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1.  Integrated Safety Assessment of 2′-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers 
Molecular Therapy  2016;24(10):1771-1782.
The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2′-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2′-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.
PMCID: PMC5112040  PMID: 27357629
2.  Clinical and Preclinical Pharmacokinetics and Pharmacodynamics of Mipomersen (Kynamro®): A Second-Generation Antisense Oligonucleotide Inhibitor of Apolipoprotein B 
Clinical Pharmacokinetics  2015;54:133-146.
Mipomersen (Kynamro®), a second-generation 2′-O-methoxyethyl chimeric antisense oligonucleotide (ASO), inhibits the synthesis of apolipoprotein B (apoB) and is indicated in the US as an adjunct therapy for homozygous familial hypercholesterolemia (HoFH) at a dose of 200 mg subcutaneously (SC) once weekly. The pharmacokinetic (PK) properties of mipomersen are generally consistent across all species studied, including mouse, rat, monkey, and humans. After SC administration, mipomersen is rapidly and extensively absorbed. It has an apparent plasma and tissue terminal elimination half-life of approximately 30 days. Mipomersen achieves steady-state tissue concentrations within approximately 4–6 months of once-weekly dosing. It does not exhibit PK-based drug–drug interactions with other concomitant medications, either involving competition for plasma protein binding or alterations in disposition of any evaluated drugs. Furthermore, mipomersen does not prolong the corrected QT (QTc) interval. There have been no ethnic- or gender-related differences in PK observed. In clinical trials, both as a single agent and in the presence of maximal lipid-lowering therapy, mipomersen has demonstrated significant dose-dependent reductions in all measured apoB-containing atherogenic lipoproteins. Overall, mipomersen has well-characterized PK and pharmacodynamic properties in both animals and humans, and is an efficacious adjunct treatment for patients with HoFH.
PMCID: PMC4305106  PMID: 25559341
3.  Effects of an Antisense Oligonucleotide Inhibitor of C‐Reactive Protein Synthesis on the Endotoxin Challenge Response in Healthy Human Male Volunteers 
C‐reactive protein (CRP) binds to damaged cells, activates the classical complement pathway, is elevated in multiple inflammatory conditions, and provides prognostic information on risk of future atherosclerotic events. It is controversial, however, as to whether inhibiting CRP synthesis would have any direct anti‐inflammatory effects in humans.
Methods and Results
A placebo‐controlled study was used to evaluate the effects of ISIS 329993 (ISIS‐CRPRx) on the acute‐phase response after endotoxin challenge in 30 evaluable subjects. Healthy adult males were randomly allocated to receive 6 injections over a 22‐day period of placebo or active therapy with ISIS 329993 at 400‐ or 600‐mg doses. Eligible subjects were subsequently challenged with a bolus of endotoxin (2 ng/kg). Inflammatory and hematological biomarkers were measured before and serially after the challenge. ISIS‐CRPRx was well tolerated with no serious adverse events. Median CRP levels increased more than 50‐fold from baseline 24 hours after endotoxin challenge in the placebo group. In contrast, the median increase in CRP levels was attenuated by 37% (400 mg) and 69% (600 mg) in subjects pretreated with ISIS‐CRPRx (P<0.05 vs. placebo). All other aspects of the acute inflammatory response were similar between treatment groups.
Pretreatment of subjects with ISIS‐CRPRx selectively reduced the endotoxin‐induced increase in CRP levels in a dose‐dependent manner, without affecting other components of the acute‐phase response. These data demonstrate the specificity of antisense oligonucleotides and provide an investigative tool to further define the role of CRP in human pathological conditions.
PMCID: PMC4310401  PMID: 25012289
acute phase response; antisense inhibitor; C‐reactive protein; endotoxin; healthy volunteers
4.  Changes in Mipomersen Dosing Regimen Provide Similar Exposure With Improved Tolerability in Randomized Placebo‐Controlled Study of Healthy Volunteers 
Mipomersen, an apolipoprotein B synthesis inhibitor, demonstrated significant reductions in low‐density lipoprotein (LDL) cholesterol, non‐high density lipoprotein cholesterol, and apolipoprotein B in 4 phase 3 studies at the FDA‐approved subcutaneous dose of 200 mg once weekly.
