The importance of concomitant mitral regurgitation for estimates of mitral valve area by pressure half time in patients with chronic rheumatic heart disease.

AIMS: Aim was to study how concomitant mitral regurgitation (MR) assessed by qualitative and quantitative methods influence mitral valve area (MVA) calculations by the pressure half time method (MVAPHT) compared to reference MVA (planimetry) in patients with rheumatic heart disease. METHODS AND RESULTS: In 72 patients with chronic rheumatic heart disease, MVAPHT was calculated as 220 divided by the pressure half time of the mitral early inflow Doppler spectrum. Direct measurement by planimetry was used as reference MVA and was mean (SD) 0.99 (0.69-1.99) cm2. Concomitant MR was present in 82%. MR severity was assessed qualitatively in all, semi-quantitatively by measuring the vena contracta width in 58 (81%), and quantitatively by calculation of the regurgitant volume in 28 (39%). MVA was significantly underestimated by MVAPHT, with increasing MR. In regression analyses MVAPHT underestimated MVA by 0.19 cm2 per higher grade of MR severity in qualitative assessment, and by 0.12-0.13 cm2 per mm larger vena contracta width and 10 ml larger regurgitant volume, respectively. The presented associations were more evident when i) MR severity was quantified compared to qualitative assessment and ii) reference measurements were made by three-dimensional transoesophageal recordings compared to transthoracic recordings. CONCLUSION: MVAPHT underestimated mitral valve area compared to planimetry in patients with MS and concomitant MR. This study highlights the importance of taking the MR severity into account when evaluating MVA based on the PHT method. Direct measurements should be included in clinical decision making.

In silico analysis provides insights for patient-specific annuloplasty in Barlow's disease.

Objective: To predict the required mitral annular area reduction in patients with Barlow's disease to obtain a predefined leaflet area index by a novel in silico modeling method. Methods: Three-dimensional echocardiography was used to create patient-specific mitral valve models of 8 patients diagnosed with Barlow's disease and bileaflet prolapse preoperatively. Six patients were also studied postoperatively in a finite element framework, to quantify the optimal coaptation area index. For the patient-specific finite element analyses, realistic papillary muscle and annular motion are incorporated, also for the in silico annuloplasty analyses. The annuloplasty ring size is reduced moderately until the optimal coaptation area index is achieved for each patient. Results: The mean mitral annular area at end-diastole was reduced by 58 ± 7% postoperatively (P < .001), resulting in a postoperative coaptation area index of 20 ± 5%. To achieve the same coaptation area index with moderate annular reductions and no leaflet resection the annular reduction was 31 ± 6% (P < .001). Conclusions: In silico analysis in selected patients diagnosed with Barlow's disease demonstrates that annuloplasty with only moderate annular reduction may be sufficient to achieve optimal coaptation as compared to conventional surgical procedures.

Mechanical behavior and collagen structure of degenerative mitral valve leaflets and a finite element model of primary mitral regurgitation.

Degenerative mitral valve disease is the main cause of primary mitral regurgitation with two phenotypes: fibroelastic deficiency (FED) often with localized myxomatous degeneration and diffuse myxomatous degeneration or Barlow's disease. Myxomatous degeneration disrupts the microstructure of the mitral valve leaflets, particularly the collagen fibers, which affects the mechanical behavior of the leaflets. The present study uses biaxial mechanical tests and second harmonic generation microscopy to examine the mechanical behavior of Barlow and FED tissue. Three tissue samples were harvested from a FED patient and one sample is from a Barlow patient. Then we use an appropriate constitutive model by excluding the collagen fibers under compression. Finally, we built an FE model based on the echocardiography of patients diagnosed with FED and Barlow and the characterized material model and collagen fiber orientation. The Barlow sample and the FED sample from the most affected segment showed different mechanical behavior and collagen structure compared to the other two FED samples. The FE model showed very good agreement with echocardiography with 2.02±1.8 mm and 1.05±0.79 mm point-to-mesh distance errors for Barlow and FED patients, respectively. It has also been shown that the exclusion of collagen fibers under compression provides versatility for the material model; it behaves stiff in the belly region, preventing excessive bulging, while it behaves very softly in the commissures to facilitate folding. STATEMENT OF SIGNIFICANCE: This study quantifies for the first time the collagen microstructure and mechanical behavior of degenerative mitral valve (DMV) leaflets. These data will then be used for the first disease-specific finite element (FE) model of DMV. While current surgical repair of DMV is based on surgical experience, FE modeling has the potential to support decision-making and make outcomes predictable. We adopt a constitutive model to exclude collagen fiber under compressions, an important consideration when modeling the mitral valve, where the leaflets are folded to ensure complete closure. The results of this study provide essential data for understanding the relationship between collagen microstructure and degenerative mitral valve mechanics.

