Targeting the Liver with Nucleic Acid Therapeutics for the Treatment of Systemic Diseases of Liver Origin.

Systemic diseases of liver origin (SDLO) are complex diseases in multiple organ systems, such as cardiovascular, musculoskeletal, endocrine, renal, respiratory, and sensory organ systems, caused by irregular liver metabolism and production of functional factors. Examples of such diseases discussed in this article include primary hyperoxaluria, familial hypercholesterolemia, acute hepatic porphyria, hereditary transthyretin amyloidosis, hemophilia, atherosclerotic cardiovascular diseases, α-1 antitrypsin deficiency-associated liver disease, and complement-mediated diseases. Nucleic acid therapeutics use nucleic acids and related compounds as therapeutic agents to alter gene expression for therapeutic purposes. The two most promising, fastest-growing classes of nucleic acid therapeutics are antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). For each listed SDLO disease, this article discusses epidemiology, symptoms, genetic causes, current treatment options, and advantages and disadvantages of nucleic acid therapeutics by either ASO or siRNA drugs approved or under development. Furthermore, challenges and future perspectives on adverse drug reactions and toxicity of ASO and siRNA drugs for the treatment of SDLO diseases are also discussed. In summary, this review article will highlight the clinical advantages of nucleic acid therapeutics in targeting the liver for the treatment of SDLO diseases. SIGNIFICANCE STATEMENT: Systemic diseases of liver origin (SDLO) contain rare and common complex diseases caused by irregular functions of the liver. Nucleic acid therapeutics have shown promising clinical advantages to treat SDLO. This article aims to provide the most updated information on targeting the liver with antisense oligonucleotides and small interfering RNA drugs. The generated knowledge may stimulate further investigations in this growing field of new therapeutic entities for the treatment of SDLO, which currently have no or limited options for treatment.

Unlocking the potential of chemically modified peptide nucleic acids for RNA-based therapeutics.

RNA therapeutics have emerged as next-generation therapy for the treatment of many diseases. Unlike small molecules, RNA targeted drugs are not limited by the availability of binding pockets on the protein, but rather utilize Watson-Crick (WC) base-pairing rules to recognize the target RNA and modulate gene expression. Antisense oligonucleotides (ASOs) present a powerful therapeutic approach to treat disorders triggered by genetic alterations. ASOs recognize the cognate site on the target RNA to alter gene expression. Nine single-stranded ASOs have been approved for clinical use and several candidates are in late-stage clinical trials for both rare and common diseases. Several chemical modifications, including phosphorothioates, locked nucleic acid, phosphorodiamidate, morpholino, and peptide nucleic acids (PNAs), have been investigated for efficient RNA targeting. PNAs are synthetic DNA mimics where the deoxyribose phosphate backbone is replaced by N-(2-aminoethyl)-glycine units. The neutral pseudopeptide backbone of PNAs contributes to enhanced binding affinity and high biological stability. PNAs hybridize with the complementary site in the target RNA and act by a steric hindrance--based mechanism. In the last three decades, various PNA designs, chemical modifications, and delivery strategies have been explored to demonstrate their potential as an effective and safe RNA-targeting platform. This review covers the advances in PNA-mediated targeting of coding and noncoding RNAs for a myriad of therapeutic applications.

New Technologies Bloom Together for Bettering Cancer Drug Conjugates.

Drug conjugates, including antibody-drug conjugates, are a step toward realizing Paul Ehrlich's idea from over 100 years ago of a "magic bullet" for cancer treatment. Through balancing selective targeting molecules with highly potent payloads, drug conjugates can target specific tumor microenvironments and kill tumor cells. A drug conjugate consists of three parts: a targeting agent, a linker, and a payload. In some conjugates, monoclonal antibodies act as the targeting agent, but new strategies for targeting include antibody derivatives, peptides, and even small molecules. Linkers are responsible for connecting the payload to the targeting agent. Payloads impact vital cellular processes to kill tumor cells. At present, there are 12 antibody-drug conjugates on the market for different types of cancers. Research on drug conjugates is increasing year by year to solve problems encountered in conjugate design, such as tumor heterogeneity, poor circulation, low drug loading, low tumor uptake, and heterogenous expression of target antigens. This review highlights some important preclinical research on drug conjugates in recent years. We focus on three significant areas: improvement of antibody-drug conjugates, identification of new conjugate targets, and development of new types of drug conjugates, including nanotechnology. We close by highlighting the critical barriers to clinical translation and the open questions going forward. SIGNIFICANCE STATEMENT: The development of anticancer drug conjugates is now focused in three broad areas: improvements to existing antibody drug conjugates, identification of new targets, and development of new conjugate forms. This article focuses on the exciting preclinical studies in these three areas and advances in the technology that improves preclinical development.

