Nucleic acid-based medicines have achieved significant advancements in recent years, with lipid nanoparticles (LNPs) being a pivotal platform for their delivery. However, the complexity of LNP presents significant challenges, requiring analytical methods to identify and quantify individual components to guide formulation development and ensure quality and safety. Current approaches often perform nucleic acid and lipid analysis separately and focus on a single type of formulation, highlighting the need for a simple platform method that can be applied to diverse formulations. We present a platform ion-pair reversed-phase HPLC method with UV and charged aerosol detection (CAD) to simultaneously separate and quantify lipid and nucleic acid components in LNPs. The method separated and quantified 12 lipid species and three types of nucleic acids (antisense oligonucleotide, single-guide RNA, and mRNA), covering a broad range of therapeutic cargoes. Notably, this can be achieved for the first time by one HPLC run with one-step facile sample preparation. Specifically, we used a simple buffer containing Triton and heparin to enable the single-step, simultaneous extraction of both nucleic acid and lipid components from LNPs, achieving quantification recoveries of 90-110 %. We further applied this method and addressed process and quality control challenges of LNPs, including the recovery rate of individual LNP components after purification and simultaneous quantification of co-loaded, different nucleic acid species for potential gene editing applications. This new platform method offers a robust and widely applicable tool to assess the quality of lipid-based nucleic acid therapies.

Delivery of exogenous nucleic acids (NAs) for gene regulation in bacteria, bypassing the barrier of the cell wall, is essential for advancing fundamental microbiology and genetic engineering, and the treatment of bacterial diseases. However, current methods that rely on electrical or chemical interventions are limited by their complexity, specialized expertise, and laboratory-specific instrumentation. This study explores the capability of gold nanoclusters (AuNCs) as carriers for delivering small-interfering RNA and antisense oligonucleotides into bacteria for targeted gene regulation while shielding them from degradation during transport. By enhancing the cytoplasmic membrane permeability, the AuNCs enable efficient internalization of NAs into both Gram-positive and Gram-negative bacteria while exerting negligible influence on bacterial activity. It is demonstrated that the rationally designed NAs can be released from the AuNCs within bacteria, enabling ~70% knockdown of mecA in Methicillin-resistant Staphylococcus aureus (MRSA). This significantly reduces MRSA's antibiotic resistance and enhances oxacillin treatment efficacy. Furthermore, the successful silencing of ligA in Escherichia coli and pilQ in Pseudomonas aeruginosa highlights the broad adaptability of the approach across diverse bacterial species. The AuNCs-based next-generation NA delivery system has the potential to transform bacterial gene regulation-previously restricted to laboratory settings-into a versatile and scalable solution for real-world application.

MECP2 duplication syndrome (MDS) is a rare X-linked neurodevelopmental disorder caused by duplications of the dosage-sensitive methyl-CpG-binding protein 2 (MECP2) gene. Developing effective therapies for MDS is particularly challenging due to the variability in MECP2 expression among patients and the potential risk of inducing Rett syndrome through excessive pharmacological intervention. Reducing dosage to optimize silencing levels often compromises durability and necessitates increased dosing frequency. We present here a series of fully chemically modified small interfering RNAs (siRNAs) designed for both isoform-selective and total MECP2 silencing. Among these, we identify six lead siRNA candidates across two distinct chemical scaffolds, achieving targeted total MECP2 expression reductions ranging from 25% to 75%, sustained for at least four months following a single administration. The efficacy and safety of human ortholog silencing were evaluated using two mouse models with distinct levels of human MECP2 transgene expression. In the severe duplication model, a single dose of the total isoform-silencing siRNA fully rescued early mortality and behavioral impairments. Additionally, we show that the isoform-selective targeting strategy may be safer in mild cases of MDS where exaggerated pharmacology may lead to Rett Syndrome. Overall, this study introduces a series of preclinical candidates with the capacity to address the varying levels of MECP2 duplication encountered in clinical settings. Furthermore, it establishes a target selection strategy that may be applied to other dosage-sensitive gene imbalances.

Pathogenic gene dysregulation can be attributed to chromatin state change that pre-transcriptionally regulates expression. Recent breakthroughs elucidating the rules governing this DNA control layer, an epigenetic code, unlock a modality in precision medicine to target gene dysregulation across myriad diseases. Here we present a modular platform to design programmable mRNA therapeutics, Epigenomic Controllers (EC), that control gene expression through directed epigenetic change. By leveraging natural mechanisms, ECs tune expression levels of one or multiple genes with durable effect of weeks-to-months in female mice following a single dose. We design and characterize ECs to multiple target genes and identify an EC that effectively inhibits the cancer- and inflammatory-disorder-associated multi-gene cluster CXCL1-8. With precision targeting of NF-kB signaling and identification of homologous murine surrogates, ECs significantly reduce neutrophil migration in vivo during acute lung inflammation in female mice. A platform approach to EC design for epigenomic modulation expands treatment frontiers for diverse gene targets, including those considered "undruggable."

