Propensities of Fatty Acid-Modified ASOs: Self-Assembly vs Albumin Binding

Abstract

We conducted a biophysical study to investigate the self-assembling and albumin-binding propensities of a series of fatty acid-modified locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers specific to the MALAT1 gene. To this end, a series of biophysical techniques were applied using label-free ASOs that were covalently modified with saturated fatty acids (FAs) of varying length, branching, and 5′/3′ attachment. Using analytical ultracentrifugation (AUC), we demonstrate that ASOs conjugated with fatty acids longer than C16 exhibit an increasing tendency to form self-assembled vesicular structures. The C16 to C24 conjugates interacted via the fatty acid chains with mouse and human serum albumin (MSA/HSA) to form stable adducts with near-linear correlation between FA-ASO hydrophobicity and binding strength to mouse albumin. This was not observed for the longer fatty acid chain ASO conjugates (>C24) under the experimental conditions applied. The longer FA-ASO however adopted self-assembled structures with increasing intrinsic stabilities proportional to the fatty acid chain length. For instance, FA chain lengths smaller than C24 readily formed self-assembled structures containing 2 (C16), 6 (C22, bis-C12), and 12 (C24) monomers, as measured by analytical ultracentrifugation (AUC). Incubation with albumin disrupted these supramolecular architectures to form FA-ASO/albumin complexes mostly with 2:1 stoichiometry and binding affinities in the low micromolar range, as determined by isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC). Binding of FA-ASOs underwent a biphasic pattern for medium-length FA chain lengths (>C16) with an initial endothermic phase of particulate disruption, followed by an exothermic binding event to the albumin. Conversely, ASO modified with di-palmitic acid (C32) formed a strong, hexameric complex. This structure was not disrupted when incubated with albumin under conditions above the critical nanoparticle concentration (CNC; <0.4 μM). It is noteworthy that the interaction of parent, fatty acid-free malat1 ASO to albumin was below detectability by ITC (K D ≫150 μM). This work demonstrates that the nature of mono- vs multimeric structures of hydrophobically modified ASOs is governed by the hydrophobic effect. Consequently, supramolecular assembly to form particulate structures is a direct consequence of the fatty acid chain length. This provides opportunities to exploit the concept of hydrophobic modification to influence pharmacokinetics (PK) and biodistribution for ASOs in two ways: (1) binding of the FA-ASO to albumin as a carrier vehicle and (2) self-assembly resulting in albumin-inert, supramolecular architectures. Both concepts create opportunities to influence biodistribution, receptor interaction, uptake mechanism, and pharmacokinetics/pharmacodynamics (PK/PD) properties in vivo, potentially enabling access to extrahepatic tissues in sufficient concentration to treat disease.

Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels

“…Despite the tight complex that L15 and PS-L15 can form with the MALAT1 SL (Figures 2B,C and 3B,C), it is not strong enough to counteract the cellular degradation machinery, because our cell-based assays showed that the levels of MALAT1 are reduced (Figure 5A). Additionally, compound 5 [32,46], 4001-6000 [38,45] and nts 2001-4000 [38] on mouse MALAT1. Gapmer ASOs targeting human MALAT1 nts 6001-8000 [40] and mouse MALAT1 nts 1-2000 [34,42], 2001-4000 [39], 4001-6000 [38].…”

“…For future investigations, it may be advantageous to design an LNA mixmer composed of natural and chemically modified nucleosides so that the increased flexibility can achieve greater stability of the MALAT1 and MENβ RNA•LNA-RNA triple helices along the Hoogsteen and Watson-Crick interfaces. [32,46], 4001-6000 [38,45] and nts 2001-4000 [38] on mouse MALAT1. Gapmer ASOs targeting human MALAT1 nts 6001-8000 [40] and mouse MALAT1 nts 1-2000 [34,42], 2001-4000 [39], 4001-6000 [38].…”

“…Although the MENβ triple helix is less studied than its counterpart in MALAT1, compounds like aurintricarboxylic acid, emodin, GW5074, mitoxantrone and rottlerin bind to the MENβ triple helix near the micromolar range, and the kinase inhibitor PIK-75 abolishes paraspeckles in the neuroblastoma cell line SH-SY5Y [30]. ASO therapeutics have been used to target and regulate the MALAT1 lncRNA in various cancer types [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. A variety of ASOs, typically 16-20 nucleotides in length, have been designed, such as small interfering RNA (siRNA) [35][36][37]; gapmers with two to three locked nucleic acids (LNAs) [31,33,34,[38][39][40]; 2 ′ -O-methylethyl groups; 2 ′ -O,4 ′ -C-ethylene-bridged nucleic acid [41] or guanidine-bridged nucleic acid [42] at the end(s); PNA-DNA chimeras [43]; and the conjugation of ASO to single-wall carbon nanotubes [44], gold nanoparticles [32], TAT peptides [32], fatty acids [45] or membrane protein-binding aptamers [46] to improve delivery and biodistribution.…”

“…ASO therapeutics have been used to target and regulate the MALAT1 lncRNA in various cancer types [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. A variety of ASOs, typically 16-20 nucleotides in length, have been designed, such as small interfering RNA (siRNA) [35][36][37]; gapmers with two to three locked nucleic acids (LNAs) [31,33,34,[38][39][40]; 2 ′ -O-methylethyl groups; 2 ′ -O,4 ′ -C-ethylene-bridged nucleic acid [41] or guanidine-bridged nucleic acid [42] at the end(s); PNA-DNA chimeras [43]; and the conjugation of ASO to single-wall carbon nanotubes [44], gold nanoparticles [32], TAT peptides [32], fatty acids [45] or membrane protein-binding aptamers [46] to improve delivery and biodistribution. Most ASO gapmers target unstructured regions of MALAT1, leading to RNase H-mediated knockdown from 2-50-fold [31,34,39,40].…”

“…Summary of therapeutic strategies and effectiveness in lowering MALAT1 levels. ASOs used to target human MALAT1 include nts 2001-4000[32,46], 4001-6000[38,45] and nts 2001-4000[38] on mouse MALAT1. Gapmer ASOs targeting human MALAT1 nts 6001-8000[40] and mouse MALAT1 nts 1-2000[34,42], 2001-4000[39], 4001-6000[38].…”