Targeted Suppression of miRNA-33 Using pHLIP Improves Atherosclerosis Regression.

BACKGROUND: miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. METHODS: We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. RESULTS: The anti-miR-33 conjugated pHLIP constructs are preferentially delivered to atherosclerotic plaque macrophages. The inhibition of miR-33 using pHLIP-directed macrophage targeting improves atherosclerosis regression by increasing collagen content and decreased lipid accumulation within vascular lesions. Single-cell RNA sequencing analysis revealed higher expression of fibrotic genes (Col2a1, Col3a1, Col1a2, Fn1, etc) and tissue inhibitor of metalloproteinase 3 (Timp3) and downregulation of Mmp12 in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. CONCLUSIONS: This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.

Role of miR-204 in segmental cardiac effects of phenylephrine and pressure overload.

Cardiotoxicity caused by adrenergic receptor agonists overdosing or stress-induced catecholamine release promotes cardiomyopathy, resembling Takotsubo cardiomyopathy (TC). TC is characterized by transient regional systolic dysfunction of the left ventricle. The animal models of TC and modalities for assessing regional wall motion abnormalities in animal models are lacking. We previously reported the protective role of a small noncoding microRNA-204-5p (miR-204) in cardiomyopathies, but its role in TC remains unknown. Here we compared the impact of miR-204 absence on phenylephrine (PE)-induced and transaortic constriction (TAC)-induced changes in cardiac muscle motion in the posterior and anterior apical, mid, and basal segments of the left ventricle using 2-dimensional speckle-tracking echocardiography (2-STE). Wildtype and miR-204-/- mice were subjected to cardiac stress in the form of PE for four weeks or TAC-induced pressure overload for five weeks. PE treatment increased longitudinal and radial motion in the apex of the left ventricle and shortened the peak motion time of all left ventricle segments. The TAC led to decreased longitudinal and radial motion in the left ventricle segments, and there was no difference in the peak motion time. Compared to wildtype mice, PE-induced peak cardiac muscle motion time in the anterior base of the left ventricle was significantly earlier in the miR-204-/- mice. There was no difference in TAC-induced peak cardiac muscle motion time between wildtype and miR-204-/- mice. Our findings demonstrate that PE and TAC induce regional wall motion abnormalities that 2-STE can detect. It also highlights the role of miR-204 in regulating cardiac muscle motion during catecholamine-induced cardiotoxicity.

Head on Comparison of Self- and Nano-assemblies of Gamma Peptide Nucleic Acid Amphiphiles.

Peptide nucleic acids (PNAs) are nucleic acid analogs with superior hybridization properties and enzymatic stability than deoxyribonucleic acid (DNA). In addition to gene targeting applications, PNAs have garnered significant attention as bio-polymer due to the Watson-Crick -based molecular recognition and flexibility of synthesis. Here, we engineered PNA amphiphiles using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA (γPNA) amphiphiles self-assemble into spherical vesicles. Further, we formulate nano-assemblies using the amphiphilic γPNA as a polymer via ethanol injection-based protocols. We perform comprehensive head-on comparison of the physicochemical and cellular uptake properties of PNA derived self- and nano-assemblies. Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) analysis reveal ellipsoidal morphology of γPNA nano-assemblies that results in superior cellular delivery compate to the spherical self-assembly. Next, we compare the functional activities of γPNA self-and nano-assemblies in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis-based assays. Overall, we establish that γPNA amphiphile is a functionally active bio-polymer to formulate nano-assemblies for a wide range of biomedical applications.