Mitral annular dynamics are influenced by left ventricular load and contractility in an acute animal model.

The purpose of this study was to investigate the effects of loading conditions and left ventricular (LV) contractility on mitral annular dynamics. In 10 anesthetized pigs, eight piezoelectric transducers were implanted equidistantly around the mitral annulus. High-fidelity catheters measured left ventricular pressures and the slope of the end-systolic pressure-volume relationship (Ees ) determined LV contractility. Adjustments of pre- and afterload were done by constriction of the inferior caval vein and occlusion of the descending aorta. Mitral annulus area indexed to body surface area (MAAi ), annular circularity index (ACI), and non-planarity angle (NPA) were calculated by computational analysis. MAAi was more dynamic in response to loading interventions than ACI and NPA. However, MAAi maximal cyclical reduction (-Δr) and average deformational velocity (- v ¯ $$ \overline{v} $$ ) did not change accordingly (p = 0.31 and p = 0.22). Reduced Ees was associated to attenuation in MAAi -Δr and MAAi - v ¯ $$ \overline{v} $$ (r2 = 0.744; p = 0.001 and r2 = 0.467; p = 0.029). In conclusion, increased cardiac load and reduced LV contractility may cause deterioration of mitral annular dynamics, likely impairing coaptation and increasing susceptibility to valvular incompetence.

Sympathetic and vagal interaction in the control of cardiac pacemaker rhythm in the guinea-pig heart: Importance of expressing heart rhythm using an appropriate metric.

There are many reports that, through pre- and post-junctional mechanisms, sympathetic and parasympathetic (vagal) nerves can interact in the control of heart rate. The predominant interaction is accentuated antagonism (AA), where the bradycardia produced by vagal stimulation (VNS) is amplified when heart rate has been increased by sympathetic stimulation (SNS) or beta-adrenergic agonists. The acetylcholine-activated potassium current (IK,Ach), is the primary driver of vagal bradycardia. To examine the participation of IK,Ach in AA, a series of experiments was performed on isolated, double innervated, guinea-pig atrial preparations. Vagal bradycardia was elicited by 10-s trains (1, 2, 5 and 7.5 Hz) or single bursts of VNS (3 stimuli at 50 Hz) before and during acceleration of HR by either SNS (1-3 Hz) or isoprenaline (ISO), in both absence and presence of tertiapin-Q (TQ-IK,Ach blocker). When expressed as an absolute change in HR (beats/min), bradycardia produced by VNS trains was amplified (AA) at all frequencies of VNS in ISO, and at 5 and 7.5 Hz during SNS. Bradycardia in response to 1 and 2 Hz VNS was reduced during SNS. In TQ, only the bradycardia produced by 5 and 7.5 Hz VNS in ISO was amplified. The bradycardia produced by a single burst of VNS was amplified in both ISO and SNS. After TQ the bradycardia in response to a VNS burst was unchanged in ISO, while it was reduced during SNS. When these data were adjusted to account for the increase in baseline HR brought about by SNS and ISO, there was no longer evidence of AA. Diminished responses to low frequencies of VNS (1 and 2 Hz) persisted, and were also seen during IK,Ach block by TQ. We applied the same adjustment to data from 20 published studies. In 8 studies all data indicated AA; 3 studies provided no evidence for AA, and in 9 studies evidence was mixed. There is no doubt that AA can occur in the control of heart rhythm during simultaneous SNS and VNS, but conditions which determine its occurrence, and the mechanisms involved in this interaction remain unclear.

Finite element analysis of mitral valve annuloplasty in Barlow's disease.

