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

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 (KD ≫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.

Impact of non-ionizable lipids and phase mixing methods on structural properties of lipid nanoparticle formulations.

Lipid nanoparticles (LNPs) have been widely investigated for nucleic acid therapeutic delivery, and demonstrated their potential in enabling new mRNA vaccines. LNPs are usually formulated with multi-lipid components and the composition variables may impact their structural properties. Here, we investigated the impact of helper lipids on physicochemical properties of LNPs using a Design of Experiments (DoE) definitive screening design. Phospholipid head group, degree of unsaturation, ratio to cholesterol as well as PEG-lipid content were varied and a series of 14 LNPs were prepared by microfluidic- and solvent-injection mixing. Solvent-injection mixing by a robotic liquid handler yielded 50-225 nm nanoparticles with highly ordered, ∼5 nm inter-lamellar spacing as measured by small angle X-ray scattering (SAXS) and confirmed by cryo-transmission electron microscopy (cryo-EM). In contrast, microfluidic mixing resulted in less ordered, notably smaller (50-75 nm) and more homogenous nanoparticles. Significant impacts of the stealth-lipid DSPE-PEG2000 on nanoparticle size, polydispersity and encapsulation efficiency of an oligonucleotide cargo were observed in LNPs produced by both methods, while varying the phospholipid type and content had only marginal effect on these physicochemical properties. These findings suggest that from a physicochemical perspective, the design space for combinations of helper lipids in LNPs may be considerably larger than anticipated based on the conservative formulation composition of the currently FDA-approved LNPs, thereby opening opportunities for screening and optimization of novel LNP formulations.

Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides.

The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.