Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology.

Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.

Safety, tolerability, pharmacokinetics and preliminary antitumour activity of an antisense oligonucleotide targeting STAT3 (danvatirsen) as monotherapy and in combination with durvalumab in Japanese patients with advanced solid malignancies: a phase 1 study.

OBJECTIVES: We assessed the safety, tolerability, pharmacokinetics, preliminary antitumour activity and pharmacodynamics of danvatirsen, an antisense oligonucleotide targeting signal transducer and activator of transcription 3 (STAT3), monotherapy and danvatirsen plus durvalumab, an antiprogrammed cell death ligand 1 monoclonal antibody, in patients with advanced solid malignancies. DESIGN: Phase 1, open-label study with two cohorts. SETTING: Two centres in Japan. PARTICIPANTS: Japanese individuals aged ≥20 years, with histologically confirmed solid malignancies, except for hepatocellular carcinoma, refractory to standard therapy. INTERVENTIONS: In cohort 1, patients received danvatirsen monotherapy; in cohort 2, patients received danvatirsen plus durvalumab combination therapy. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoint was safety and tolerability based on adverse events (AEs). Secondary endpoints were pharmacokinetics, immunogenicity, antitumour activity and pharmacodynamics. RESULTS: Eleven patients were assigned to treatment and included in the analysis. Danvatirsen dose reductions were only required in cohort 2 for hepatic function abnormal (alanine aminotransferase (ALT)/ aspartate aminotransferase (AST)/gamma-glutamyl transferase (γGT) increased), neutrophil count decreased and platelet count decreased. One patient experienced grade 3 ALT/AST increased and new appearance of eosinophilia as a dose-limiting toxicity. AEs were reported in 90.9% (10/11) patients. Commonly reported AEs causally related to the danvatirsen were platelet count decreased (60% (3/5)) and ALT/AST/γGT increased (50% (3/6)) in cohorts 1 and 2, respectively; none was causally related to durvalumab. One serious AE occurred in cohort 1 (pancreatitis; unrelated to study treatment). One case of ALT/AST/γGT increased occurred in cohort 2, leading to discontinuation. No AEs led to death. Danvatirsen did not accumulate in plasma after multiple dosing. In cohort 2, three patients had disease control at 12 weeks and one had unconfirmed partial response. STAT3 expression tended to decrease regardless of monotherapy or combination therapy. CONCLUSIONS: Danvatirsen was well tolerated by Japanese patients with advanced solid tumours as monotherapy and combined with durvalumab. No new safety signals arose. TRIAL REGISTRATION NUMBER: NCT03394144; ClinicalTrials.gov.

Validation and application of hybridization liquid chromatography-tandem mass spectrometry methods for quantitative bioanalysis of antisense oligonucleotides.

Background: Antisense oligonucleotide (ASO), an emerging modality in drug research and development, demands accurate and sensitive bioanalysis to understand its pharmacokinetic and pharmacodynamic properties. Results: By combining the advantages of both ligand binding and liquid chromatography-mass spectrometry/tandem mass (LC-MS/MS), hybridization LC-MS/MS methods were successfully developed and validated/qualified in a good lab practice (GLP) environment for the quantitation of an ASO drug candidate in monkey serum, cerebrospinal fluid (CSF) and tissues in the range of 0.5-500 ng/ml. Special treatment of CSF samples was employed to mitigate nonspecific binding, improve long-term storage stability and enable the usage of artificial CSF as a more accessible surrogate matrix. The method was also qualified and applied to ASO quantitation in various monkey tissue samples using a cocktail tissue homogenate as a surrogate matrix. Conclusion: This work was the first reported GLP validation and application of ASO bioanalysis using the hybridization LC-MS/MS platform.

Restoration of Nusinersen Levels Following Treatment Interruption in People With Spinal Muscular Atrophy: Simulations Based on a Population Pharmacokinetic Model.

