title: 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 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.
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.
authors.forename: Kariem
authors.initials: K
authors.lastname: Ezzat
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.|●Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo 187-8551, Japan.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Aoki
authors.affiliation: †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.
authors.forename: Taeyoung
authors.initials: T
authors.lastname: Koo
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.
authors.forename: Graham
authors.initials: G
authors.lastname: McClorey
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.
authors.forename: Leif
authors.initials: L
authors.lastname: Benner
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.
authors.forename: Anna
authors.initials: A
authors.lastname: Coenen-Stass
authors.affiliation: ∥Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors.forename: Liz
authors.initials: L
authors.lastname: O'Donovan
authors.affiliation: ⊥Department of Laboratory Medicine, Karolinska Institute, Stockholm SE-171 77, Sweden.
authors.forename: Taavi
authors.initials: T
authors.lastname: Lehto
authors.affiliation: #Clarendon Laboratory, Department of Physics, University of Oxford, OX13PU, Oxford, United Kingdom.
authors.forename: Antonio
authors.initials: A
authors.lastname: Garcia-Guerra
authors.affiliation: ⊥Department of Laboratory Medicine, Karolinska Institute, Stockholm SE-171 77, Sweden.
authors.forename: Joel
authors.initials: J
authors.lastname: Nordin
authors.affiliation: ∥Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors.forename: Amer F
authors.initials: AF
authors.lastname: Saleh
authors.affiliation: ∇Integrated DNA Technologies (IDT), Coralville, Iowa 55241, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Behlke
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Morris
authors.affiliation: ○Université de Versailles Saint Quentin, Montigny le Bretonneux 78180, France.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Goyenvalle
authors.affiliation: ◆Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland.
authors.forename: Branislav
authors.initials: B
authors.lastname: Dugovic
authors.affiliation: ◆Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Leumann
authors.affiliation: ¶Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, Oxford, United Kingdom.
authors.forename: Siamon
authors.initials: S
authors.lastname: Gordon
authors.affiliation: ∥Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.|⊥Department of Laboratory Medicine, Karolinska Institute, Stockholm SE-171 77, Sweden.
authors.forename: Samir
authors.initials: S
authors.lastname: El-Andaloussi
authors.affiliation: †Department of Physiology, Anatomy, and Genetics, University of Oxford, OX13QX, Oxford, United Kingdom.
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D015316:Genetic Therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008823:Micelles; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy; D053758:Nanoparticles / Q000737:chemistry*; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics* / Q000627:therapeutic use; D051117:Scavenger Receptors, Class A / Q000235:genetics / Q000378:metabolism*
chemical_list: D008823:Micelles; D016376:Oligonucleotides, Antisense; D051117:Scavenger Receptors, Class A
keywords: Antisense oligonucleotides; delivery; nanoparticles; scavenger receptors; self-assembly; uptake mechanism