Methods and Results
A short‐term phase 1 study in healthy volunteers was conducted to evaluate the relative bioavailability, safety, and tolerability of mipomersen in 2 test dose regimens in reference to the 200 mg weekly dose regimen. Eighty‐four adults were randomized to 1 of 3 cohorts (30 mg once daily, 70 mg 3 times weekly, or 200 mg once weekly) and then mipomersen or placebo (3:1 ratio) for 3 weeks of treatment. Comparable mipomersen post‐distribution phase plasma concentrations were observed across the 3 dose regimens suggesting similar tissue exposure. Injection site reactions were reported, but did not lead to treatment discontinuation. The median incidence of these responses per injection was decreased by lowering the dose. Signals from a diverse panel of systemic inflammation markers were essentially indistinguishable between dose regimens and placebo treatment. The one exception was a modest transient post‐dose elevation of C‐reactive protein (CRP) in the mipomersen 200 mg weekly group. This elevation was not associated with an increase in other proinflammatory markers.
This study demonstrated a similar drug exposure and overall safety profile between the 3 dosing regimens. Exploratory assessment of a diverse panel of biomarkers found no indication of a systemic inflammatory response to mipomersen treatment. These results support assessment of alternative dose regimens in longer‐term studies.
Clinical Trial Registration
URL: Unique identifier: NCT01061814.
PMCID: PMC4187476  PMID: 24627419
dosing; inhibitor; pharmacokinetics; randomized controlled trial; safety
5.  Antisense Technology: An Emerging Platform for Cardiovascular Disease Therapeutics 
Antisense oligonucleotides and small interfering RNAs, which suppress the translation of specific mRNA target proteins, are emerging as important therapeutic modalities for the treatment of cardiovascular disease. Over the last 25 years, the advances in all aspects of antisense technology, as well as a detailed understanding of the mechanism of action of antisense drugs, have enabled their use as therapeutic agents. These advancements culminated in the FDA approval of the first chronically administered cardiovascular antisense therapeutic, mipomersen, which targets hepatic apolipoprotein B mRNA. This review provides a brief history of antisense technology, highlights the progression of mipomersen from preclinical studies to multiple Phase III registration trials, and gives an update on the status of other cardiovascular antisense therapeutics currently in the clinic.
PMCID: PMC3838598  PMID: 23856914
Antisense oligonucleotides; Small interfering RNAs; Apolipoprotein B; Atherosclerosis; Dyslipidemia; Coagulation; Thrombosis
6.  Mipomersen, an apolipoprotein B synthesis inhibitor, lowers low-density lipoprotein cholesterol in high-risk statin-intolerant patients: a randomized, double-blind, placebo-controlled trial 
European Heart Journal  2012;33(9):1142-1149.
A randomized, double-blind, placebo-controlled study was conducted to investigate the safety and efficacy of mipomersen, an apolipoprotein B-100 (apoB) synthesis inhibitor, in patients who are statin intolerant and at high risk for cardiovascular disease (CVD).
Methods and results
Thirty-three subjects, not receiving statin therapy because of statin intolerance, received a weekly subcutaneous dose of 200 mg mipomersen or placebo (2:1 randomization) for 26 weeks. The primary endpoint was per cent change in LDL cholesterol (LDL-c) from the baseline to Week 28. The other efficacy endpoints were per cent change in apoB and lipoprotein a [Lp(a)]. Safety was determined using the incidence of treatment-emergent adverse events (AEs) and clinical laboratory evaluations. After 26 weeks of mipomersen administration, LDL-c was reduced by 47 ± 18% (P < 0.001 vs. placebo). apoB and Lp(a) were also significantly reduced by 46 and 27%, respectively (P < 0.001 vs. placebo). Four mipomersen (19%) and two placebo subjects (17%) discontinued dosing prematurely due to AEs. Persistent liver transaminase increases ≥3× the upper limit of normal were observed in seven (33%) subjects assigned to mipomersen. In selected subjects, liver fat content was assessed, during and after treatment, using magnetic resonance spectroscopy. Liver fat content in these patients ranged from 0.8 to 47.3%. Liver needle biopsy was performed in two of these subjects, confirming hepatic steatosis with minimal inflammation or fibrosis.
The present data suggest that mipomersen is a potential therapeutic option in statin-intolerant patients at high risk for CVD. The long-term follow-up of liver safety is required.
Clinical Trial Registration: identifier: NCT00707746
PMCID: PMC3751967  PMID: 22507979
Lipoproteins; Hypercholesterolaemia; Hepatic steatosis; Antisense oligonucleotides; Statin intolerance
7.  A Selective Inhibitor of Human C-reactive Protein Translation Is Efficacious In Vitro and in C-reactive Protein Transgenic Mice and Humans 
Observational studies of patients with established rheumatoid arthritis (RA) document a positive correlation between C-reactive protein (CRP) blood concentration and worsening of RA symptoms, but whether this association is causal or not is not known. Using CRP transgenic mice (CRPTg) with collagen-induced arthritis (CIA; a rodent model of RA), we explored causality by testing if CRP lowering via treatment with antisense oligonucleotides (ASOs) targeting human CRP mRNA was efficacious and of clinical benefit. We found that in CRPtg with established CIA, ASO-mediated lowering of blood human CRP levels improved the clinical signs of arthritis. In addition, in healthy human volunteers the ASO was well tolerated and efficacious i.e., treatment achieved significant CRP lowering. ASOs targeting CRP should provide a specific and effective way to lower human CRP levels, which might be an effective therapy in patients with established RA.
