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Paper in Nano Letters

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

Ezzat K., Aoki Y., Koo T., McClorey G., Benner L., Coenen-Stass A., O´Donovan L., Lehto T., Garcia-Guerra A., Nordin J., Saleh A.F., Behike M., Morris J., Goyenvalle A., Dugovic B., Leumann C., Gordon S., Gait M.J., El‑Andaloussi S., Wood M.J.A.

Nano Lett., 2015, June 4, doi10.1021/acs.nanolett.5b00490

  • Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom 
  • Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea
  • Functional Genomics, University of Science and Technology, Daejeon 305-338, South Korea
  • Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
  • Department of Laboratory Medicine, Karolinska Institute, Stockholm SE-171 77, Sweden
  • Clarendon Laboratory, Department of Physics, University of Oxford, OX13PU, Oxford, United Kingdom
  • Integrated DNA Technologies (IDT), Coralville, Iowa 55241, United States
  • Université de Versailles Saint Quentin, Montigny le Bretonneux 78180, France
  • Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland
  • Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, Oxford, United Kingdom
  • Department of Molecular Therapy, National Institute of Neuroscience, National Center of
  • Neurology and Psychiatry (NCNP), Tokyo 187-8551, Japan

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 knockout 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 colocalization 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.