Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle

Prakash, Thazha P.; Mullick, Adam E.; Lee, Richard G.; Yu, Jinghua; Yeh, Shu-Hui; Low, Audrey; Chappell, Alfred E.; Østergaard, Michael E.; Murray, Sue; Gaus, Hans; Swayze, Eric E.; Seth, Punit P.

doi:10.1093/nar/gkz354

Cited by 109 publications

(105 citation statements)

References 46 publications

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“…This study of splice-switching oligonucleotide conjugates complements recent investigations of the pharmacokinetic and pharmacodynamic properties of antisense and siRNA conjugates (26,27). The results reinforce the notion that increased delivery does not necessarily translate to improved potency.…”

Section: Introductionsupporting

confidence: 74%

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

Halloy

Iyer

Ćwiek

et al. 2020

Preprint

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ABSTRACTErythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favours aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

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Section: Introductionsupporting

confidence: 74%

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

Halloy

Iyer

Ćwiek

et al. 2020

Preprint

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“…A range of different conjugates have been explored in vivo : GalNAc delivers specifically to hepatocytes, while hydrophobic modifications enable delivery to a wide range of tissues beyond the liver. 5 , 43 , 44 …”

Section: Discussionmentioning

confidence: 99%

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy

Echeverria

Sousa

et al. 2020

Molecular Therapy - Nucleic Acids

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Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo . Here, we systematically evaluated the impact of number, structure, and sequence context of PS-modified backbones on cellular uptake and RNAi-mediated silencing efficacy. We find that PS enhances cellular internalization in a sequence-dependent manner but only when present in a single-stranded but not double-stranded region. Furthermore, the observed increase in cellular internalization did not correlate with functional silencing improvement, indicating that PS-mediated uptake may drive compounds to non-productive sinks. Thus, the primary contributing factor of PS modifications to functional efficacy is likely stabilization rather than enhanced cellular uptake. A better understanding of the relative impact of different chemistries on productive versus non-productive uptake will assist in improved design of therapeutic RNAs.

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“…The other promising strategy for solving this problem is to conjugate nucleic acids with lipophilic molecules, since these interact in the bloodstream with lipoproteins whose receptors are expressed throughout the body [ 18 ]. Moreover, the binding of lipophilic conjugates with lipoproteins reduces their excretion by the kidneys and increases their circulation time in the bloodstream [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Transport Oligonucleotides—A Novel System for Intracellular Delivery of Antisense Therapeutics

et al. 2020

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Biological activity of antisense oligonucleotides (asON), especially those with a neutral backbone, is often attenuated by poor cellular accumulation. In the present proof-of-concept study, we propose a novel delivery system for asONs which implies the delivery of modified antisense oligonucleotides by so-called transport oligonucleotides (tON), which are oligodeoxyribonucleotides complementary to asON conjugated with hydrophobic dodecyl moieties. Two types of tONs, bearing at the 5′-end up to three dodecyl residues attached through non-nucleotide inserts (TD series) or anchored directly to internucleotidic phosphate (TP series), were synthesized. tONs with three dodecyl residues efficiently delivered asON to cells without any signs of cytotoxicity and provided a transfection efficacy comparable to that achieved using Lipofectamine 2000. We found that, in the case of tON with three dodecyl residues, some tON/asON duplexes were excreted from the cells within extracellular vesicles at late stages of transfection. We confirmed the high efficacy of the novel and demonstrated that MDR1 mRNA targeted asON delivered by tON with three dodecyl residues significantly reduced the level of P-glycoprotein and increased the sensitivity of KB-8-5 human carcinoma cells to vinblastine. The obtained results demonstrate the efficacy of lipophilic oligonucleotide carriers and shows they are potentially capable of intracellular delivery of any kind of antisense oligonucleotides.

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Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle

Cited by 109 publications

References 46 publications

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy

Transport Oligonucleotides—A Novel System for Intracellular Delivery of Antisense Therapeutics

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