. In addition, recent advances in the delivery of nucleic acid compounds across the blood-brain barrier and challenges facing PD clinical trials are also reviewed.

RNA Interference Nanotherapeutics for Treatment of Glioblastoma Multiforme.

Nucleic acid therapeutics for RNA interference (RNAi) are gaining attention in the treatment and management of several kinds of the so-called "undruggable" tumors via targeting specific molecular pathways or oncogenes. Synthetic ribonucleic acid (RNAs) oligonucleotides like siRNA, miRNA, shRNA, and lncRNA have shown potential as novel therapeutics. However, the delivery of such oligonucleotides is significantly hampered by their physiochemical (such as hydrophilicity, negative charge, and instability) and biopharmaceutical features (in vivo serum stability, fast renal clearance, interaction with extracellular proteins, and hindrance in cellular internalization) that markedly reduce their biological activity. Recently, several nanocarriers have evolved as suitable non-viral vectors for oligonucleotide delivery, which are known to either complex or conjugate with these oligonucleotides efficiently and also overcome the extracellular and intracellular barriers, thereby allowing access to the tumoral micro-environment for the better and desired outcome in glioblastoma multiforme (GBM). This Review focuses on the up-to-date advancements in the field of RNAi nanotherapeutics utilized for GBM treatment.

The RNA-binding protein Mex-3B plays critical roles in the development of steroid-resistant neutrophilic airway inflammation.

While most asthma can be treated with steroids, about 10%, called severe asthma, is refractory to steroids. It has recently been shown that in a subgroup of severe asthma cases, neutrophils that infiltrate into the airways play an important role in inflammation. However, the mechanisms underlying this increased neutrophil infiltration are not well understood. Here, using a mouse model of steroid-resistant neutrophilic inflammation, we show that mice deficient for the RNA-binding protein Mex-3B have significantly less neutrophil infiltration in the airways than wild-type mice. We further demonstrate that Mex-3B post-transcriptionally upregulates CXCL2, a chemokine that induces neutrophil chemotaxis and migration. Moreover, we show that treatment with either anti-CXCL2 antibody or anti-Mex-3B antisense oligonucleotide suppresses neutrophilic allergic airway inflammation. These results suggest that Mex-3B-mediated induction of CXCL2 is crucial for steroid-resistant neutrophilic allergic airway inflammation. Our findings suggest new strategies for therapeutic intervention in steroid-resistant severe asthma.

Development of "CLAN" Nanomedicine for Nucleic Acid Therapeutics.

Nucleic acid-based macromolecules have paved new avenues for the development of therapeutic interventions against a spectrum of diseases; however, their clinical translation is limited by successful delivery to the target site and cells. Therefore, numerous systems have been developed to overcome delivery challenges to nucleic acids. From the viewpoint of clinical translation, it is highly desirable to develop systems with clinically validated materials and controllability in synthesis. With this in mind, a cationic lipid assisted PEG-b-PLA nanoparticle (CLAN) is designed that is capable of protecting nucleic acids via encapsulation inside the aqueous core, and delivers them to target cells, while maintaining or improving nucleic acid function. The system is formulated from clinically validated components (PEG-b-PLA and its derivatives) and can be scaled-up for large scale manufacturing, offering potential for its future use in clinical applications. Here, the development and working mechanisms of CLANs, the ways to improve its delivery efficacy, and its application in various disease treatments are summarized. Finally, a prospective for the further development of CLAN is also discussed.

Synthesis of 2'-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides.

The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.

RNA-Mediated Gene Activation: Identifying a Candidate RNA for Preclinical Development.

The ability to activate gene expression would provide new opportunities for drug development for diseases caused by inadequate or nonexistent expression of a therapeutic protein. Duplex RNAs that target gene promoters are one strategy for enhancing the expression of target genes. This chapter summarizes our understanding of mechanism behind gene activation by small RNAs that bind noncoding transcripts at gene promoters. We describe a path for choosing candidate genes for therapeutic development.

Stimuli-responsive liposomes for the delivery of nucleic acid therapeutics.