Despite the significance of antibiotics in treating bacterial infections, antibiotic resistance is continuously increasing, thus posing a significant threat. In addition to strains resistant to individual drugs, multidrug-resistant (MDR) and pandrug-resistant strains, are emerging. Salmonella, a primary cause of global foodborne illness, is often transmitted through animal products. Antibiotic treatment is crucial for immunocompromised individuals, such as older adults and patients with weakened immune systems, due to their increased susceptibility to severe effects. MDR Salmonella, which can arise following antibiotic use in food animals, may transfer to humans, leading to significant health challenges. The emergence of Salmonella strains resistant to carbapenems, often considered a last-resort antibiotic class, is particularly concerning. Salmonella neutralizes antibiotics through mechanisms, such as horizontal gene transfer via plasmids, efflux/influx system regulation, and enzyme production that deactivate or alter antibiotics. The rise of megaplasmids in Salmonella is particularly alarming, as it may enable resistance to a broader range of antibiotics. This review summarizes the current state of the growing threat of MDR Salmonella and underscores the urgent need for a coordinated response.

Genome and transcriptome engineering have emerged as powerful tools in modern biotechnology, driving advancements in precision medicine and novel therapeutics. In this review, we provide a comprehensive overview of the current methodologies, applications, and future directions in genome and transcriptome engineering. Through this, we aim to provide a guide for tool selection, critically analyzing the strengths, weaknesses, and best use cases of these tools to provide context on their suitability for various applications. We explore standard and recent developments in genome engineering, such as base editors and prime editing, and provide insight into tool selection for change of function (knockout, deletion, insertion, substitution) and change of expression (repression, activation) contexts. Advancements in transcriptome engineering are also explored, focusing on established technologies like antisense oligonucleotides (ASOs) and RNA interference (RNAi), as well as recent developments such as CRISPR-Cas13 and adenosine deaminases acting on RNA (ADAR). This review offers a comparison of different approaches to achieve similar biological goals, and consideration of high-throughput applications that enable the probing of a variety of targets. This review elucidates the transformative impact of genome and transcriptome engineering on biological research and clinical applications that will pave the way for future innovations in the field.

Oligonucleotides are a rapidly emerging class of therapeutics. Their most well-known examples are informational drugs that modify gene expression by binding mRNA. Despite inducing proximity between biological machinery and mRNA when applied to modulating gene expression, oligonucleotides are not typically labeled as "proximity-inducing" in literature. Yet, they have recently been explored as building blocks for multispecific proximity-inducing drugs (MPIDs). MPIDs are unique because they can direct endogenous biological machinery to destroy targeted molecules and cells, in contrast to traditional drugs that inhibit only their functions. The unique mechanism of action of MPIDs has enabled the targeting of previously "undruggable" molecular entities that cannot be effectively inhibited. However, the development of MPIDs must ensure that these molecules will selectively direct a potent, destruction-based mechanism of action toward intended targets over healthy tissues to avoid causing life-threatening toxicities. Oligonucleotides have emerged as promising building blocks for the design of MPIDs because they are sequence-controlled molecules that can be rationally designed to program multispecific binding interactions. In this Review, we examine the emergence of oligonucleotide-containing MPIDs in the proximity induction space, which has been dominated by antibody and small molecule MPID modalities. Moreover, examples of oligonucleotides developed as MPID candidates in immunotherapy and protein degradation are discussed to demonstrate the utility of oligonucleotides in expanding the scope and selectivity of the MPID toolbox. Finally, we discuss the utility of programming "AND" gates into oligonucleotide scaffolds to encode conditional responses that have the potential to be incorporated into MPIDs, which can further enhance their selectivity, thus increasing the scope of this drug category.

Antisense oligonucleotide (ASO) was established as a viable therapeutic option for genetic disorders. ASOs can target RNAs implicated in various diseases, including upregulated mRNA and pre-mRNA undergoing abnormal alternative splicing events. Therapeutic applications of ASOs have been proven with the Food and Drug Administration approval of several drugs in recent years. Earlier enzymatic stability and delivery remains a big challenge for ASOs. Introducing new chemical modifications and new formulations resolving the issues related to the nuclease stability and delivery of the ASOs. Excitingly, ASOs-based bioconjugates that target the hepatocyte have gained much attraction. Efforts are ongoing to increase the therapeutic application of the ASOs to the extrahepatic tissue as well.

We have briefly discussed the mechanism of ASOs, the development of new chemistries, and delivery strategies for ASO-based drug discovery and development. The discussion focuses more on the already approved ASOs and those in the clinical development stage.

To expand the clinical application of ASOs, continuous effort is required to develop precise delivery strategies for targeting extrahepatic tissue to minimize the off-target effects.

Gene therapy has recently become a reality in the treatment of cardiovascular diseases. Strategies to modulate gene expression using antisense oligonucleotides or small interfering RNA are proving to be safe and effective in the clinic. Adeno-associated viral vector-based gene delivery and CRISPR-Cas9-based genome editing have emerged as efficient strategies for gene delivery and repair in humans. Overall, gene therapy holds the promise not only of expanding current treatment options, but also of intervening in previously untackled causal disease mechanisms with little side effects. This scientific statement provides a comprehensive overview of the various modalities of gene therapy used to treat heart failure and some of its risk factors, and their application in the clinical setting. It discusses specifically the possibilities of gene therapy for hereditary heart diseases and (non)-genetic heart failure. Furthermore, it addresses safety and clinical trial design issues and challenges for future regulatory strategies.