Absorption, Distribution, Metabolism, and Excretion of US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

Absorption, distribution, metabolism, and excretion (ADME) are the key biologic processes for determination of a drug's pharmacokinetic parameters, which have direct impacts on efficacy and adverse drug reactions (ADRs). The chemical structures, dosage forms, and sites and routes of administration are the principal determinants of ADME profiles and consequent impacts on their efficacy and ADRs. Newly developed large molecule biologic antisense oligonucleotide (ASO) drugs have completely unique ADME that is not fully defined. ASO-based drugs are single-stranded synthetic antisense nucleic acids with diverse modes of drug actions from induction of mRNA degradation, exon skipping and restoration, and interactions with proteins. ASO drugs have a great potential to treat certain human diseases that have remained untreatable with small molecule-based drugs. The ADME of ASO drugs contributes to their unique set of ADRs and toxicity. In this review, to better understand their ADME, the 10 US Food and Drug Administration (FDA)-approved ASO drugs were selected: fomivirsen, pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. A meta-analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization through endocytosis and nucleolytic degradation by endonucleases and exonucleases are major ADME features of the ASO drugs that differ from small-molecule drugs. The information summarized here provides comprehensive ADME characteristics of FDA-approved ASO drugs, leading to a better understanding of their therapeutic efficacy and their potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distribution, are identified, and future perspectives and directions are discussed. SIGNIFICANCE STATEMENT: Through a systematic analysis of the existing information of absorption, distribution, metabolism, and excretion (ADME) parameters for 10 US Food and Drug Administration (FDA)-approved antisense oligonucleotide (ASO) drugs, this review provides an overall view of the unique ADME characteristics of ASO drugs, which are distinct from small chemical drug ADME. This knowledge is useful for discovery and development of new ASO drugs as well as clinical use of current FDA-approved ASO drugs.

Adverse Drug Reactions and Toxicity of the Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

The market for large molecule biologic drugs has grown rapidly, including antisense oligonucleotide (ASO) drugs. ASO drugs work as single-stranded synthetic oligonucleotides that reduce production or alter functions of disease-causing proteins through various mechanisms, such as mRNA degradation, exon skipping, and ASO-protein interactions. Since the first ASO drug, fomivirsen, was approved in 1998, the U.S. Food and Drug Administration (FDA) has approved 10 ASO drugs to date. Although ASO drugs are efficacious in treating some diseases that are untargetable by small-molecule chemical drugs, concerns on adverse drug reactions (ADRs) and toxicity cannot be ignored. Illustrative of this, mipomersen was recently taken off the market due to its hepatotoxicity risk. This paper reviews ADRs and toxicity from FDA drug labeling, preclinical studies, clinical trials, and postmarketing real-world studies on the 10 FDA-approved ASO drugs, including fomivirsen and pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. Unique and common ADRs and toxicity for each ASO drug are summarized here. The risk of developing hepatotoxicity, kidney toxicity, and hypersensitivity reactions co-exists for multiple ASO drugs. Special precautions need to be in place when certain ASO drugs are administrated. Further discussion is extended on studying the mechanisms of ADRs and toxicity of these drugs, evaluating the existing physiologic and pathologic states of patients, optimizing the dose and route of administration, and formulating personalized treatment plans to improve the clinical utility of FDA-approved ASO drugs and discovery and development of new ASO drugs with reduced ADRs. SIGNIFICANCE STATEMENT: The current review provides a comprehensive analysis of unique and common ADRs and the toxicity of FDA-approved ASO drugs. The information can help better manage the risk of severe hepatotoxicity, kidney toxicity, and hypersensitivity reactions in the usage of currently approved ASO drugs and the discovery and development of new and safer ASO drugs.

Simultaneous Targeting of Multiple oncomiRs with Phosphorothioate or PNA-Based Anti-miRs in Lymphoma Cell Lines.

