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Emerging clinical data show that three ceramide molecules, Cer d18:1/16:0, Cer d18:1/24:1, and Cer d18:1/24:0, are biomarkers of a fatal outcome in patients with cardiovascular disease. This finding raises basic questions about their metabolic origin, their contribution to disease pathogenesis, and the utility of targeting the underlying enzymatic machinery for treatment of cardiometabolic disorders. Here, we outline the development of a potent N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. We demonstrate that this compound reduces the ceramide synthase 2 mRNA level and that this translates into efficient lowering of protein expression and activity as well as Cer d18:1/24:1 and Cer d18:1/24:0 levels in liver. Intriguingly, we discover that the hepatocyte-specific antisense oligonucleotide also triggers a parallel modulation of blood plasma ceramides, revealing that the biomarkers predictive of cardiovascular death are governed by ceramide biosynthesis in hepatocytes. Our work showcases a generic therapeutic framework for targeting components of the ceramide enzymatic machinery to disentangle their roles in disease causality and to explore their utility for treatment of cardiometabolic disorders. ispartof: MOLECULAR THERAPY vol:30 issue:4 pages:1661-1674 ispartof: location:United States status: published

Liver and kidney uptake and antisense activity is studied for a series of Locked Nucleic Acid (LNA) oligonucleotides with fully stereo-defined, internucleoside linkages. These stereo-specific phosphorothioates are made with a newly developed synthesis method and are being analyzed both theoretically and experimentally. Their structures are obtained theoretically by using many-body Schrödinger equations applied to a group of 11 stereo-defined LNA antisense oligonucleotides selected for biological experiments. The fully converged electronic structures were obtained from ab initio quantum calculations providing the specific electronic structures. One important result was the observation that the calculated electronic structure, represented by the iso-surface area of the electron density in Å2, correlated linearly with LNA oligonucleotide uptake in the liver and kidney. This study also shows that more complex biological phenomena, such as drug activity, will require more molecular and cellular identifiers than used here before a correlation can be found. Establishing biological correlations between quantum mechanical (QM) calculated structures and antisense oligonucleotides is novel, and this method may constitute new tools in drug discovery.

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.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3′ thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3′ and 5′ thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

Drug discovery with phosphorothioate oligonucleotides is an area of intensive research. In this study we have controlled the stereochemistry of the phosphorothioate backbone of LNA oligonucleotides to investigate the differences in safety profile, target mRNA knock down, and cellular uptake in vitro. The study reveals that controlling only four stereocenters in an isomeric phosphorothioate mixture can improve the therapeutic index significantly by improving safety without compromising activity.

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.

An efficient route detailing the construction of the central core of pierisformaside C, the first grayanane-type diterpene to possess three central double bonds, is reported.

Udgivelsesdato: 2007-Aug-7 The triplex-forming abilities of some conformationally restricted nucleotide analogs are disclosed and compared herein. 2'-Amino-LNA monomers proved to be less stabilising to triplexes than LNA monomers when incorporated into a triplex-forming third strand. N2'-functionalisation of 2'-amino-LNA monomers with a glycyl unit induced the formation of exceptionally stable triplexes. Nucleotide analogs containing a C2',C3'-oxymethylene linker (E-type furanose conformation) or a C2',C4'-propylene linker (N-type furanose conformation) had no significant effect on triplex stability proving that conformational restriction per se is insufficient to stabilise triplexes.

An attempt of preparing a carbocyclic LNA-analogue using different RCM-methods failed. However, a compound with a hemiacetal linker between the C2′ and the C4′-positions was isolated and found to be a suitable substrate for making a conformationally restricted double-headed nucleoside. This contains two uracil nucleobases organized on a bicyclic skeleton and is locked in an N -type conformation.

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.

Two locked nucleic acid (LNA) analogues with three-carbon 2'-4' linkages, saturated or unsaturated, are synthesized using a ring-closing metathesis based strategy. Strongly stabilized duplexes with complementary RNA and slightly destabilized duplexes with complementary DNA are observed. CD-spectroscopy indicates a less pronounced shift toward A-type duplexes compared to LNA. These results combining a strong N-type conformation with the absence of a 2'-oxygen demonstrate a stronger importance of minor groove hydration in an intermediate duplex type than in an A-type duplex.

Ring-closing metathesis (RCM) is applied as a new and powerful technology in the construction of nucleoside analogues that are conformationally restricted in S-type conformations due to additional 3',4'- and/or 3',5'-linkages.