Nucleic acid therapeutics (NATs) are valuable tools in the modulation of gene expression in a highly specific manner. So far, NATs have been actively pursued in both pre-clinical and clinical studies to treat diseases such as cancer, infectious and inflammatory diseases. However, the clinical application of NATs remains a considerable challenge owing to their limited cellular uptake, low biological stability, off-target effect, and unfavorable pharmacokinetics. One concept to address these issues is to deliver NATs within stimuli-responsive liposomes, which release their contents of NATs upon encountering environmental changes such as temperature, pH, and ion strength. In this case, before reaching the targeted tissue/organ, NATs are protected from degradation by enzymes and immune system. Once at the area of interest, localized and targeted delivery can be achieved with minimal influence to other parts of the body. Here, we discuss the latest developments and existing challenges in this field. FROM THE CLINICAL EDITOR: Nucleic acid therapeutics have been shown to enhance or eliminate specific gene expression in experimental research. Unfortunately, clinical applications have so far not been realized due to problems of easy degradation and possible toxicity. The use of nanosized carriers such as liposomes to deliver nucleic acids is one solution to overcome these problems. In this review article the authors describe and discuss the potentials of various trigger-responsive "smart" liposomes, with a view to help other researchers to design better liposomal nucleic acid delivery systems.

Self-assembling micelle-like nanoparticles with detachable envelopes for enhanced delivery of nucleic acid therapeutics.

In spite of the great potential of nucleic acids as therapeutic agents, the clinical application of nucleic acid therapeutics requires the development of effective systemic delivery strategies. In an effort to develop effective nucleic acid delivery systems suitable for clinical application, we previously reported a self-assembling micelle-like nanoparticle that was based on phospholipid-polyethylenimine conjugates, i.e., "micelle-like nanoparticles" (MNPs). In this study, we aimed to improve the system by enhancing the efficiency of intracellular delivery of the payload via pH-responsive detachment of the monolayer envelope and release of the nucleic acid therapeutics upon reaching the target tissues with an acidic pH, e.g., tumors. The acid-cleavable phospholipid-polyethylenimine conjugate was synthesized via hydrazone bond, and acid-cleavable MNPs were then prepared and characterized as before. We evaluated the acid-cleavable MNP construct for in vitro and in vivo nucleic acid delivery efficiency using cultured tumor cells and tumor-bearing mice. The acid-cleavable nanocarrier showed an enhanced cellular delivery at pH 6.5 as compared to pH 7.4, whereas the noncleavable nanocarrier did not show any differences. Tail vein injections also led to enhanced intracellular uptake of the acid-cleavable nanocarrier compared to the noncleavable nanocarrier into tumor cells of tumor-bearing mice although no significant difference was observed in total tumor accumulation.

Generalizable anchor aptamer strategy for loading nucleic acid therapeutics on exosomes.

Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXOEAA-PMO). EXOEAA-PMO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXOEAA-PMO elicited therapeutic levels of dystrophin restoration and functional improvements in mdx mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.

A microRNA focus on acne.

Acne (syn. acne vulgaris) is a common inflammatory skin disorder associated with puberty and adolescence. The disease is characterized by comedoneous lesions, papules, pustules, and nodules that are mostly found on the face. These lesions are caused by intricate interactions between the pilosebaceous unit and the Cutibacterium acnes (C. acnes) bacteria. Unhealthy acne and its aftereffects, like pigment changes and scarring, have a detrimental impact on one's quality of life. Recent years have seen a sharp increase in the approval of nucleic acid therapies (NATs), such as antisense oligonucleotides and short-interfering RNA medications, for rare diseases for which there are few or no effective treatments. These developments suggest that NATs may be useful in acne treatment plans down the road, as do clinical trials for microRNA (miRNA) modulation in skin contexts. We highlight promising miRNA targets for anti-acne therapy in this review. We outline the pathophysiology of acne in brief and emphasize the functions of C. acnes. Next, we concentrate on the distinct impacts of biofilm and planktonic C. acnes on a Toll-like receptor 2 axis that spans miR-146a-5p, which was recently discovered. Before discussing the potential contributions of miR-21-5p, miR-233-3p, and miR-150-5p to inflammatory axes in acne, we evaluate miR-146a-5p in sebocytes. Finally, we address patient involvement in miRNA-related acne research and translational perspectives.