PURPOSE: MicroRNAs (miRNAs) are short (~ 22 nts) RNAs that regulate gene expression via binding to mRNA. MiRNAs promoting cancer are known as oncomiRs. Targeting oncomiRs is an emerging area of cancer therapy. OncomiR-21 and oncomiR-155 are highly upregulated in lymphoma cells, which are dependent on these oncomiRs for survival. Targeting specific miRNAs and determining their effect on cancer cell progression and metastasis have been the focus of various studies. Inhibiting a single miRNA can have a limited effect, as there may be other overexpressed miRNAs present that may promote tumor proliferation. Herein, we target miR-21 and miR-155 simultaneously using nanoparticles delivered two different classes of antimiRs: phosphorothioates (PS) and peptide nucleic acids (PNAs) and compared their efficacy in lymphoma cell lines. METHODS: Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NPs) containing PS and PNA-based antimiR-21 and -155 were formulated, and comprehensive NP characterizations: morphology (scanning electron microscopy), size (differential light scattering), and surface charge (zeta potential) were performed. Cellular uptake analysis was performed using a confocal microscope and flow cytometry analysis. The oncomiR knockdown and the effect on downstream targets were confirmed by gene expression (real time-polymerase chain reaction) assay. RESULTS: We demonstrated that simultaneous targeting with NP delivered PS and PNA-based antimiRs resulted in significant knockdown of miR-21 and miR-155, as well as their downstream target genes followed by reduced cell viability ex vivo. CONCLUSIONS: This project demonstrated that targeting miRNA-155 and miR-21 simultaneously using nanotechnology and a diverse class of antisense oligomers can be used as an effective approach for lymphoma therapy.

Serine-γPNA, Invader probes, and chimeras thereof: three probe chemistries that enable sequence-unrestricted recognition of double-stranded DNA.

Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence in situ hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), i.e., single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with (R)-hydroxymethyl moieties, (ii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of 2'-O-(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, i.e., duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, with Invader probes resulting in the most favorable and consistent characteristics (signals in >90% of interphase nuclei against a low background and no signal in negative control experiments). These probe chemistries constitute valuable additions to the molecular toolbox needed for DNA-targeting applications.

Evidence that miR-152-3p is a positive regulator of SETDB1-mediated H3K9 histone methylation and serves as a toggle between histone and DNA methylation.

SETDB1 is a histone methyltransferase that converts H3K9me2 to H3K9me3. SETDB1 activity and H3K9me3 are crucial for the formation of obligately silenced heterochromatin such as that of centromeres. Here we show that a microRNA, miR-152-3p, is involved in the regulation of SETDB1 protein levels, but surprisingly, miR-152-3p plays a positive regulatory role for SETDB1 expression. Inhibition of miR-152-3p by anti-miR treatment resulted in a robust reduction in SETDB1 protein levels, though SETDB1 mRNA levels were unaffected. This was also accompanied by a blockade of the biochemical pathway proceeding from H3K9me2 to H3K9me3 as evidenced by quantitative nucleosome ELISA assays that showed that H3K9me2 accumulates in cells treated with an anti-miR that targets miR-152-3p. In addition, the action of a miR-152-3p mimic increased flux of the reaction leading to H3K9me3. We also performed site-directed mutagenesis of three predicted miR-152-3p target recognition sequences to yield three precise deletions. Deletion of one of the three sites recapitulated the positive regulatory aspect of the action of miR-152-3p upon SETDB1 expression in a luciferase reporter assay. Previous studies have shown that miR-152-3p negatively regulates DNMT1, the sole maintenance DNA methyltransferase which is required for levels of 5-methylcytosine levels within DNA. Our results shown that miR-152-3p positively regulates the production of H3K9me3 by regulating the production of SETDB1. Therefore, our findings provide strong evidence that miR-152-3p can serve as a toggle switch that regulates the balance between DNA methylation and H3K9 histone methylation in constitutive heterochromatin.

MicroRNA silencing for cancer therapy targeted to the tumour microenvironment.

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

Tumor-Targeted, Cytoplasmic Delivery of Large, Polar Molecules Using a pH-Low Insertion Peptide.