BACKGROUND: Nusinersen is approved for the treatment of spinal muscular atrophy. The most common approved dosing regimen is four intrathecal loading doses of nusinersen 12 mg; the first three are administered at 14-day intervals followed by a fourth dose 30 days later, and then 12-mg maintenance doses are administered every 4 months thereafter. Interruption of nusinersen treatment in the maintenance dosing phase might occur for a number of clinical reasons. OBJECTIVE: The objective of this report is to describe dosing regimens that allow for the most rapid restoration of steady-state concentrations of nusinersen in the cerebrospinal fluid (CSF) following a treatment interruption during maintenance dosing. METHODS: Population pharmacokinetic models using integrated pharmacokinetic data from ten nusinersen clinical trials that included a broad range of participants with spinal muscular atrophy treated with intrathecal nusinersen were used to investigate different durations of treatment interruptions during maintenance treatment. Potential dosing regimens for re-initiation of nusinersen were evaluated, with the goal of achieving the quickest restoration of steady-state nusinersen CSF concentrations without exceeding maximal CSF exposures observed during the initial loading period. RESULTS: Our pharmacokinetic modeling indicates the following regimen will lead to optimal restoration of nusinersen CSF levels after treatment interruption: two doses of nusinersen should be administered at 14-day intervals following treatment interruptions of ≥ 8 to < 16 months since the last dose, and three doses of nusinersen at 14-day intervals for treatment interruptions of ≥ 16 to < 40 months since the last maintenance dose, with subsequent maintenance dosing every 4 months in both instances. After treatment interruptions of ≥ 40 months, the full loading regimen will rapidly restore nusinersen CSF levels. CONCLUSIONS: Prolonged treatment interruptions lead to suboptimal CSF levels of nusinersen. The optimal regimen to restore nusinersen CSF levels depends on the interval since the last maintenance dose was administered.

Nusinersen is a drug used to treat people of all ages who have spinal muscular atrophy. Nusinersen is injected with a thin needle into the lower back, a procedure known as a lumbar puncture. People initially receive three doses of nusinersen 12 mg each 14 days apart. They receive a fourth dose 1 month later, and then injections every 4 months (known as maintenance dosing). This treatment plan allows nusinersen to build up to effective levels in the fluid surrounding the spinal cord and brain. Some people may miss dose(s) or may stop nusinersen treatment at some point during maintenance dosing and then may want to continue treatment. This study used information from ten clinical trials to find out the best way to restart treatment to build up nusinersen to effective levels. People with a treatment break of ≥ 8 to < 16 months since the last dose need two doses of nusinersen at 14-day intervals before receiving maintenance dosing. People with a treatment break of ≥ 16 to < 40 months since the last dose need three doses of nusinersen at 14-day intervals before receiving maintenance dosing. If people stopped treatment for ≥ 40 months, they would need four doses before starting maintenance treatment. Results from this study showed that the number of doses that people needed before starting maintenance treatment depended on how long the treatment break was.

Antisense Oligonucleotide In Vitro Protein Binding Determination in Plasma, Brain, and Cerebral Spinal Fluid Using Hybridization LC-MS/MS.

The development of quantitative models for prediction of drug pharmacokinetics based on in vitro data has transformed early drug discovery. Drug unbound fraction (ƒu) characterization is a key consideration in pharmacokinetic and pharmacodynamic (PK/PD) modeling, assuming only unbound drug can interact with the target, and therefore has direct implications in the efficacy and potential toxicity of the drug. The current study describes the implementation of a hybridization liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform for the direct quantitation of antisense oligonucleotide (ASO) ƒu The method provides substantial improvements, including minimal matrix effects and high specificity when compared with previously used oligonucleotide ƒu detection methods such as ligand binding assays or liquid scintillation. The hybridization LC-MS/MS platform was integrated with ultracentrifugation, ultrafiltration, and equilibrium dialysis, and method performance for each technique was evaluated. Although ASO protein binding has been previously characterized in plasma, there were no studies that quantitated ASO ƒu in brain or cerebral spinal fluid (CSF). As ASOs continue to undergo clinical trials for neurologic and neuromuscular indications, ƒu characterization in brain and CSF can provide invaluable information about ASO distribution and target engagement in the central nervous system, therefore providing support for in vivo PK/PD data characterization. SIGNIFICANCE STATEMENT: A novel hybridization LC-MS/MS-based approach was successfully developed for the determination of ASO in vitro protein binding in plasma, and for the first time brain and CSF. Ultrafiltration, equilibrium dialysis, and ultracentrifugation were assessed for the separation of unbound ASO from biological matrices. The hybridization LC-MS/MS platform provided unique advantages, including minimal matrix effects and high specificity, compared with traditional ligand binding assays or liquid scintillation approaches, which enabled efficient and reliable in vitro protein binding assay.

Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides.

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.

Synthesis and Exon-Skipping Properties of a 3'-Ursodeoxycholic Acid-Conjugated Oligonucleotide Targeting DMD Pre-mRNA: Pre-Synthetic versus Post-Synthetic Approach.

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides.

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.

Delivering Antisense Oligonucleotides across the Blood-Brain Barrier by Tumor Cell-Derived Small Apoptotic Bodies.

The blood-brain barrier (BBB) is the most restrictive and complicated barrier that keeps most biomolecules and drugs from the brain. An efficient brain delivery strategy is urgently needed for the treatment of brain diseases. Based on the studies of brain-targeting extracellular vesicles (EVs), the potential of using small apoptotic bodies (sABs) from brain metastatic cancer cells for brain-targeting drug delivery is explored. It is found that anti-TNF-α antisense oligonucleotide (ASO) combined with cationic konjac glucomannan (cKGM) can be successfully loaded into sABs via a transfection/apoptosis induction process and that the sABs generated by B16F10 cells have an extraordinarily high brain delivery efficiency. Further studies suggest that ASO-loaded sABs (sCABs) are transcytosed by b. End3 (brain microvascular endothelial cells, BMECs) to penetrate the BBB, which is mediated by CD44v6, and eventually taken up by microglial cells in the brain. In a Parkinson's disease (PD) mouse model, sCABs dramatically ameliorate PD symptoms via the anti-inflammatory effect of ASO. This study suggests that sABs from brain metastatic cancer cells are excellent carriers for brain-targeted delivery, as they have not only an extraordinary delivery efficiency but also a much higher scale-up production potential than other EVs.

PK/PD modeling of FXI antisense oligonucleotides to bridge the dose-FXI activity relation from healthy volunteers to end-stage renal disease patients.

IONIS-FXIRX (BAY2306001) is an antisense oligonucleotide that inhibits the synthesis of coagulation factor XI (FXI) and has been investigated in healthy volunteers and patients with end-stage renal disease (ESRD). FXI-LICA (BAY2976217) shares the same RNA sequence as IONIS-FXIRX but contains a GalNAc-conjugation that facilitates asialoglycoprotein receptor (ASGPR)-mediated uptake into hepatocytes. FXI-LICA has been studied in healthy volunteers and is currently investigated in patients with ESRD on hemodialysis. We present a model-informed bridging approach that facilitates the extrapolation of the dose-exposure-FXI relationship from IONIS-FXIRX to FXI-LICA in patients with ESRD and, thus, supports the selection of FX-LICA doses being investigated in patients with ESRD. A two-compartment pharmacokinetic (PK) model, with mixed first- and zero-order subcutaneous absorption and first-order elimination, was combined with an indirect response model for the inhibitory effect on the FXI synthesis rate via an effect compartment. This PK/pharmacodynamic model adequately described the median trends, as well as the interindividual variabilities for plasma drug concentration and FXI activity in healthy volunteers of IONIS-FXIRX and FXI-LICA, and in patients with ESRD of IONIS-FXIRX . The model was then used to predict dose-dependent steady-state FXI activity following repeat once-monthly doses of FXI-LICA in a virtual ESRD patient population. Under the assumption of similar ASGPR expression in patients with ESRD and healthy volunteers, doses of 40 mg, 80 mg, and 120 mg FXI-LICA are expected to cover the target range of clinical interest for steady-state FXI activity in the phase IIb study of FXI-LICA in patients with ESRD undergoing hemodialysis.

A physiologically-based pharmacokinetic model to describe antisense oligonucleotide distribution after intrathecal administration.