PMCID: PMC3511672  PMID: 23629027
antisense therapy; CRP; rheumatoid arthritis
8.  Enhancement of SMN2 Exon 7 Inclusion by Antisense Oligonucleotides Targeting the Exon 
PLoS Biology  2007;5(4):e73.
Several strategies have been pursued to increase the extent of exon 7 inclusion during splicing of SMN2 (survival of motor neuron 2) transcripts, for eventual therapeutic use in spinal muscular atrophy (SMA), a genetic neuromuscular disease. Antisense oligonucleotides (ASOs) that target an exon or its flanking splice sites usually promote exon skipping. Here we systematically tested a large number of ASOs with a 2′-O-methoxy-ethyl ribose (MOE) backbone that hybridize to different positions of SMN2 exon 7, and identified several that promote greater exon inclusion, others that promote exon skipping, and still others with complex effects on the accumulation of the two alternatively spliced products. This approach provides positional information about presumptive exonic elements or secondary structures with positive or negative effects on exon inclusion. The ASOs are effective not only in cell-free splicing assays, but also when transfected into cultured cells, where they affect splicing of endogenous SMN transcripts. The ASOs that promote exon 7 inclusion increase full-length SMN protein levels, demonstrating that they do not interfere with mRNA export or translation, despite hybridizing to an exon. Some of the ASOs we identified are sufficiently active to proceed with experiments in SMA mouse models.
Author Summary
Spinal muscular atrophy (SMA) is a severe genetic disease that causes motor-neuron degeneration. SMA patients lack a functional SMN1 (survival of motor neuron 1) gene, but they possess an intact SMN2 gene, which though nearly identical to SMN1, is only partially functional. The defect in SMN2 gene expression is at the level of pre-mRNA splicing (skipping of exon 7), and the presence of this gene in all SMA patients makes it an attractive target for potential therapy. Here we have surveyed a large number of antisense oligonucleotides (ASOs) that are complementary to different regions of exon 7 in the SMN2 mRNA. A few of these ASOs are able to correct the pre-mRNA splicing defect, presumably because they bind to regions of exon 7 that form RNA structures, or provide protein-binding sites, that normally weaken the recognition of this exon by the splicing machinery in the cell nucleus. We describe optimal ASOs that promote correct expression of SMN2 mRNA and, therefore, normal SMN protein, in cultured cells from SMA patients. These ASOs can now be tested in mouse models of SMA, and may be useful for SMA therapy.
Mutations inSMN1 cause spinal muscular atrophy; a nearly identical gene is not functional, but becomes functional in vitro and in vivo after addition of antisense oligos.
PMCID: PMC1820610  PMID: 17355180
9.  Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides 
Nucleic Acids Research  2000;28(2):582-592.
Phosphorothioate oligodeoxynucleotides (P=S ODNs) are frequently used as antisense agents to specifically interfere with the expression of cellular target genes. However, the cell biological properties of P=S ODNs are poorly understood. Here we show that P=S ODNs were able to continuously shuttle between the nucleus and the cytoplasm and that shuttling P=S ODNs retained their ability to act as antisense agents. The shuttling process shares characteristics with active transport since it was inhibited by chilling and ATP depletion in vivo. Transport was carrier-mediated as it was saturable, and nuclear pore complex-mediated as it was sensitive to treatment with wheatgerm agglutinin. Oligonucleotides without a P=S backbone chemistry were only weakly restricted in their migration by chilling, ATP depletion and wheatgerm agglutinin and thus moved by diffusion. P=S ODN shuttling was only moderately affected by disruption of the Ran/RCC1 system. We propose that P=S ODNs shuttle through their binding to yet unidentified cellular molecules that undergo nucleocytoplasmic transport via a pathway that is not as strongly dependent on the Ran/RCC1 system as nuclear export signal-mediated protein export, U-snRNA, tRNA and mRNA export. The shuttling property of P=S ODNs must be taken into account when considering the mode and site of action of these antisense agents.
PMCID: PMC102511  PMID: 10606658
10.  Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies 
Molecular Biology of the Cell  1998;9(5):1007-1023.
Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.
PMCID: PMC25326  PMID: 9571236

Results 1-10 (10)