Barlow's Disease affects the entire mitral valve apparatus causing mitral regurgitation. Standard annuloplasty procedures lead to an average of 55% annular area reduction of the end diastolic pre-operative annular area in Barlow's diseased valves. Following annular reduction, mitral valvuloplasty may be needed, usually with special focus on the posterior leaflet. An in silico pipeline to perform annuloplasty by utilizing the pre- and -postoperative 3D echocardiographic recordings was developed. Our objective was to test the hypothesis that annuloplasty ring sizes based on a percentage (10%-25%) decrease of the pre-operative annular area at end diastole can result in sufficient coaptation area for the selected Barlow's diseased patient. The patient specific mitral valve geometry and finite element model were created from echocardiography recordings. The post-operative echocardiography was used to obtain the artificial ring geometry and displacements, and the motion of the papillary muscles after surgery. These were used as boundary conditions in our annuloplasty finite element analyses. Then, the segmented annuloplasty ring was scaled up to represent a 10%, 20% and 25% reduction of the pre-operative end diastolic annular area and implanted to the end diastolic pre-operative finite element model. The pre-operative contact area decrease was shown to be dependent on the annular dilation at late systole. Constraining the mitral valve from dilating excessively can be sufficient to achieve proper coaptation throughout systole. The finite element analyses show that the selected Barlow's diseased patient may benefit from an annuloplasty ring with moderate annular reduction alone.

Mitral Annular Elasticity Determines Severity of Regurgitation in Barlow's Mitral Valve Disease.

OBJECTIVES: Barlow's mitral valve disease with late systolic mitral regurgitation provides diagnostic and therapeutic challenges. The mechanisms of the regurgitation are still unclear. We hypothesized that the onset and the severity of late systolic regurgitation are determined by annulus dynamics and the mechanical stresses imposed by the left ventricle. METHODS: Ten patients with Barlow's mitral valve disease and mitral annulus disjunction (MAD) were compared with 10 healthy controls. Resting blood pressure was measured, and transthoracic three-dimensional echocardiography was analyzed using a holographic display that allows tracking and measurements of mitral annulus surface area (ASA) throughout the cardiac cycle. A novel annulus elastance index (dASA/dP) was calculated between aortic valve opening and onset of mitral regurgitation. Severity of MAD was quantified as the disjunction index (mm × degree). Leaflet coaptation area was calculated using a finite element model. RESULTS: Peak systolic ASAs in controls and patients were 9.3 ± 0.6 and 21.1 ± 3.1 cm2, respectively (P < .001). In patients, the ASA increased rapidly during left ventricular ejection, and onset of mitral regurgitation coincided closely with peak upslope of annulus area change (dASA/dt). The finite element model showed a close association between rapid annulus displacement and coaptation area deficit in Barlow's mitral valve disease. Systolic annulus elastance index (0.058 ± 0.036 cm2/mm Hg) correlated strongly with disjunction index (r = 0.91, P < .0001). Moreover, regurgitation volume showed a positive correlation with systolic blood pressure (r = 0.80, P < .01). CONCLUSION: The present pilot study supports the hypothesis that annulus dilatation may accentuate mitral valve regurgitation in patients with Barlow's mitral valve disease. A novel annulus elastance index may predict the severity of mitral valve regurgitation in selected patients.

Short-term outcome after open-heart surgery for severe chronic rheumatic heart disease in a low-income country, with comparison with an historical control group: an observational study.

OBJECTIVES: Rheumatic heart disease (RHD) is a major burden in low-income and middle-income countries (LMICs). Cardiac surgery is the only curative treatment. Little is known about patients with severe chronic RHD operated in LMICs, and challenges regarding postoperative follow-up are an important issue. At Tikur Anbessa Specialised Hospital, Addis Ababa, Ethiopia, we aimed to evaluate the course and 12-month outcome of patients with severe chronic RHD who received open-heart surgery, as compared with the natural course of controls waiting for surgery and undergoing only medical treatment. METHODS: Clinical data and outcome measures were registered in 46 patients operated during five missions from March 2016 to November 2019, and compared with the first-year course in a cohort of 49 controls from the same hospital's waiting list for surgery. Adverse events were death or complications such as stroke, other thromboembolic events, bleeding, hospitalisation for heart failure and infectious endocarditis. RESULTS: Survival at 12 months was 89% and survival free from complications was 80% in the surgical group. Despite undergoing open-heart surgery, with its inherent risks, outcome measures of the surgical group were non-inferior to the natural course of the control group in the first year after inclusion on the waiting list (p≥0.45). All except six surgical patients were in New York Heart Association class I after 12 months and 84% had resumed working. CONCLUSIONS: Cardiac surgery for severe chronic RHD is feasible in LMICs if the service is structured and planned. Rates of survival and survival free from complications were similar to those of controls at 12 months. Functional level and resumption of work were high in the surgical group. Whether the patients who underwent cardiac surgery will have better long-term prognosis, in line with what is known in high-income countries, needs to be evaluated in future studies.

A novel case of impaired C-reactive protein response following open-heart surgery: A case report and review of the literature.