Tumor-targeted drug delivery systems offer not only the advantage of an enhanced therapeutic index, but also the possibility of overcoming the limitations that have largely restricted drug design to small, hydrophobic, "drug-like" molecules. Here, we explore the ability of a tumor-targeted delivery system centered on the use of a pH-low insertion peptide (pHLIP) to directly deliver moderately polar, multi-kDa molecules into tumor cells. A pHLIP is a short, pH-responsive peptide capable of inserting across a cell membrane to form a transmembrane helix at acidic pH. pHLIPs target the acidic tumor microenvironment with high specificity, and a drug attached to the inserting end of a pHLIP can be translocated across the cell membrane during the insertion process. We investigate the ability of wildtype pHLIP to deliver peptide nucleic acid (PNA) cargoes of varying sizes across lipid membranes. We find that pHLIP effectively delivers PNAs up to ∼7 kDa into cells in a pH-dependent manner. In addition, pHLIP retains its tumor-targeting capabilities when linked to cargoes of this size, although the amount delivered is reduced for PNA cargoes greater than ∼6 kDa. As drug-like molecules are traditionally restricted to sizes of ∼500 Da, this constitutes an order-of-magnitude expansion in the size range of deliverable drug candidates.

Applications of PNA-laden nanoparticles for hematological disorders.

Safe and efficient genome editing has been an unmitigated goal for biomedical researchers since its inception. The most prevalent strategy for gene editing is the use of engineered nucleases that induce DNA damage and take advantage of cellular DNA repair machinery. This includes meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems. However, the clinical viability of these nucleases is marred by their off-target cleavage activity (≥ 50% in RNA-guided endonucleases). In addition, in vivo applications of CRISPR require systemic administration of Cas9 protein, mRNA, or DNA, which presents a significant delivery challenge. The development of nucleic acid probes that can recognize specific double-stranded DNA (dsDNA) regions and activate endogenous DNA repair machinery holds great promise for gene editing applications. Triplex-forming oligonucleotides (TFOs), which were introduced more than 25 years ago, are among the most extensively studied oligomeric dsDNA-targeting agents. TFOs bind duplex DNA to create a distorted helical structure, which can stimulate DNA repair and the exchange of a nearby mutated region-otherwise leading to an undesired phenotype-for a short single-stranded donor DNA that contains the corrective nucleotide sequence. Recombination can be induced within several hundred base-pairs of the TFO binding site and has been shown to depend on triplex-induced initiation of the nucleotide excision repair pathway and engagement of the homology-dependent repair pathway. Since TFOs do not possess any direct nuclease activity, their off-target effects are minimal when compared to engineered nucleases. This review comprehensively covers the advances made in peptide nucleic acid-based TFOs for site-specific gene editing and their therapeutic applications.

Role of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy.

The field of gene therapy has experienced an insurgence of attention for its widespread ability to regulate gene expression by targeting genomic DNA, messenger RNA, microRNA, and short-interfering RNA for treating malignant and non-malignant disorders. Numerous nucleic acid analogs have been developed to target coding or non-coding sequences of the human genome for gene regulation. However, broader clinical applications of nucleic acid analogs have been limited due to their poor cell or organ-specific delivery. To resolve these issues, non-viral vectors based on nanoparticles, liposomes, and polyplexes have been developed to date. This review is centered on non-viral vectors mainly comprising of cationic lipids and polymers for nucleic acid-based delivery for numerous gene therapy-based applications.

Investigation of PLGA nanoparticles in conjunction with nuclear localization sequence for enhanced delivery of antimiR phosphorothioates in cancer cells in vitro.

Numerous first generation phosphorothioates (PS) and their derivatives have shown promise targeting mRNA for therapeutic applications and also gained market approval for their use as a drug. However, PS have not been explored for targeting microRNAs (miRNAs or miRs). In particular, efficient delivery remains a critical cog in PS-based antimiR applications. In this study, we tested and characterized a series of poly-lactic-co-glycolic-acid (PLGA) polymers of different molecular weights that can encapsulate the optimum amount of antimiR-155 PS with uniform morphology and surface charge density. We found that nuclear localization sequence substantially increases loading of antimiR-155 PS in PLGA nanoparticles. Further, in a battery of cell culture studies, we confirmed that PLGA nanoparticles encapsulated nuclear localization sequence/antimiR-155 PS combination undergoes significant intracellular delivery and results in reduced expression of miR-155. In conclusion, we successfully demonstrate the feasibility and promise of optimized PLGA nanoparticles based PS delivery in combination with nuclear localization sequence for antimiRs based therapeutics.