Functional cure of chronic hepatitis B (CHB)-defined as sustained seroclearance of hepatitis B surface antigen (HBsAg) with unquantifiable hepatitis B virus (HBV) DNA at 24 weeks off treatment, is an optimal treatment endpoint. Nonetheless, it cannot be consistently attained by current treatment modalities. RNA interference (RNAi) is a novel treatment strategy using small-interfering RNA (siRNA) or antisense oligonucleotide (ASO) to target HBV post-transcriptional RNA, in turn suppressing viral protein production and replication. Hence, RNAi has indirect effects in promoting host immune reconstitution against HBV. Multiple RNAi therapeutics have entered phase II/III clinical trials, demonstrating potent, dose-dependent, and sustainable effects in suppressing HBsAg. Incidences of HBsAg seroclearance, particularly with the use of ASO, have also been documented. The combination of RNAi with other antivirals/immunomodulators (e.g. pegylated interferon), have shown promising results in potentiating RNAi effects and enhancing treatment durability. Further research will be required to establish predictors of response, optimal treatment protocols, and long-term outcomes in patients on RNAi. RNAi therapeutics have shown promising results and will likely be the keystone of future HBV treatment.

The αLβ2 integrin LFA-1 plays a key role in T-cell adhesion to the endothelial vasculature and migration into both secondary lymphoid organs and peripheral tissues via interactions with its target protein ICAM-1, but the pathways that regulate LFA-1-mediated T-cell polarity and migration are not fully understood. In this study we screened two RNAi libraries targeting G protein-coupled receptors (GPCR)/GPCR-associated proteins and kinases in a HuT 78 T cell line model of LFA-1-stimulated T-cell migration. Based on staining of the actin cytoskeleton, multiple parameters to measure cell morphology were used to assess the contribution of 1109 genes to LFA-1-mediated T-cell polarity and migration. These RNAi screens identified a number of both novel and previously identified genes that either increased or decreased the polarity and migratory capacity of these cells. Following multiparametric analysis, hierarchical clustering and pathway analysis, three of these genes were characterized in further detail using primary human T cells, revealing novel roles for the heterotrimeric G protein subunit Gβ1 and Casein Kinase 2 in LFA-1-mediated T-cell polarity and migration in vitro. Our studies also highlighted a new role for ICAP-1, an adaptor protein previously described to be associated with β1 integrins, in β2 integrin LFA-1-directed migration in T cells. Knockdown of ICAP-1 expression in primary T cells revealed a role in cell polarity, cell velocity and transmigration towards SDF-1 for this adaptor protein. This study therefore uncovers new roles for GPCR/GPCR-associated proteins and kinases in T-cell migration and provides potential novel targets for modulation of the T-cell immune response.

RNA degradation in mammalian cells is performed by multiple enzymes and cofactors making it difficult to identify the specific impact of each of them separately. The auxin-inducible degron system enables direct depletion of a protein of interest limiting the time of depletion and thus reducing secondary effects due to cell adaptation. In this chapter, using XRN1 as an example of cytoplasmic RNA decay enzyme, we describe a combination of methods to introduce the auxin-inducible degron by CRISPR-Cas9, together with downstream analyses of RNA levels after protein depletion.

Three types of highly promising small RNA therapeutics, namely, small interfering RNAs (siRNAs), microRNAs (miRNAs) and the RNA subtype of antisense oligonucleotides (ASOs), offer advantages over small-molecule drugs. These small RNAs can target any gene product, opening up new avenues of effective and safe therapeutic approaches for a wide range of diseases. In preclinical research, synthetic small RNAs play an essential role in the investigation of physiological and pathological pathways as silencers of specific genes, facilitating discovery and validation of drug targets in different conditions. Off-target effects of small RNAs, however, could make it difficult to interpret experimental results in the preclinical phase and may contribute to adverse events of small RNA therapeutics. Out of the two major types of off-target effects we focused on the hybridization-dependent, especially on the miRNA-like off-target effects. Our main aim was to discuss several approaches, including sequence design, chemical modifications and target prediction, to reduce hybridization-dependent off-target effects that should be considered even at the early development phase of small RNA therapy. Because there is no standard way of predicting hybridization-dependent off-target effects, this review provides an overview of all major state-of-the-art computational methods and proposes new approaches, such as the possible inclusion of network theory and artificial intelligence (AI) in the prediction workflows. Case studies and a concise survey of experimental methods for validating in silico predictions are also presented. These methods could contribute to interpret experimental results, to minimize off-target effects and hopefully to avoid off-target-related adverse events of small RNA therapeutics. LINKED ARTICLES: This article is part of a themed issue Non-coding RNA Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.2/issuetoc.

Antisense oligonucleotides (ASOs) are promising drugs capable of modulating the protein expression of virtually any target with high specificity and high affinity through complementary base pairing. However, this requires a deep understanding of the target sequence and significant effort in designing the correct complementary drug. In addition, ASOs have been demonstrated to be well tolerated during their clinical use. Indeed, they are already used in many diseases due to pathogenic RNAs of known sequences and in several neurodegenerative diseases and metabolic diseases, for which they were given marketing authorizations (MAs) in Europe and the United States. Their use in oncology is gaining momentum with several identified targets, promising preclinical and clinical results, and recent market authorizations in the US. However, many challenges remain for their clinical use in cancer. It seems necessary to take a step back and review our knowledge of ASOs and their therapeutic uses in oncology. The objectives of this review are (i) to summarize the current state of the art of ASOs; (ii) to discuss the therapeutic use of ASOs in cancer; and (iii) to focus on ASO usage in glioblastoma, the challenges, and the perspective ahead.

