The precise delivery of modern molecular medicines to their intended cellular target is necessary to realize their full potential. The site-specific delivery of therapeutic antisense oligonucleotides (ASOs) is a challenging task. To improve the delivery and circulation of gamer ASOs, specific modifications are needed.
Fatty acid-modified gapmers represent a conjugation strategy for enhancing the therapeutic potential of antisense oligonucleotides by improving their circulation through the human body to reach their intended RNA targets.
Fatty acid-modified gapmers are antisense oligonucleotides (ASOs) chemically modified with fatty acid chains. The conjugation of fatty acid moieties enhances their pharmacokinetic properties, particularly their circulatory half-life and biodistribution within the human body.
Hvam et al. (2017) employed a fatty acid modification strategy to enhance endogenous albumin binding for the tunable pharmacokinetics of gapmer ASOs.
What did the researchers observe?
- Hvam et al. found that the addition of fatty acids, palmitic or myristic acid, to gapmer antisense ASOs improves the pharmacokinetics of the ASOs. The researchers found that a fatty acid-modified gapmer interacts with human serum albumin (HSA) to self-assemble into molecular constructs that have favorable pharmacokinetics. The research group introduced fatty acids via N2’-functionalized amino-LNA monomers at specific locations within the gapmer sequence.
- The binding of these modified gapmers to HSA resulted in increased blood circulation in mice (t1/2 increased from 23 to 49 min for phosphodiester [PO] gapmer ASOs and from 28 to 66 min for PS gapmer ASOs with two palmitic acid modifications) and a shift toward a broader biodistribution for PS compared with PO gapmer ASOs. The addition of two palmitoyl groups to the ASOs shifted the biodistribution to resemble that of natural albumin.
- Additionally, the research group discovered that the number, position, and fatty acid modification of the ligand influence its binding affinity to albumin.
- The binding affinity increases with the addition of more fatty acid modifications.
- The introduction of PS backbone linkages also increases the binding affinity of fatty acid modified ASOs to albumin. However, the increase was much less significant compared with that of fatty acid modifications.
Prakash et al. (2019) synthesized several saturated and unsaturated fatty acid ASO conjugates to determine their binding affinity to plasma proteins in a mouse model.
What did the researchers observe?
- ASO conjugates containing fatty acid chain lengths from 16 to 22 carbons exhibited the best binding affinities. The degree of unsaturation or conformation of the double bond appeared not to influence protein binding or activity of ASO fatty acid conjugates.
- The activity of fatty acid ASO conjugates correlated with the affinity to albumin. The best albumin binder showed the most significant improvement in muscle activity.
- Palmitic acid conjugation increases ASO plasma Cmax and improves delivery of ASO to the interstitial space of mouse muscle.
- The conjugation of palmitic acid improved the potency of DMPK, Cav3, CD36, and Malat-1 ASOs (3- to 7-fold) in mouse muscle.
- Palmitoyl conjugation improves the potency of cEt BNA Malat-1 ASO 3–6-fold in muscle.
- The palmitoyl moiety can be attached using a phosphodiester d(TCA) linker, which is metabolized in tissues to release the ASO.
- Palmitoyl conjugation enhances the potency of ASO targeting DMPK mRNA in mice.
- Palmitoyl conjugation enhances the potency of ASO targeting Cav3 mRNA more than 5-fold in mouse skeletal and cardiac muscle.
| Palmitic Acid, C16 | Myristic Acid, C14 |
|  |  |
| Wiki-Palmitic_acid; Palmitic-Acid C16H32O2; Mw 256.430 g/mol | Wiki/Myristic_acid; Myristic-Acid C14H28O2; Mw 228.376 g·mol−1 |
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Example of an antisense oligonucleotide conjugated to palmitic acid. The selected fatty acid can be connected to the ASO or gapmer ASO internally, at the 5’- or the 2’-end. |
What is a Gapmer?
A gapmer is a short, synthetic, single-stranded DNA molecule designed to bind to a specific messenger RNA (mRNA) or other selected RNA target. Gapmers typically have a central "gap" region made of DNA (often with phosphorothioate modifications for stability) flanked by "wings" made of RNA mimics (like bridged (BNA) or locked nucleic acids (LNA) or 2'-O-methoxyethyl (2'-MOE) modifications).
Gapmer-Design Example
When a gapmer binds to its complementary RNA target, it forms a DNA-RNA hybrid duplex that is recognized and cleaved by RNase H. The targeted mRNA is cleaved and degraded, preventing the production of the protein it codes for and effectively silencing the gene. Gapmers offer the development of therapeutics for various diseases, including cancers, viral infections, and genetic disorders.
Fatty Acid Modification
Fatty acid modification utilizes the natural transport mechanisms of serum albumin, the most abundant protein in blood plasma. The binding of serum albumin to fatty acids results in a long circulatory half-life, approximately 19 days in humans, due to its interaction with the neonatal Fc receptor (FcRn), which recycles it back into circulation. By attaching fatty acids, such as palmitic acid or myristic acid, to a gapmer antisense oligonucleotide (ASO), the ASO can self-assemble with serum albumin in the bloodstream. The binding to albumin significantly increases the ASO's blood circulation time.
Advantages of Fatty Acid-Modified Gapmers
Conjugating fatty acids to ASOS results in improved pharmacokinetics and prolonged circulation half-life. By binding to albumin, the fatty acid-modified gapmer prevents rapid clearance from the body, for example, by the kidneys, allowing the ASO to remain in circulation longer and reach target tissues more effectively.
The interaction with albumin also influences how the ASO distributes in the body, leading to better delivery to specific tissues beyond the liver and kidneys. The liver and kidneys are often the primary sites of uptake for unmodified ASOs.
A longer half-life allows the administration of the drug less frequently, improving patient convenience and compliance.
The type, number, and position of fatty acid modifications, as well as backbone modifications such as phosphorothioate linkages, enable fine-tuning of the binding affinity to albumin and, consequently, the pharmacokinetic profile.
Fatty acid conjugation offers a simpler alternative to more complex, pre-formulated delivery systems, such as lipid nanoparticles, by utilizing the human body's natural transport mechanisms.
References
Fatty-acid-conjugated-BNA-antisense-oligonucleotides-exhibit-enhanced-uptake-into-muscle
Hvam ML, Cai Y, Dagnæs-Hansen F, Nielsen JS, Wengel J, Kjems J, Howard KA. Fatty Acid-Modified Gapmer Antisense Oligonucleotide and Serum Albumin Constructs for Pharmacokinetic Modulation. Mol Ther. 2017 Jul 5;25(7):1710-1717. [PMC]
Palmitoyl-c16-modification
Prakash TP, Mullick AE, Lee RG, Yu J, Yeh ST, Low A, Chappell AE, Østergaard ME, Murray S, Gaus HJ, Swayze EE, Seth PP. Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle. Nucleic Acids Res. 2019 Jul 9;47(12):6029-6044. [PMC]
Regiospecific-modification
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Bio-Synthesis provides a full spectrum of oligonucleotide and peptide synthesis including bio-conjugation services as well as high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.
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