Design of a "Mini" Nucleic Acid Probe for Cooperative Binding of an RNA-Repeated Transcript Associated with Myotonic Dystrophy Type 1.

Toxic RNAs containing expanded trinucleotide repeats are the cause of many neuromuscular disorders, one being myotonic dystrophy type 1 (DM1). DM1 is triggered by CTG-repeat expansion in the 3'-untranslated region of the DMPK gene, resulting in a toxic gain of RNA function through sequestration of MBNL1 protein, among others. Herein, we report the development of a relatively short miniPEG-γ peptide nucleic acid probe, two triplet repeats in length, containing terminal pyrene moieties, that is capable of binding rCUG repeats in a sequence-specific and selective manner. The newly designed probe can discriminate the pathogenic rCUGexp from the wild-type transcript and disrupt the rCUGexp-MBNL1 complex. The work provides a proof of concept for the development of relatively short nucleic acid probes for targeting RNA-repeat expansions associated with DM1 and other related neuromuscular disorders.

RNA-Templated Concatenation of Triplet Nucleic-Acid Probe.

Template-directed synthesis offers several distinct benefits over conventional laboratory creation, including unsurpassed reaction rate and selectivity. Although it is central to many biological processes, such an approach has rarely been applied to the in situ synthesis and recognition of biomedically relevant target. Towards this goal, we report the development of a three-codon nucleic-acid probe containing a C-terminal thioester group and an N-terminal cysteine that is capable of undergoing template-directed oligomerization in the presence of an RNA target and self-deactivation in its absence. The work has implications for the development of millamolecular nucleic-acid probes for targeting RNA-repeated expansions associated with myotonic dystrophy type 1 and other related neuromuscular and neurodegenerative disorders.

Therapeutic Peptide Nucleic Acids: Principles, Limitations, and Opportunities.

Since their invention in 1991, peptide nucleic acids (PNAs) have been used in a myriad of chemical and biological assays. More recently, peptide nucleic acids have also been demonstrated to hold great potential as therapeutic agents because of their physiological stability, affinity for target nucleic acids, and versatility. While recent modifications in their design have further improved their potency, their preclinical development has reached new heights due to their combination with recent advancements in drug delivery. This review focuses on recent advances in PNA therapeutic applications, in which chemical modifications are made to improve PNA function and nanoparticles are used to enhance PNA delivery.

Mechanisms of Action of the US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

Antisense oligonucleotides (ASOs) are single stranded nucleic acids that target RNA. The US Food and Drug Administration has approved ASOs for several diseases. ASOs utilize three principal modes of action (MOA). The first MOA is initiated by base-pairing between the ASO and its target mRNA, followed by RNase H-dependent mRNA degradation. The second MOA is triggered by ASOs that occlude splice acceptor sites in pre-mRNAs leading to skipping of a mutation-bearing exon. The third MOA involves ASOs that sterically hinder mRNA function, often inhibiting translation. ASOs contain a variety of modifications to the sugar-phosphate backbone and bases that stabilize the ASO or render them resistant to RNase activity. RNase H-dependent ASOs include inotersen and eplontersen (for hereditary transthyretin amyloidosis), fomiversen (for opportunistic cytomegalovirus infection), mipomersen (for familial hypercholesterolemia), and tofersen [for amyotrophic lateral sclerosis (ALS)]. Splice modulating ASOs include nursinersen (for spinal muscular atrophy) and eteplirsen, golodirsen, viltolarsen, and casimersen (all for the treatment of Duchenne muscular dystrophy). In addition, a designer ASO, milasen, was used to treat a single individual afflicted with Batten disease. Since ASO design relies principally upon knowledge of mRNA sequence, the bench to bedside pipeline for ASOs is expedient compared with protein-directed drugs. [Graphical abstract available.].