2. Academic Validation
  3. A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity

A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity

  • Cell Rep Methods. 2023 Dec 23:100673. doi: 10.1016/j.crmeth.2023.100673.
Amy E Byrnes 1,  Filip Roudnicky 2,  Alvin Gogineni 3,  Allison L Soung 1,  Monica Xiong 1,  Margaret Hayne 1,  Tiffany Heaster-Ford 3,  Whitney Shatz-Binder 4,  Sara L Dominguez 1,  Jose Imperio 1,  Sarah Gierke 5,  Jasmine Roberts 1,  Jinglong Guo 6,  Soumitra Ghosh 1,  Charles Yu 7,  Merone Roose-Girma 7,  Justin Elstrott 3,  Amy Easton 1,  Casper C Hoogenraad 8
Affiliations

Affiliations

  • 1 Department of Neuroscience, Genentech, Inc., South San Francisco, CA 94080, USA.
  • 2 Pharmaceutical Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
  • 3 Department of Translational Imaging, Genentech, Inc., South San Francisco, CA 94080, USA.
  • 4 Pharmaceutical Development, Genentech, Inc., South San Francisco, CA 94080, USA.
  • 5 Department of Pathology, Genentech, Inc., South San Francisco, CA 94080, USA; Center for Advanced Light Microscopy, Genentech, Inc., South San Francisco, CA 94080, USA.
  • 6 Department of Cancer Immunology, Genentech, Inc., South San Francisco, CA 94080, USA.
  • 7 Molecular Biology, Genentech, Inc., South San Francisco, CA 94080, USA.
  • 8 Department of Neuroscience, Genentech, Inc., South San Francisco, CA 94080, USA. Electronic address: [email protected].
PMID: 38171361 DOI: 10.1016/j.crmeth.2023.100673
Abstract

While Antisense Oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity in vivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types. We then assayed the impact of lipid nanoparticle delivery on ASO activity after intravenous administration. Robust EGFP fluorescence was observed in the liver and EGFP+ cells were successfully isolated using fluorescence-activated cell sorting. Together, these results show the utility of this animal model in quantifying both cell-type- and organ-specific ASO delivery, which can be used to advance ASO therapeutics for many disease indications.

Keywords

CP: Biotechnology; antisense oligonucleotide; central nervous system; delivery; fluorescence; knockin mouse; lipid nanoparticle; splice-switching.

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