Response of plasma microRNAs to nusinersen treatment in patients with SMA.

OBJECTIVE: Spinal muscular atrophy (SMA) is a common genetic cause of infant mortality. Nusinersen treatment ameliorates the clinical outcome of SMA, however, some patients respond well, while others have limited response. We investigated microRNAs in blood samples from SMA patients and their response to nusinersen treatment evaluating the potential of circulating microRNAs as biomarkers for SMA. METHODS: In a discovery cohort study, microRNA next-generation sequencing was performed in blood samples from SMA patients (SMA type 2, n = 10; SMA type 3, n = 10) and controls (n = 7). The dysregulated microRNAs were further analysed in the therapeutic response cohort comprised of SMA type 1 patients (n = 22) who had received nusinersen treatment, at three time points along the treatment course (baseline, 2 and 6 months of treatment). The levels of the studied microRNAs were correlated to the SMA clinical outcome measures. RESULTS: In the discovery cohort, 69 microRNAs were dysregulated between SMA patients and controls. In the therapeutic response cohort, the baseline plasma levels of miR-107, miR-142-5p, miR-335-5p, miR-423-3p, miR-660-5p, miR-378a-3p and miR-23a-3p were associated with the 2 and 6 months response to nusinersen treatment. Furthermore, the levels of miR-107, miR-142-5p, miR-335-5p, miR-423-3p, miR-660-5p and miR-378-3p at 2 months of treatment were associated with the response after 6 months of nusinersen treatment. INTERPRETATION: Blood microRNAs could be used as biomarkers to indicate SMA patients' response to nusinersen and to monitor the efficacy of the therapeutic intervention. In addition, some of these microRNAs provide insight into processes involved in SMA that could be exploited as novel therapeutic targets.

G4 Matters-The Influence of G-Quadruplex Structural Elements on the Antiproliferative Properties of G-Rich Oligonucleotides.

G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich sequences of DNA or RNA that have attracted increased attention as anticancer agents. This systematic study aimed to investigate the anticancer potential of five G4-forming, sequence-related DNA molecules in terms of their thermodynamic and structural properties, biostability and cellular uptake. The antiproliferative studies revealed that less thermodynamically stable G4s with three G-tetrads in the core and longer loops are more predisposed to effectively inhibit cancer cell growth. By contrast, highly structured G4s with an extended core containing four G-tetrads and longer loops are characterized by more efficient cellular uptake and improved biostability. Various analyses have indicated that the G4 structural elements are intrinsic to the biological activity of these molecules. Importantly, the structural requirements are different for efficient cancer cell line inhibition and favorable G4 cellular uptake. Thus, the ultimate antiproliferative potential of G4s is a net result of the specific balance among the structural features that are favorable for efficient uptake and those that increase the inhibitory activity of the studied molecules. Understanding the G4 structural features and their role in the biological activity of G-rich molecules might facilitate the development of novel, more potent G4-based therapeutics with unprecedented anticancer properties.

Age and baseline values predict 12 and 24-month functional changes in type 2 SMA.

The aim of this retrospective study was to establish the range of functional changes at 12 and 24-month in 267 type 2 Spinal Muscular Atrophy (SMA) patients with multiple assessments. We included 652 Hammersmith Functional Motor Scale Expanded (HFMSE) assessments at 12 month- and 305 at 24 month- intervals. The cohort was subdivided by functional level, Survival of Motor Neuron copy number and age. Stable scores (± 2 points) were found in 68% at 12 months and in 55% at 24 months. A decrease ≥2 points was found in 21% at 12 months and in 35% at 24 months. An increase ≥2 points was found in 11% at 12 months and 9.5% at 24 months. The risk of losing ≥2 points increased with age and HFMSE score at baseline both at 12 and 24-month. For each additional HFMSE point at baseline, the relative risk of a >2 point decline at 12 months increases by 5% before age 5 years (p = 0.023), by 8% between 5 and 13 (p<0.001) and by 26% after 13 years (p = 0.003). The combination of age and HFMSE scores at baseline increased the ability to predict progression in type 2 SMA.

Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

Population Pharmacokinetic-Pharmacodynamic Modeling of Inotersen, an Antisense Oligonucleotide for Treatment of Patients with Hereditary Transthyretin Amyloidosis.