Breast cancer, a pervasive and complex disease, continues to pose significant challenges in the field of oncology. Its heterogeneous nature and diverse molecular profiles necessitate a nuanced understanding of the underlying mechanisms driving tumorigenesis and progression. MicroRNA-21 (miR-21) has emerged as a crucial player in breast cancer development and progression by modulating apoptosis, a programmed cell death mechanism that eliminates aberrant cells. MiR-21 overexpression is a hallmark of breast cancer, and it is associated with poor prognosis and resistance to conventional therapies. This miRNA exerts its oncogenic effects by targeting various pro-apoptotic genes, including Fas ligand (FasL), programmed cell death protein 4 (PDCD4), and phosphatase and tensin homolog (PTEN). By suppressing these genes, miR-21 promotes breast cancer cell survival, proliferation, invasion, and metastasis. The identification of miR-21 as a critical regulator of apoptosis in breast cancer has opened new avenues for therapeutic intervention. This review investigates the intricate mechanisms through which miR-21 influences apoptosis, offering insights into the molecular pathways and signaling cascades involved. The dysregulation of apoptosis is a hallmark of cancer, and understanding the role of miR-21 in this context holds immense therapeutic potential. Additionally, the review highlights the clinical significance of miR-21 as a diagnostic and prognostic biomarker in breast cancer, underscoring its potential as a therapeutic target.

Direct-acting antiviral (DAA) drugs have been shown to effectively reduce viral load and cure a high proportion of hepatitis C virus (HCV) infections. However, costs associated with the course of therapy and any possible adverse effects should also be considered. It is important to acknowledge, moreover, that certain groups may not be eligible for treatment. Given that there is currently no approved vaccine for HCV infection, the need for an effective, safe, and accessible treatment remains a crucial priority. The aim of this study is to develop an antisense oligonucleotide (ASO)-based therapeutic drug that can inhibit HCV capsid. After analyzing 817 HCV capsid protein mRNA sequences using the NCBI Virus Data Portal, a conserved region of 7 nucleotides (nt) was identified in all genotypes (1-7). However, because of its high GC% content, this region is not a suitable target for ASO. Conversely, the other highly conserved region, which is only 8 nt long, was preserved in 801 datasets after removing missing and differing sequence data. The candidate ASO was then investigated using computer simulations to assess its potential. Thus, it is possible that the ASO sequence consisting of 8 nt could be a viable therapeutic target for the inhibition of HCV capsid. Furthermore, the 7 nt sequence, which is conserved in all datasets, may be targeted using alternative strategies in lieu of ASO-based targeting.

Small interfering RNAs (siRNAs) represent a novel class of drugs capable of potent and sustained modulation of genes across various tissues. Preclinical development of siRNAs necessitates assessing efficacy and toxicity in animal models. While identifying therapeutic leads with cross-species activity can expedite development, it may compromise efficacy and be infeasible for certain gene targets. Here, we investigate whether deriving species-active siRNAs from potent human-targeting leads-an approach termed mismatch conversion-can yield potent compounds. We systematically altered potent siRNAs targeting human genes associated with diseases-SOD1 (ALS), JAK1 (inflammation), and HTT (HD)-to generate species-matching variants with full complementarity to their target in NHPs, mice, rats, sheep, and dogs. Variants potency and efficacy were measured in corresponding cell lines. We demonstrate that sequence, position, and number of mismatches significantly influence the ability to generate potent species-active compounds via mismatch conversion. Across tested sequences, mismatch conversion strategy ability to identify a species-active lead varied from 0% to 70%. For SOD1, lead compounds identified from species-focus screening in mouse and dog cells were more potent than leads obtained from mismatch conversion. Thus, a focused screening of therapeutic lead and model compounds may represent a more reliable strategy for the clinical advancement of siRNAs.

Colorectal cancer (CRC) being one of the most common malignancies, has a significant death rate, especially when detected at an advanced stage. In most cases, the fundamental aetiology of CRC remains unclear despite the identification of several environmental and intrinsic risk factors. Numerous investigations, particularly in the last ten years, have indicated the involvement of epigenetic variables in this type of cancer. The development, progression, and metastasis of CRC are influenced by long non-coding RNAs (lncRNAs), which are significant players in the epigenetic pathways. LncRNAs are implicated in diverse pathological processes in CRC, such as liver metastasis, epithelial to mesenchymal transition (EMT), inflammation, and chemo-/radioresistance. It has recently been determined that CRC cells and tissues exhibit dysregulation of tens of oncogenic and tumor suppressor lncRNAs. Serum samples from CRC patients exhibit dysregulated expressions of several of these transcripts, offering a non-invasive method of detecting this kind of cancer. In this review, we outlined the typical paradigms of the deregulated lncRNA which exert significant role in the underlying molecular mechanisms of CRC initiation and progression. We comprehensively discuss the role of lncRNAs as innovative targets for CRC prognosis and treatment.