BACKGROUND: C-reactive protein (CRP) is expected to increase in response to a range of inflammatory stimuli such as infections or extensive tissue trauma. CASE REPORT: We present a novel case of severely impaired CRP response following NSTEMI, influenza A infection and open-heart surgery in which serum CRP concentrations remained < 1 mg/L during an observational period of 28 days. CONCLUSION: To our knowledge, no previous publications exists describing patients with a lack of CRP response following cardiothoracic surgery. We believe this to be a novel finding warranting further investigations regarding the etiology and prevalence of this phenomenon.

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo.

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 µM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

Locked nucleic acid: modality, diversity, and drug discovery.

Over the past 20 years, the field of RNA-targeted therapeutics has advanced based on discoveries of modified oligonucleotide chemistries, and an ever-increasing understanding of how to apply cellular assays to identify oligonucleotides with improved pharmacological properties in vivo. Locked nucleic acid (LNA), which exhibits high binding affinity and potency, is widely used for this purpose. Our understanding of RNA biology has also expanded tremendously, resulting in new approaches to engage RNA as a therapeutic target. Recent observations indicate that each oligonucleotide is a unique entity, and small structural differences between oligonucleotides can often lead to substantial differences in their pharmacological properties. Here, we outline new principles for drug discovery exploiting oligonucleotide diversity to identify rare molecules with unique pharmacological properties.

From the Cover: The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides.

Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.

Antisense-mediated reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9): a first-in-human randomized, placebo-controlled trial.

AIMS: LDL-receptor expression is inhibited by the protease proprotein convertase subtilisin/kexin type 9 (PCSK9), which is considered a pharmacological target to reduce LDL-C concentrations in hypercholesterolaemic patients. We performed a first-in-human trial with SPC5001, a locked nucleic acid antisense inhibitor of PCSK9. METHODS: In this randomized, placebo-controlled trial, 24 healthy volunteers received three weekly subcutaneous administrations of SPC5001 (0.5, 1.5 or 5 mg kg(-1)) or placebo (SPC5001 : placebo ratio 6 : 2). End points were safety/tolerability, pharmacokinetics and efficacy of SPC5001. RESULTS: SPC5001 plasma exposure (AUC(0,24 h)) increased more than dose-proportionally. At 5 mg kg(-1), SPC5001 decreased target protein PCSK9 (day 15 to day 35: -49% vs. placebo, P < 0.0001), resulting in a reduction in LDL-C concentrations (maximal estimated difference at day 28 compared with placebo -0.72 mmol l(-1), 95% confidence interval - 1.24, -0.16 mmol l(-1); P < 0.01). SPC5001 treatment (5 mg kg(-1)) also decreased ApoB (P = 0.04) and increased ApoA1 (P = 0.05). SPC5001 administration dose-dependently induced mild to moderate injection site reactions in 44% of the subjects, and transient increases in serum creatinine of ≥20 µmol l(-1) (15%) over baseline with signs of renal tubular toxicity in four out of six subjects at the highest dose level. One subject developed biopsy-proven acute tubular necrosis. CONCLUSIONS: SPC5001 treatment dose-dependently inhibited PCSK9 and decreased LDL-C concentrations, demonstrating human proof-of-pharmacology. However, SPC5001 caused mild to moderate injection site reactions and renal tubular toxicity, and clinical development of SPC5001 was terminated. Our findings underline the need for better understanding of the molecular mechanisms behind the side effects of compounds such as SPC5001, and for sensitive and relevant renal toxicity monitoring in future oligonucleotide studies.

The relationship between irritable bowel syndrome, functional dyspepsia, chronic fatigue and overactive bladder syndrome: a controlled study 6 years after acute gastrointestinal infection.

BACKGROUND: To investigate in a cohort with previous gastrointestinal infection and a control group the prevalence of overactive bladder syndrome (OAB), and how it was associated with three other functional disorders; irritable bowel syndrome (IBS), functional dyspepsia (FD) and chronic fatigue (CF). METHODS: Controlled historic cohort study including 724 individuals with laboratory confirmed giardiasis six years earlier, and 847 controls matched by gender and age. Prevalence and odds ratios (OR) with 95 % confidence intervals (CI) were calculated. RESULTS: The prevalence of OAB was 18.7 % (134/716) in the exposed group and 13.6 % (113/833) in the control group (p = 0.007). The association between OAB and IBS was strong in the control group (OR: 2.42; 95 % CI: 1.45 to 4.04), but insignificant in the Giardia exposed (OR: 1.29; 95 % CI: 0.88 to 1.88). The association between OAB and FD was weak in both groups. CF was strongly associated with OAB (OR: 2.73; 95 % CI: 1.85 to 4.02 in the exposed and OR: 2.79; 95 % CI: 1.69 to 4.62 in the controls), and this association remained when comorbid conditions were excluded. CONCLUSIONS: Sporadic IBS was associated with increased risk of OAB, whereas post-infectious IBS was not. An apparent association between OAB and previous Giardia infection can be ascribed to comorbid functional disorders.