Antisense oligonucleotides (ASOs) are promising therapeutic agents for a variety of neurodegenerative and neuromuscular disorders, e.g., Alzheimer's, Parkinson's and Huntington's diseases, spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), caused by genetic abnormalities or increased protein accumulation. The blood-brain barrier (BBB) represents a challenge to the delivery of systemically administered ASOs to the relevant sites of action within the central nervous system (CNS). Intrathecal (IT) delivery, in which drugs are administered directly into the cerebrospinal fluid (CSF) space, enables to bypass the BBB. Several IT-administered ASO therapeutics have already demonstrated clinical effect, e.g., nusinersen (SMA) and tofersen (ALS). Due to novelty of IT dosing for ASOs, very limited pharmacokinetic (PK) data is available and only a few modeling reports have been generated. The objective of this work is to advance fundamental understanding of whole-body distribution of IT-administered ASOs. We propose a physiologically-based pharmacokinetic modeling approach to describe the distribution along the neuroaxis based on PK data from non-human primate (NHP) studies. We aim to understand the key processes that drive and limit ASO access to the CNS target tissues. To elucidate the trade-off between parameter identifiability and physiological plausibility of the model, several alternative model structures were chosen and fitted to the NHP data. The model analysis of the NHP data led to important qualitative conclusions that can inform projection to human. In particular, the model predicts that the maximum total exposure in the CNS tissues, including the spinal cord and brain, is achieved within two days after the IT injection, and the maximum amount absorbed by the CNS tissues is about 4% of the administered IT dose. This amount greatly exceeds the CNS exposures delivered by systemic administration of ASOs. Clearance from the CNS is controlled by the rate of transfer from the CNS tissues back to CSF, whereas ASO degradation in tissues is very slow and can be neglected. The model also describes local differences in ASO concentration emerging along the spinal CSF canal. These local concentrations need to be taken into account when scaling the NHP model to human: due to the lengthier human spinal column, inhomogeneity along the spinal CSF may cause even higher gradients and delays potentially limiting ASO access to target CNS tissues.

Distribution and biotransformation of therapeutic antisense oligonucleotides and conjugates.

Drug properties of antisense oligonucleotides (ASOs) differ significantly from those of traditional small-molecule therapeutics. In this review, we focus on ASO disposition, mainly as characterized by distribution and biotransformation, of nonconjugated and conjugated ASOs. We introduce ASO chemistry to allow the following in-depth discussion on bioanalytical methods and determination of distribution and elimination kinetics at low concentrations over extended periods of time. The resulting quantitative data on the parent oligonucleotide, and the identification and quantification of formed metabolites define the disposition. Proper quantitative understanding of disposition is pivotal for nonclinical to clinical predictions, supports communication with health agencies, and increases the probability of delivering optimal ASO therapy to patients.

Pharmacokinetic-pharmacodynamic modeling for hepatic delivery and efficacy of antisense oligonucleotides with lipophilic ligands in mice.

Conjugation with lipophilic ligands such as cholesterol and α-tocopherol dramatically improves the delivery and efficacy of antisense oligonucleotides (ASOs) in the liver. To estimate the hepatic ASO concentration and the efficacy of ASOs conjugated with lipophilic ligands in mice, we constructed a pharmacokinetic-pharmacodynamic (PK-PD) model that consisted of a two-linear compartment model for the plasma and the hepatic ASO concentration, and two indirect response models for the hepatic apolipoprotein B (Apo-B) mRNA and plasma total cholesterol. The model provided a good fit of the hepatic ASO concentration although it showed an overprediction of Apo-B mRNA and an underprediction of the plasma total cholesterol within 2-fold at a later time after single intravenous administration of ASOs conjugated with lipophilic ligands. In addition, the model simulations indicated that the efficacy at a dose regimen of ASOs conjugated with lipophilic ligands (0.2 mg/kg, once a week) in mice was comparable to that at an effective dose of unchanged ASO (2.5 mg/kg, once a week). Although further studies are required to refine the parameters of the PK-PD model, this approach could be used to guide dose-ranging pharmacological studies for ASOs conjugated with lipophilic ligands in mice.

Advances in the Design of (Nano)Formulations for Delivery of Antisense Oligonucleotides and Small Interfering RNA: Focus on the Central Nervous System.

RNA-based therapeutics have emerged as one of the most powerful therapeutic options used for the modulation of gene/protein expression and gene editing with the potential to treat neurodegenerative diseases. However, the delivery of nucleic acids to the central nervous system (CNS), in particular by the systemic route, remains a major hurdle. This review will focus on the strategies for systemic delivery of therapeutic nucleic acids designed to overcome these barriers. Pathways and mechanisms of transport across the blood-brain barrier which could be exploited for delivery are described, focusing in particular on smaller nucleic acids including antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). Approaches used to enhance delivery including chemical modifications, nanocarrier systems, and target selection (cell-specific delivery) are critically analyzed. Learnings achieved from a comparison of the successes and failures reported for CNS delivery of ASOs versus siRNA will help identify opportunities for a wider range of nucleic acids and accelerate the clinical translation of these innovative therapies.