A population pharmacokinetic (PK) and pharmacodynamic (PD) model was developed for inotersen to evaluate exposure-response relationships and to optimize therapeutic dosing regimen in patients with hereditary transthyretin (TTR) amyloidosis polyneuropathy (hATTR-PN). Inotersen PK and TTR level (PD) data were composed of one Phase 1 study in healthy subjects, one Phase 2/3 study in hATTR patients, and its one open-label extension study. Effects of intrinsic and extrinsic factors (covariates) on PK and PK/PD of inotersen were evaluated using a full model approach. Inotersen PK was characterized by a two-compartment model with elimination from the central compartment. The population PK analysis identified disease status and lean body mass (LBM) as significant covariates for inotersen PK. Nonetheless, the contribution of disease status and LBM on PK was small, as the difference in clearance (CL/F) was 11.1% between healthy subjects and patients with hATTR-PN and 38% between the lowest and highest LBM quartiles of the patient population. Age, race, sex, baseline renal function estimated glomerular filtration rate, and hepatic function markers (baseline albumin, bilirubin, and alanine aminotransferase values) were not statistically significant covariates affecting inotersen PK. An inhibitory effect indirect-response model (inhibition of TTR production) was used to describe the drug effect on TTR-time profiles, with baseline TTR included as a covariate. The overall population Imax and IC50, together with 95% confidence interval, was estimated to be 0.913 (0.899-0.925) and 9.07 (8.08-10.1) ng/mL, respectively. V30M mutation showed no effect on the estimated IC50 value for hATTR patients. The final population PK and PK/PD model was used to simulate four different treatment regimens. The population PK/PD model developed well described the PK and PD of inotersen in patients with hATTR-PN and has been used for label recommendation and trial simulations.

Multiplexed Continuous Biosensing by Single-Molecule Encoded Nanoswitches.

Single-molecule techniques have become impactful in bioanalytical sciences, though the advantages for continuous biosensing are yet to be discovered. Here we present a multiplexed, continuous biosensing method, enabled by an analyte-sensitive, single-molecular nanoswitch with a particle as a reporter. The nanoswitch opens and closes under the influence of single target molecules. This reversible switching yields binary transitions between two highly reproducible states, enabling reliable quantification of the single-molecule kinetics. The multiplexing functionality is encoded per particle via the dissociation characteristics of the nanoswitch, while the target concentration is revealed by the association characteristics. We demonstrate by experiments and simulations the multiplexed, continuous monitoring of oligonucleotide targets, at picomolar concentrations in buffer and in filtered human blood plasma.

LC-MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction.

Aim: Quantitative LC-MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. Methodology & results: A set of 20 single stranded antisense oligonucleotides (ASO) and five siRNAs were extracted from plasma and tissue homogenates. Anion Exchange (AEX) SPE was selected as generic extraction approach, resulting in recoveries from plasma >70%. Extraction from tissue homogenates showed often more variation and lower recoveries. A proof of concept of a novel tailored hybridization extraction is demonstrated for two 16-mer reference OGNs. Conclusion: Two methods for extraction of OGNs were investigated and applied for quantitative analysis. The AEX-SPE is considered a more generic approach preferred when multiple compounds are evaluated. Hybridization extraction has great potential but critical reagents per analyte are needed.

Oligonucleotide quantification and metabolite profiling by high-resolution and accurate mass spectrometry.

Aim: Advancements in RNA interference therapeutics have triggered development of improved bioanalytical methods for oligonucleotide metabolite profiling and high-throughput quantification in biological matrices. Results & methodology: HPLC coupled with high-resolution mass spectrometry (LC-HRMS) methods were developed to investigate the metabolism of a REVERSIR™ molecule in vivo. Plasma and tissue samples were extracted using solid-phase extraction followed by LC-HRMS analysis for metabolite profiling and quantification. The method was qualified from 10 to 5000 ng/ml (plasma) and 100 to 50000 ng/g (liver and kidney). In rat liver, intra and interday accuracy ranged from 80.9 to 118.5% and 88.4 to 111.9%, respectively, with acceptable precision (<20% CV). Conclusion: The LC-HRMS method can be applied for metabolite profiling and quantification of oligonucleotides in biological matrices.

Importance of probe design for bioanalysis of oligonucleotides using hybridization-based LC-fluorescence assays.

Aim: The importance of the length and/or structure of fluorescently labeled PNA (peptide nucleic acid) probes for quantitative determination of oligodeoxynucleotides (ODNs) is demonstrated in human plasma using hybridization-based LC-fluorescence assays. The length of the PNA probes impacts the peak shape and chromatographic separation of the resulting PNA/ODN hybridization complexes and affects assay sensitivity, dynamic range and carryover. Methods: For quantitative determination of an 18-mer phosphodiester ODN (DNL1818) in human plasma, an assay utilizing an Atto dye-labeled 12-mer PNA probe provided a linear quantitation range of 0.1-50 ng/ml with excellent accuracy and precision (within -5.3-7.73%). Conclusion: This method provides a convenient method for sensitive and specific quantification of ODNs in biological matrix with limited sample volume and no special extraction.