Nucleic acid (NA)-based therapeutics have shown great potential for downregulating or augmenting gene expression, and for promising applications, e.g., protein-replacement therapy and vaccination, a comprehensive understanding of the requirements for their targeted delivery to specific tissues or cells is needed.

In this review, we discuss clinical applications of four representative types of NA-based therapeutics, i.e. antisense oligonucleotides, small interfering RNA, messenger RNA, and circular RNA, with a focus on the lipid nanoparticle (LNP) technology used for intracellular delivery. The in vivo fate of LNPs is discussed to improve the understanding of trafficking of nanomedicines at the systemic and cellular levels. In addition, NA-based vaccines are discussed, focusing on targeting antigen-presenting cells and immune activation.

Optimization of delivery systems for NA-based therapeutics is mainly focused on the standard requirements of prolonged systemic circulation and enhancing endosomal escape. Depending on the final destination in specific target tissues or cells, strategies should be adjusted to achieve the desired biodistribution of NA-based payloads. More studies relating to the pharmacokinetics of both cargo and carrier are encouraged, because their in vivo fates may differ, considering the possibility of premature cargo release before reaching the target.

Dysregulation of master regulator c-MYC (MYC) plays a central role in hepatocellular carcinoma (HCC) and other cancers but remains an elusive target for therapeutic intervention. MYC expression is epigenetically modulated within naturally occurring DNA loop structures, Insulated Genomic Domains (IGDs). We present a therapeutic approach using an epigenomic controller (EC), a programmable epigenomic mRNA medicine, to precisely modify MYC IGD sub-elements, leading to methylation of MYC regulatory elements and durable downregulation of MYC mRNA transcription. Significant antitumor activity is observed in preclinical models of HCC treated with the MYC-targeted EC, as monotherapy or in combination with tyrosine kinase or immune checkpoint inhibitors. These findings pave the way for clinical development of MYC-targeting epigenomic controllers in HCC patients and provide a framework for programmable epigenomic mRNA therapeutics for cancer and other diseases.

Gapmer antisense oligonucleotides (ASOs) hold therapeutic promise for allele-specific silencing, but face challenges in distinguishing between mutant and wild-type transcripts. This study explores new design strategies to enhance ASO specificity, focusing on a common dominant mutation in COL6A3 gene associated with Ullrich congenital muscular dystrophy. Initial gapmer ASO design exhibited high efficiency but poor specificity for the mutant allele. We then adopted a mixmer design, incorporating additional RNA bases based on computational predictions of secondary structures for both mutant and wild-type alleles, aiming to enhance ASO accessibility to mutant transcripts. The mixmer ASO design demonstrated up to a 3-fold increase in specificity compared with the classical gapmer design. Further refinement involved introducing a nucleotide mismatch as a structural modification, resulting in a 10-fold enhancement in specificity compared with the gapmer design and a 3-fold over the mixmer design. Additionally, we identified for the first time a potential role of the RNA-induced silencing complex (RISC), alongside RNase H1, in gapmer-mediated silencing, in contrast with what was observed with mixmer ASOs, where only RNase H1 was involved. In conclusion, this study presents a novel design concept for allele-specific ASOs leveraging mRNA secondary structures and nucleotide mismatching and suggests a potential involvement of RISC in gapmer-mediated silencing.

Oncogenic microRNA (miRNA), especially miRNA-21 upregulation in triple-negative breast cancer (TNBC), suggests a new class of therapeutic targets. In this study, we aimed to create GE11 peptide-conjugated small interfering RNA-loaded chitosan nanoparticles (GE11-siRNA-CSNPs) for the targeting of EGFR overexpressed TNBC and selectively inhibit miRNA-21 expression. A variety of in-silico and in vitro cellular and molecular studies were conducted to investigate the binding affinities of specific targets used as well as the anticancer efficacies and mechanisms of GE11-siRNA-CSNPs in TNBC cells. An in-silico assessment reveals a distinct binding affinity of miRNA-21 with siRNA as well as between the extracellular domain of EGFR and synthesized peptides. Notably, the in vitro results showed that GE11-siRNA-CSNPs were revealed to have better cytotoxicity against TNBC cells. It significantly inhibits miRNA-21 expression, cell migration, and colony formation. The results also indicated that GE11-siRNA-CSNPs impeded cell cycle progression. It induces cell death by reducing the expression of the antiapoptotic gene Bcl-2 and increasing the expression of the proapoptotic genes Bax, Caspase 3, and Caspase 9. Additionally, the docking analysis and immunoblot investigations verified that GE1-siRNA-CSNPs, which specifically target TNBC cells and suppress miRNA-21, can prevent the effects of miRNA-21 on the proliferation of TNBC cells via controlling EGFR and subsequently inhibiting the PI3K/AKT and ERK1/2 signaling axis. The GE11-siRNA-CSNPs design, which specifically targets TNBC cells, offers a novel approach for the treatment of breast cancer with improved effectiveness. This study suggests that GE11-siRNA-CSNPs could be a promising candidate for further assessment as an additional strategy in the treatment of TNBC.

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies.