Biological activity and biotechnological aspects of locked nucleic acids.

Locked nucleic acid (LNA) is one of the most promising new nucleic acid analogues that has been produced under the past two decades. In this chapter, we have tried to cover many of the different areas, where this molecule has been used to improve the function of synthetic oligonucleotides (ONs). The use of LNA in antisense ONs, including gapmers, splice-switching ONs, and siLNA, as well as antigene ONs, is reviewed. Pharmacokinetics as well as pharmacodynamics of LNA ONs and a description of selected compounds in, or close to, clinical testing are described. In addition, new LNA modifications and the adaptation of enzymes for LNA incorporation are reviewed. Such enzymes may become important for the development of stabilized LNA-containing aptamers.

Treatment of HCV infection by targeting microRNA.

BACKGROUND: The stability and propagation of hepatitis C virus (HCV) is dependent on a functional interaction between the HCV genome and liver-expressed microRNA-122 (miR-122). Miravirsen is a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide that sequesters mature miR-122 in a highly stable heteroduplex, thereby inhibiting its function. METHODS: In this phase 2a study at seven international sites, we evaluated the safety and efficacy of miravirsen in 36 patients with chronic HCV genotype 1 infection. The patients were randomly assigned to receive five weekly subcutaneous injections of miravirsen at doses of 3 mg, 5 mg, or 7 mg per kilogram of body weight or placebo over a 29-day period. They were followed until 18 weeks after randomization. RESULTS: Miravirsen resulted in a dose-dependent reduction in HCV RNA levels that endured beyond the end of active therapy. In the miravirsen groups, the mean maximum reduction in HCV RNA level (log10 IU per milliliter) from baseline was 1.2 (P=0.01) for patients receiving 3 mg per kilogram, 2.9 (P=0.003) for those receiving 5 mg per kilogram, and 3.0 (P=0.002) for those receiving 7 mg per kilogram, as compared with a reduction of 0.4 in the placebo group. During 14 weeks of follow-up after treatment, HCV RNA was not detected in one patient in the 5-mg group and in four patients in the 7-mg group. We observed no dose-limiting adverse events and no escape mutations in the miR-122 binding sites of the HCV genome. CONCLUSIONS: The use of miravirsen in patients with chronic HCV genotype 1 infection showed prolonged dose-dependent reductions in HCV RNA levels without evidence of viral resistance. (Funded by Santaris Pharma; ClinicalTrials.gov number, NCT01200420.).

A locked nucleic acid oligonucleotide targeting microRNA 122 is well-tolerated in cynomolgus monkeys.

MicroRNA 122 (miR-122) is liver specific, fine-tunes lipid metabolism, and is required for hepatitis C virus (HCV) abundance. Miravirsen, an oligonucleotide with locked nucleic acid, binds to miR-122, potently inhibiting its activity. We aimed at determining the safety of the miR-122 antagonism in vivo in 6 to 10 cynomolgus monkeys/group intravenously treated with a range of dose levels twice weekly for 4 weeks. Survival, body weights, clinical signs, and cardiovascular and ophthalmologic parameters were unaffected. Anticipated hypolipidemia due to the inhibition of miR-122 was observed in all treated animals. Only the highest dose level produced distinct transient prolongations of clotting times, slight alternative complement pathway activation, and a reversible increase of hepatic transaminases. Distribution half-life was 10-20 minutes, and accumulation was mainly in the kidney and liver with slow elimination. Microscopic examinations revealed granulated Kupffer cells and lymph node macrophages, cytoplasmic vacuolation in proximal renal tubules, and hepatocytes. The granules were most likely phagolysosomes containing miravirsen. A slightly increased incidence of hepatocyte apoptosis was observed in some monkeys given the highest dose; otherwise, there was no evidence of treatment-related degenerative changes in any organ. In conclusion, the maximal inhibition of miR-122 was associated with limited phenotypic changes, indicating that the clinical assessment of miravirsen as host factor antagonist for treatment of HCV infections is warranted.

PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.

The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.