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

2'-O-(N-(Aminoethyl)carbamoyl)methyl (2'-O-AECM)-modified oligonucleotides (ONs) and their mixmers with 2'-O-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. It is therefore particularly interesting and unprecedented that the 2'-O-AECM ONs are shown to have efficient splice-switching activity even without inclusion of PS linkages and found to be as effective as 2'-OMe PS ONs. Importantly, the PS linkages can be partially included, without any significant reduction in splice-switching efficacy. This suggests that AECM modification has the potential to be used in balancing the PS content of ONs. Furthermore, conjugation of 2'-O-AECM ONs to an endosomal escape peptide significantly increased splice-switching suggesting that this effect could possibly be due to an increase in uptake of ON to the site of action.

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. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.

An oral antisense oligonucleotide for PCSK9 inhibition.

Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.

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). In oligonucleotide therapies, affinity and efficacy can be both easily evaluated by gene expression analysis. Although more advanced techniques can be used, this chapter presents a protocol to evaluate splice switching oligonucleotide efficacy that can be easily applied in a molecular biology laboratory without the need of advanced equipment. It describes all steps from sample preparation to data analysis.

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. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best dendrimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum-free conditions. Interestingly, addition of sucrose to the formulation enabled - for the first time - peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on transfection efficacy.

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. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.

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. To address this, we have developed a small "tissue" library of new, stable, pLuc/705 splice-switching reporter cell lines (named HuH7_705, U-2 OS_705, C2C12_705, and Neuro-2a_705). Our data show that, indeed, the cell type used in activity screenings influences the efficiency of ONs of different chemistry (phosphorothioate with locked nucleic acid or 2'-O-methyl with or without N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine). Likewise, the delivery method, Lipofectamine® 2000, PepFect14 nanoparticles, or "naked" uptake, also demonstrates cell-type-dependent outcomes. Taken together, these cell lines can potentially become useful tools for future in vitro evaluation of new nucleic acid-based oligomers as well as delivery compounds for splice-switching approaches and cell-specific therapies.

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). From these, the proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising pharmacologic target. The objective of this work is to develop a new approach to inactivate PCSK9 by splice-switching oligonucleotides (SSOs), converting the normal splice form to a natural, less abundant and inactive, splice variant. For this purpose, a new RNA therapeutic approach for hypercholesterolemia based on SSOs was developed for modulation of the splice pattern of human PCSK9 pre-mRNA. Our results show an increase of the selected splice form at both the mRNA and protein level when compared to non-treated Huh7 and HepG2 cell lines, with concomitant increase of the protein level of the low-density lipoprotein receptor (LDLR) demonstrating the specificity and efficiency of the system. In vivo, full conversion to the splice form was achieved in a reporter system when mice were treated with the specific oligonucleotide, thus further indicating the therapeutic potential of the approach. In conclusion, PCSK9 activity can be modulated by splice-switching through an RNA therapeutic approach. The tuning of the natural active to non-active isoforms represents a physiological way of regulating the cholesterol metabolism, by controlling the amount of LDL receptor available and the rate of LDL-cholesterol clearance.

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. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.