title: Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides.
abstract: Splice switching oligonucleotides (SSOs) induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, "ZEN™") to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2'-O-methyl (2'OMe) oligonucleotide, increasing melting temperature and potency over unmodified 2'OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2'OMe phosphorothioate (PS) oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2'OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Suzan M
authors.initials: SM
authors.lastname: Hammond
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Graham
authors.initials: G
authors.lastname: McClorey
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden.
authors.forename: Joel Z
authors.initials: JZ
authors.lastname: Nordin
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Godfrey
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden.
authors.forename: Sofia
authors.initials: S
authors.lastname: Stenler
authors.affiliation: Integrated DNA Technologies, Inc, Coralville, Iowa, USA.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Lennox
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden.
authors.forename: C I Edvard
authors.initials: CI
authors.lastname: Smith
authors.affiliation: Integrated DNA Technologies, Inc, Coralville, Iowa, USA.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Jacobi
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Varela
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Integrated DNA Technologies, Inc, Coralville, Iowa, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood
authors.affiliation: 1] Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK [2] Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden.
authors.forename: Samir E L
authors.initials: SE
authors.lastname: Andaloussi