Safety, Pharmacokinetic, and Pharmacodynamic Evaluation of a 2'-(2-Methoxyethyl)-D-ribose Antisense Oligonucleotide-Triantenarry N-Acetyl-galactosamine Conjugate that Targets the Human Transmembrane Protease Serine 6.

Cellular uptake of antisense oligonucleotides (ASOs) is one of the main determinants of in vivo activity and potency. A significant advancement in improving uptake into cells has come through the conjugation of ASOs to triantenarry N-acetyl-galactosamine (GalNAc3), a ligand for the asialoglycoprotein receptor on hepatocytes. The impact for antisense oligonucleotides, which are already taken up into hepatocytes, is a 10-fold improvement in potency in mice and up to a 30-fold potency improvement in humans, resulting in overall lower effective dose and exposure levels. 2'-Methoxyethyl-modified antisense oligonucleotide conjugated to GalNAc3 (ISIS 702843) is specific for human transmembrane protease serine 6 and is currently in clinical trials for the treatment of ß-thalassemia. This report summarizes a chronic toxicity study of ISIS 702843 in nonhuman primates (NHPs), including pharmacokinetic and pharmacology assessments. Suprapharmacologic doses of ISIS 702843 were well tolerated in NHPs after chronic dosing, and the data indicate that the overall safety profile is very similar to that of the unconjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) ASOs. Notably, the GalNAc3 moiety did not cause any new toxicities nor exacerbate the known nonspecific class effects of the 2'-MOE ASOs. This observation was confirmed with multiple GalNAc3-MOE conjugates by querying a data base of monkey studies containing both GalNAc3-conjugated and unconjugated 2'-MOE ASOs. SIGNIFICANCE STATEMENT: This report documents the potency, pharmacology, and overall tolerability profile of a triantenarry N-acetyl-galactosamine (GalNAc3)-conjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) antisense oligonucleotide (ASO) specific to transmembrane protease serine 6 after chronic treatment in the cynomolgus monkey. Collective analysis of 15 independent GalNAc3-conjugated and unconjugated 2'-MOE ASOs shows the consistency in the dose response and character of hepatic and platelet tolerability across sequences that will result in much larger safety margins for the GalNAc3-conjugated 2'-MOE ASOs when compared with the unconjugated 2'-MOE ASOs given the increased potency.

Preclinical Nanomedicines for Polyglutamine-Based Neurodegenerative Diseases.

Polyglutamine (polyQ) diseases, such as Huntington's disease and several types of spinocerebellar ataxias, are dominantly inherited progressive neurodegenerative disorders and characterized by the presence of expanded CAG trinucleotide repeats in the respective disease locus of the patient genomes. Patients with polyQ diseases currently need to rely on symptom-relieving treatments because disease-modifying therapeutic interventions remain scarce. Many disease-modifying therapeutic agents are now under clinical testing for treating polyQ diseases, but their delivery to the brain is often too invasive (e.g., intracranial injection) or inefficient, owing to in vivo degradation and clearance by physiological barriers (e.g., oral and intravenous administration). Nanoparticles provide a feasible solution for improving drug delivery to the brain, as evidenced by an increasing number of preclinical studies that document the efficacy of nanomedicines for polyQ diseases over the past 5-6 years. In this review, we present the pathogenic mechanisms of polyQ diseases, the common animal models of polyQ diseases for evaluating the efficacy of nanomedicines, and the common administration routes for delivering nanoparticles to the brain. Next, we summarize the recent preclinical applications of nanomedicines for treating polyQ diseases and improving neurological conditions in vivo, placing emphasis on antisense oligonucleotides, small peptide inhibitors, and small molecules as the disease-modifying agents. We conclude with our perspectives of the burgeoning field of "nanomedicines for polyQ diseases", including the use of inorganic nanoparticles and potential drugs as next-generation nanomedicines, development of higher-order animal models of polyQ diseases, and importance of "brain-nano" interactions.

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration.

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS).

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides.

The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.

Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency.

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.