2'--(-(Aminoethyl)carbamoyl)methyl Modification Allows for Lower Phosphorothioate Content in Splice-Switching Oligonucleotides with Retained Activity.

2'--(-(Aminoethyl)carbamoyl)methyl (2'--AECM)-modified oligonucleotides (ONs) and their mixmers with 2'--methyl oligonucleotides (2'-OMe ONs) with phosphodiester linkers as well as with partial and full phosphorothioate (PS) inclusion were synthesized and functionally evaluated as splice-switching oligonucleotides in several different reporter cell lines originating from different tissues. This was enabled by first preparing the AECM-modified A, C, G and U, which required a different strategy for each building block. The AECM modification has previously been shown to provide high resistance to enzymatic degradation, even without PS linkages.

Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation.

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood.

Antisense Oligonucleotides for Splice Modulation: Assessing Splice Switching Efficacy.

Today, there are emerging numbers of oligonucleotide therapies being approved by the governmental authorities. These types of therapies present a different mode of action when compared to the traditional small molecules, acting at the RNA level instead of the protein level. In drug development, drug potency is defined by the drug affinity to the target biomolecule (target engagement), together with the ability to initiate a response at the molecular, cellular, tissue, or system level (efficacy).

Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity.

Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity.

Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)n Repeats by PNA or LNA Targeting.

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich's ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions.

Four Novel Splice-Switch Reporter Cell Lines: Distinct Impact of Oligonucleotide Chemistry and Delivery Vector on Biological Activity.

New advances in oligonucleotide (ON) chemistry emerge continuously, and over the last few years, several aspects of ON delivery have been improved. However, clear knowledge regarding how certain chemistries behave alone, or in combination with various delivery vectors, is limited. Moreover, characterization is frequently limited to a single reporter cell line and, when different cell types are studied, experiments are commonly not carried out under similar conditions, hampering comparative analysis.

RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form.

Hypercholesterolemia is a medical condition often characterized by high levels of low-density lipoprotein cholesterol (LDL-C) in the blood. Despite the available therapies, not all patients show sufficient responses, especially those with very high levels of LDL-C or those with familial hypercholesterolemia. Regulation of plasma cholesterol levels is very complex and several proteins are involved (both receptors and enzymes).

Nuclease resistant oligonucleotides with cell penetrating properties.

2'-O-AECM modified oligonucleotides provide an unusual combination of remarkable properties. This includes the combination of high resistance towards enzymatic degradation and the spontaneous cellular uptake of AECM oligonucleotides.

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA.

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA.