Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides

Abstract

Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2’Omethyl phosphorothioate (2’OMe) and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knock-out mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2’OMe as shown by competitive inhibition and co-localization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2’OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.

Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA

“…Clustering or assembly of oligonucleotides often correlates with enhanced uptake and activity, across multiple size scales and likely via multiple mechanisms 48, 49. Nevertheless, in our case, despite the efficient self-assembly observed for the lipid tail conjugates, the biostable hexadecyloxypropyl conjugates showed reduced silencing efficacy in cells (Figure 5C).…”

Peptides for nucleic acid delivery

“…However, we have recently shown that due to their amphipathic nature, PPMOs can self-assemble into micellar structures that are then taken up via receptor-mediated endocytosis [117]. Additionally, the net charge of the conjugates was dependent on the concentration.…”

“…In gel electrophoresis experiments, the migration of the conjugates was reversed between high and low concentrations displaying a net negative charge at the low concentrations and a net positive charge at high concentrations. We speculated that this was due to the change in the balance between the ions in the medium and the charges on the conjugates enabling better shielding with counterions at lower concentrations [117]. Furthermore, self-assembly into nanoparticles was also proposed as mechanism of uptake for PNAOligo(bicycloguanidinium) conjugates [118] and the propensity to form particles was demonstrated to enhance cellular uptake even for newly developed negatively-charged ON chemistry, tcDNA [57] Different mechanisms have been proposed for CPP conjugates and nanoparticles and distinct pathways have been postulated from direct penetration to receptor-mediated endocytosis.…”

“…We would like to speculate that the apparent discrepancies might arise from different aspects of uptake of CPPs. Recent research has demonstrated that the demarcation line between conjugates and nanocomplexes is not very clear, since both PPMO and PPNA conjugates were shown to form nanoparticles [117,118]. The formation of nanoparticles might be important for the uptake based on the particle-wrapping model of endocytosis, where certain threshold of particle size should be reached for optimal uptake [119].…”

Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers

“…Third, our in vivo experiments, as well as other prior studies, show a mild but measurable effect of the control (Scrambled) PPMO despite a lack of in vitro activity. It is unclear whether this is related to nonspecific immune activation of the peptide itself; however, the recently reported scavenger receptor binding of PPMOs in eukaryotic cells (40) could make this a possible explanation. Recent studies by our group show that, at least in a sepsis infection model, the control PPMO acts similarly to a PBS control in terms of serum proinflammatory cytokine stimulation, and only the active PPMO significantly reduced serum proinflammatory cytokines (interleukin-6 and tumor necrosis factor alpha), presumably due to pathogen inhibition.…”