An oligonucleotide bioanalytical LC-SRM methodology entirely liberated from ion-pairing.

Aim: Reliable quantitative LC-MS methodology has been established and validated for an oligonucleotide in plasma in a fresh and unique fashion, free of ion-pairing reagents and the various associated deleterious effects from primary solution preparation through sample preparation and extraction to the LC-MS analytical end point, offering a highly selective mixed-mode solid-phase extraction with hydrophilic-interaction liquid chromatography as the chromatographic element prior to SRM detection. Results: Inter- and intra-assay accuracy and precision ranged from 97.9 to 111% and 2.75 to 9.66%, respectively. Recoveries of 50% were attained, and there was no significant matrix effect manifestation. Conclusion: The method demonstrated rugged performance and reliability under the optimized conditions, indicating a possible exciting new avenue, free of ion-pairing, for general application in oligonucleotide quantitative LC-MS.

NiO Nanoparticles for Exceptionally Stable DNA Adsorption and Its Extraction from Biological Fluids.

Selective extraction of a small amount of nucleic acids from complex biological samples containing a high concentration of proteins is critical for bioanalytical chemistry. A number of previously published studies have focused on long, double-stranded DNA such as plasmid DNA. On the other hand, we are interested in short oligonucleotides. Nucleic acids have a negatively charged phosphate backbone that interacts with metal oxides strongly, and this may be used to distinguish them from proteins. In this work, a few metal oxide nanoparticles were screened, including NiO, CoO, ZnO, TiO2, CeO2, and Fe3O4 for DNA recovery. NiO had the highest DNA adsorption efficiency from mixtures containing bovine serum albumin or human blood serum. The adsorption of DNA by NiO was further characterized as a function of the pH, salt concentration, DNA length, and DNA sequence. The adsorption mechanism was studied by adding competing chemicals or denaturing agents. A striking observation was the extremely high adsorption affinity of NiO, much higher than that of the other tested oxides. Polyphosphate was the most effective agent for displacing adsorbed DNA, whereas simple inorganic phosphate was less effective. NiO was able to concentrate DNA from a serum mixture by 33- to 55-fold, depending on the serum concentration. NiO is thus a promising candidate for extracting DNA from biological samples.

Quantitative analysis of imetelstat in plasma with LC-MS/MS using solid-phase or hybridization extraction.

AIM: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC-MS/MS. Calibration curves were established (0.1-50 µg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC-MS/MS were comparable with a validated ELISA. CONCLUSION: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC-MS/MS. The hybridization extraction in combination with LC-MS/MS is a novel extraction approach.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

Population Pharmacokinetics of Nusinersen in the Cerebral Spinal Fluid and Plasma of Pediatric Patients With Spinal Muscular Atrophy Following Intrathecal Administrations.

Nusinersen is an antisense oligonucleotide intended for the treatment of spinal muscular atrophy. The pharmacokinetics of nusinersen, following intrathecal administrations, in the cerebrospinal fluid (CSF) and plasma of 72 pediatric patients (3 months to 17 years) with spinal muscular atrophy across 5 clinical trials was analyzed via population-based modeling. With sparse data in the CSF and profile data in the plasma, a linear 4-compartment model simultaneously described the time-concentration profiles in both matrices. The typical population parameters were: Qp = 0.572 L/h, QCSF = 0.069 L/h, CLp = 2.50 L/h, CLCSF = 0.133 L/hr, VCSF = 0.441 L, Vp = 32.0 L, Vsystemic_tissue = 429 L, and VCNS_tissue = 258 L. A full covariate modeling approach identified baseline body weight to be a statistically and clinically relevant covariate on VCSF , Vp , and CLp . The model predicted that the CSF volume of distribution increased steadily with age from 0 to 2 years but became relatively steady for children >2 years. Simulations from the final model showed that age-based dosing in children under 2 years ensured a more comparable exposure (peak concentration and area under the concentration-time curve) across subjects in the population relative to a fixed dosing scheme. However, because no dose-limiting toxicity has been reported in any of the trials, a fixed-dose scheme (12 mg across all age groups) was recommended. The median terminal half-life of nusinersen in the CSF was determined from the model to be 163 days, which supported infrequent dosing, once every 4 to 6 months in pediatric patients with spinal muscular atrophy.

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation.

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides that target the BRAF V600E oncogene, with a library of rationally designed peptides employing CuAAC "click" chemistry. The peptide sequence has an influence on the specificity and affinity of target DNA/RNA binding. We also investigated the impact of locked nucleic acids (LNAs) on the latter. Lysine residues improve binding of POCs to target DNA and RNA, whereas the distance to lysine correlates exclusively with a decrease in binding of mismatched RNA targets. Glycine and tyrosine residues affect target binding as well. Importantly, the resistance of POCs to enzymatic degradation is dramatically improved by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.