Pain, a complex and debilitating condition affecting millions globally, is a significant concern, especially in the context of post-operative recovery. This comprehensive review explores the complexity of pain and its global impact, emphasizing the modulation of voltage-gated sodium channels (VGSC or NaV channels) as a promising avenue for pain management with the aim of reducing reliance on opioids. The article delves into the role of specific NaV isoforms, particularly NaV 1.7, NaV 1.8, and NaV 1.9, in pain process and discusses the development of sodium channel blockers to target these isoforms precisely. Traditional local anesthetics and selective NaV isoform inhibitors, despite showing varying efficacy in pain management, face challenges in systemic distribution and potential side effects. The review highlights the potential of nanomedicine in improving the delivery of local anesthetics, toxins and selective NaV isoform inhibitors for a targeted and sustained release at the site of pain. This innovative strategy seeks to improve drug bioavailability, minimize systemic exposure, and optimize therapeutic outcomes, holding significant promise for secure pain management and enhancing the quality of life for individuals recovering from surgical procedures or suffering from chronic pain.

One of the most widely used techniques for the quantification of small interfering ribonucleic acid (siRNA) is the ultraviolet (UV) spectroscopy method. However, due to uncertainties in the extinction coefficient affecting the accuracy of the method and a sample preparation including several dilution steps, the purpose of this study was to explore the possibility of determining the content of siRNA by a platform method using quantitative 31P nuclear magnetic resonance (31P-qNMR) and the internal standard method. In this paper, acquisition time, selection of a suitable internal certified reference material, signal selection used for quantification, relaxation delay, and precision are discussed. In addition, the robustness of the method and the ability to apply this platform method to both drug substance (DS) and drug product samples is also discussed. Quantifications of siRNA determined by the 31P-qNMR platform method were on average 98.5%w/w when adjusting for the sodium and water contents. The data confirmed the applicability of 31P-qNMR in siRNA content determinations. The quantifications were compared to quantifications determined by the traditional UV spectroscopy method by F- and t-tests. The statistical tests showed that the platform 31P-qNMR method provided more accurate results (mass balance close to 100% w/w) compared to the traditional UV spectroscopy method when analyzing DS samples.

Gene therapy in pediatrics represents a cutting-edge therapeutic strategy for treating a range of genetic disorders that manifest in childhood. Gene therapy involves the modification or correction of a mutated gene or the introduction of a functional gene into a patient's cells. In general, it is implemented through two main modalities namely ex vivo gene therapy and in vivo gene therapy. Currently, a noteworthy array of gene therapy products has received valid market authorization, with several others in various stages of the approval process. Additionally, a multitude of clinical trials are actively underway, underscoring the dynamic progress within this field. Pediatric genetic disorders in the fields of hematology, oncology, vision and hearing loss, immunodeficiencies, neurological, and metabolic disorders are areas for gene therapy interventions. This review provides a comprehensive overview of the evolution and current progress of gene therapy-based treatments in the clinic for pediatric patients. It navigates the historical milestones of gene therapies, currently approved gene therapy products by the U.S. Food and Drug Administration (FDA) and/or European Medicines Agency (EMA) for children, and the promising future for genetic disorders. By providing a thorough compilation of approved gene therapy drugs and published results of completed or ongoing clinical trials, this review serves as a guide for pediatric clinicians to get a quick overview of the situation of clinical studies and approved gene therapy products as of 2023.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition, with 20% of familial and 2-3% of sporadic cases linked to mutations in the cytosolic superoxide dismutase (SOD1) gene. Mutant SOD1 protein is toxic to motor neurons, making SOD1 gene lowering a promising approach, supported by preclinical data and the 2023 FDA approval of the GapmeR ASO targeting SOD1, tofersen. Despite the approval of an ASO and the optimism it brings to the field, the pharmacodynamics and pharmacokinetics of therapeutic SOD1 modulation can be improved. Here, we developed a chemically stabilized divalent siRNA scaffold (di-siRNA) that effectively suppresses SOD1 expression in vitro and in vivo. With optimized chemical modification, it achieves remarkable CNS tissue permeation and SOD1 silencing in vivo. Administered intraventricularly, di-siRNASOD1 extended survival in SOD1-G93A ALS mice, surpassing survival previously seen in these mice by ASO modalities, slowed disease progression, and prevented ALS neuropathology. These properties offer an improved therapeutic strategy for SOD1-mediated ALS and may extend to other dominantly inherited neurological disorders.

Long non-coding RNAs (lncRNAs) have emerged as critical players in brain development and disease. These non-coding transcripts, which once considered as "transcriptional junk," are now known for their regulatory roles in gene expression. In brain development, lncRNAs participate in many processes, including neurogenesis, neuronal differentiation, and synaptogenesis. They employ their effect through a wide variety of transcriptional and post-transcriptional regulatory mechanisms through interactions with chromatin modifiers, transcription factors, and other regulatory molecules. Dysregulation of lncRNAs has been associated with certain brain diseases, including Alzheimer's disease, Parkinson's disease, cancer, and neurodevelopmental disorders. Altered expression and function of specific lncRNAs have been implicated with disrupted neuronal connectivity, impaired synaptic plasticity, and aberrant gene expression pattern, highlighting the functional importance of this subclass of brain-enriched RNAs. Moreover, lncRNAs have been identified as potential biomarkers and therapeutic targets for neurological diseases. Here, we give a comprehensive review of the existing knowledge of lncRNAs. Our aim is to provide a better understanding of the diversity of lncRNA structure and functions in brain development and disease. This holds promise for unravelling the complexity of neurodevelopmental and neurodegenerative disorders, paving the way for the development of novel biomarkers and therapeutic targets for improved diagnosis and treatment.

RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.

Neurodevelopment is a highly organized and complex process involving lasting and often irreversible changes in the central nervous system. Inherited disorders of neurotransmission (IDNT) are a group of genetic disorders where neurotransmission is primarily affected, resulting in abnormal brain development from early life, manifest as neurodevelopmental disorders and other chronic conditions. In principle, IDNT (particularly those of monogenic causes) are amenable to gene replacement therapy via precise genetic correction. However, practical challenges for gene replacement therapy remain major hurdles for its translation from bench to bedside. We discuss key considerations for the development of gene replacement therapies for IDNT. As an example, we describe our ongoing work on gene replacement therapy for succinic semialdehyde dehydrogenase deficiency, a GABA catabolic disorder.

Colorectal cancer (CRC) is the third most common cancer and has the second highest mortality rate among cancers. The development of CRC involves both genetic and epigenetic abnormalities, and recent research has focused on exploring the ex-transcriptome, particularly post-transcriptional modifications. RNA-binding proteins (RBPs) are emerging epigenetic regulators that play crucial roles in post-transcriptional events. Dysregulation of RBPs can result in aberrant expression of downstream target genes, thereby affecting the progression of colorectal tumors and the prognosis of patients. Recent studies have shown that RBPs can influence CRC pathogenesis and progression by regulating various components of the tumor microenvironment (TME). Although previous research on RBPs has primarily focused on their direct regulation of colorectal tumor development, their involvement in the remodeling of the TME has not been systematically reported. This review aims to highlight the significant role of RBPs in the intricate interactions within the CRC tumor microenvironment, including tumor immune microenvironment, inflammatory microenvironment, extracellular matrix, tumor vasculature, and CRC cancer stem cells. We also highlight several compounds under investigation for RBP-TME-based treatment of CRC, including small molecule inhibitors such as antisense oligonucleotides (ASOs), siRNAs, agonists, gene manipulation, and tumor vaccines. The insights gained from this review may lead to the development of RBP-based targeted novel therapeutic strategies aimed at modulating the TME, potentially inhibiting the progression and metastasis of CRC.

Antisense oligonucleotides (ASOs) were the first modality to pioneer targeted gene knockdown in the treatment of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1). RNA interference (RNAi) is another mechanism of gene silencing in which short interfering RNAs (siRNAs) effectively degrade complementary transcripts. However, delivery to extrahepatic tissues like the CNS has been a bottleneck in the clinical development of RNAi. Herein, we identify potent siRNA duplexes for the knockdown of human SOD1 in which medicinal chemistry and conjugation to an accessory oligonucleotide (ACO) enable activity in CNS tissues. Local delivery via intracerebroventricular or intrathecal injection into SOD1G93A mice delayed disease progression and extended animal survival with superior efficacy compared with an ASO resembling tofersen in sequence and chemistry. Treatment also prevented disease-related declines in motor function, including improvements in animal mobility, muscle strength, and coordination. The ACO itself does not target any specific complementary nucleic acid sequence; rather, it imparts benefits conducive to bioavailability and delivery through its chemistry. The complete conjugate (i.e., siRNA-ACO) represents a novel modality for delivery of duplex RNA (e.g., siRNA) to the CNS that is currently being tested in the clinic for treatment of ALS.

The last decade (2013-2023) has seen unprecedented successes in the clinical translation of therapeutic antisense oligonucleotides (ASOs). Eight such molecules have been granted marketing approval by the United States Food and Drug Administration (US FDA) during the decade, after the first ASO drug, fomivirsen, was approved much earlier, in 1998. Splice-modulating ASOs have also been developed for the therapy of inborn errors of metabolism (IEMs), due to their ability to redirect aberrant splicing caused by mutations, thus recovering the expression of normal transcripts, and correcting the deficiency of functional proteins. The feasibility of treating IEM patients with splice-switching ASOs has been supported by FDA permission (2018) of the first "N-of-1" study of milasen, an investigational ASO drug for Batten disease. Although for IEM, owing to the rarity of individual disease and/or pathogenic mutation, only a low number of patients may be treated by ASOs that specifically suppress the aberrant splicing pattern of mutant precursor mRNA (pre-mRNA), splice-switching ASOs represent superior individualized molecular therapeutics for IEM. In this work, we first summarize the ASO technology with respect to its mechanisms of action, chemical modifications of nucleotides, and rational design of modified oligonucleotides; following that, we precisely provide a review of the current understanding of developing splice-modulating ASO-based therapeutics for IEM. In the concluding section, we suggest potential ways to improve and/or optimize the development of ASOs targeting IEM.