The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified oligonucleotides in serum samples by liquid chromatography coupled with tandem mass spectrometry.

Present study highlights the usage of various ion-pairing agents and their impact on the process of separation and ionization of oligonucleotides in the fortified human serum samples. What is more, retention studies involved different stationary phases, including: octadecyl, phenyl, pentafluorophenyl groups and ligands with embedded polar groups. It was proved that retention of oligonucleotides strongly depends on the alkyl chain branching in the structure of ion pairing reagent. Furthermore ion-pairing agents build of straight alkyl chain are more easily adsorbed on the stationary phase modified with octadecyl groups, while branching of alkyl chain caused more effective adsorption of studied compounds at phenyl groups compared to octadecyl ones. The lowest limit of quantification values were obtained for 5mMN,N-dimethylbutylamine, while the highest values are characteristic for hexylamine. Moreover it was shown that a 2-fold increase of ion-pairing agent concentration results in higher LOQ. The greatest sensitivity was obtained for 2.5mMN,N-dimethylbutylamine/150mM hexafluoroisopropanol. On the other hand Hypersil GOLD aQ column was the most appropriate in terms of time and separation effectiveness. The developed method was successfully used for the determination of studied oligonucleotides and their metabolites in human serum samples. The compounds were separated in just 3.5min with high sensitivity (0.09-0.16ng).

Controlling DNA-nanoparticle serum interactions.

Understanding the interaction of molecularly assembled nanoparticles with physiological fluids is critical to their use for in vivo delivery of drugs and contrast agents. Here, we systematically investigated the factors and mechanisms that govern the degradation of DNA on the nanoparticle surface in serum. We discovered that a higher DNA density, shorter oligonucleotides, and thicker PEG layer increased protection of DNA against serum degradation. Oligonucleotides on the surface of nanoparticles were highly resistant to DNase I endonucleases, and degradation was carried out exclusively by protein-mediated exonuclease cleavage and full-strand desorption. These results enabled the programming of the degradation rates of the DNA-assembled nanoparticle system from 0.1 to 0.7 h-1 and the engineering of superstructures that can release two different preloaded dye molecules with distinct kinetics and half-lives ranging from 3.3 to 9.8 h. This study provides a general framework for investigating the serum stability of DNA-containing nanostructures. The results advance our understanding of engineering principles for designing nanoparticle assemblies with controlled in vivo behavior and present a strategy for storage and multistage release of drugs and contrast agents that can facilitate the diagnosis and treatment of cancer and other diseases.

No effect on QT intervals of mipomersen, a 2'-O-methoxyethyl modified antisense oligonucleotide targeting ApoB-100 mRNA, in a phase I dose escalation placebo-controlled study, and confirmed by a thorough QT (tQT) study, in healthy subjects.

PURPOSE: The aim of this study to evaluate the effect of mipomersen on QT intervals in a phase I dose escalation, placebo-controlled study, and a thorough QT (tQT) study in healthy subjects. METHODS: In the initial phase I study, 29 healthy subjects received either single or multiple (for 4 weeks) ascending doses of mipomersen (50-400 mg) administered subcutaneously (SC) or via a 2-h intravenous (IV) infusion, and 7 subjects received placebo. In the confirmative tQT study, 58 healthy subjects received placebo, 400 mg IV moxifloxacin, 200 mg SC, or 200 mg IV of mipomersen in a double-blind, 4-way crossover design with a minimum 5-day washout between treatments. ECG measurements were performed at baseline and selected time points (including Tmax). The correlation between QTcF intervals corrected for baseline and time-matched placebo when available with PK plasma exposure was evaluated by linear regression analysis. RESULTS: In the phase I study, no positive correlation between the PK exposure and ∆QTcF or ∆∆QTcF was observed within the wide dose or exposure range tested. Similar results were observed in the tQT study, where the predicted ΔΔQTcF and its upper bound of the 90% CI at Cmax of therapeutic and supratherapeutic dose were approximately -1.7 and 2.9 ms, respectively. CONCLUSIONS: Mipomersen showed no effect on QT intervals in both the phase I dose escalation study and the tQT study. These results support the proposal that QT assessment can be made in a phase I dose escalation study, and no tQT study may be necessary if the phase I dose escalation study showed a negative QT effect.

Local and systemic tolerability of a 2'O-methoxyethyl antisense oligonucleotide targeting interleukin-4 receptor-α delivery by inhalation in mouse and monkey.

Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.