Ocular diseases present a unique challenge and opportunity for therapeutic development. The eye has distinct advantages as a therapy target given its accessibility, compartmentalization, immune privilege, and size. Various methodologies for therapeutic delivery in ocular diseases are under investigation that impact long-term efficacy, toxicity, invasiveness, and delivery range. While gene, cell, and antibody therapy and nanoparticle delivery directly treat regions that have been damaged by disease, they can be limited in the duration of the therapeutic delivery and have a focal effect. In contrast, contact lenses and ocular implants can more effectively achieve sustained and widespread delivery of therapies; however, they can increase dilution of therapeutics, which may result in reduced effectiveness. Current therapies either offer a sustained release or a broad therapeutic effect, and future directions should aim toward achieving both. This review discusses current ocular therapy delivery systems and their applications, mechanisms for delivering therapeutic products to ocular tissues, advantages and challenges associated with each delivery system, current approved therapies, and clinical trials. Future directions for the improvement in existing ocular therapies include combination therapies, such as combined cell and gene therapies, as well as AI-driven devices, such as cortical implants that directly transmit visual information to the cortex.

Three-dimensional (3D) cell culture using tumor spheroids provides a crucial platform for replicating tissue microenvironments. However, effective gene modulation via nanoparticle-based transfection remains a challenge, often facing delivery hurdles. Gold nanoparticles (AuNPs) with their tailored synthesis and biocompatibility, have shown promising results in two-dimensional (2D) cultures, nevertheless, they still require a comprehensive evaluation before they can reach its full potential on 3D models. While 2D cultures offer simplicity and affordability, they lack physiological fidelity. In contrast, 3D spheroids better capture in vivo conditions, enabling the study of cell interactions and nutrient distribution. These models are essential for investigating cancer behavior, drug responses, and developmental processes. Nevertheless, transitioning from 2D to 3D models demands an understanding of altered internalization mechanisms and microenvironmental influences. This study assessed ASO-AuNP conjugates for silencing the c-MYC oncogene in 2D cultures and 3D tumor spheroids, revealing distinctions in gene silencing efficiency and highlighting the microenvironment's impact on AuNP-mediated gene modulation. Herein, we demonstrate that increasing the number of AuNPs per cell by 2.6 times, when transitioning from a 2D cell model to a 3D spheroid, allows to attain similar silencing efficiencies. Such insights advance the development of targeted gene therapies within intricate tissue-like contexts.

This article covers the triazole-linked nucleic acids where the triazole linkage (TL) replaces the natural phosphate backbone. The replacement is done at either a few selected linkages or all the phosphate linkages. Two triazole linkages, the four-atom TL1 and the six-atom TL2, have been discussed in detail. These triazole-modified oligonucleotides have found a wide range of applications, from therapeutics to synthetic biology. For example, the triazole-linked oligonucleotides have been used in the antisense oligonucleotide (ASO), small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology as therapeutic agents. Due to the ease of the synthesis and a wide range of biocompatibility, the triazole linkage TL2 has been used to assemble a functional 300-mer DNA from alkyne- and azide-functionalized 100-mer oligonucleotides as well as an epigenetically modified variant of a 335 base-pair gene from ten short oligonucleotides. These outcomes highlight the potential of triazole-linked nucleic acids and open the doors for other TL designs and artificial backbones to fully exploit the vast potential of artificial nucleic acids in therapeutics, synthetic biology and biotechnology.

Vaccines for the deadliest brain tumor - glioblastoma (GBM) - are generally based on targeting growth factors or their receptors, often using antibodies. The vaccines described in the review were prepared to suppress the principal cancer growth factor - IGF-I, using anti-gene approaches either of antisense (AS) or of triple helix (TH) type. Our objective was to increase the median survival of patients treated with AS and TH cell vaccines.

The cells were transfected in vitro by both constructed IGF-I AS and IGF-I TH expression episomal vectors; part of these cells was co-cultured with plant phytochemicals, modulating IGF-I expression. Both AS and TH approaches completely suppressed IGF-I expression and induced MHC-1 / B7 immunogenicity related to the IGF-I receptor signal.

This immunogenicity proved to be stronger in IGF-I TH than in IGF-I AS-prepared cell vaccines, especially in TH / phytochemical cells. The AS and TH vaccines generated an important TCD8+ and TCD8+CD11b- immune response in treated GBM patients and increased the median survival of patients up to 17-18 months, particularly using TH vaccines; in some cases, 2- and 3-year survival was reported. These clinical results were compared with those obtained in therapies targeting other growth factors.

The anti-gene IGF-I vaccines continue to be applied in current GBM personalized medicine. Technical improvements in the preparation of AS and TH vaccines to increase MHC-1 and B7 immunogenicity have, in parallel, allowed to increase in the median survival of patients.

Many preclinical and clinical studies support the use of antisense oligonucleotides (ASOs) as effective therapeutic strategies. However, acquiring ASOs for research purposes may be limited by partnerships with the pharmaceutical companies. Our lab previously developed an effective ASO strategy to lower human tau and reverse pathology in aged tauopathy model mice. Testing the efficacy of purchased tau lowering ASOs would provide support for these reagents as broad research tools. Purchased mouse and human tau lowering ASOs were infused or injected intracerebroventricularly into wildtype and tau transgenic mice. Following treatment, brain tissue evaluated for ASO distribution and levels of tau mRNA, protein, and phosphorylated tau. We show that purchased ASOs enter cell types of the brain and effectively decrease mouse or human tau mRNA and protein levels. Human tau lowering ASO treatment in PS19 mice decreased phosphorylated tau and gliosis relative to saline-treated PS19 mice, consistent with our previous study using a non-commercial tau lowering ASO. The results of this study demonstrate the efficacy of purchased tau targeting ASOs in vivo to support their broad use by researchers.