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Kilchrist V. Kilchrist, Ashwath Jayagopal, Faye Drawnel; Engineering of locked nucleic acids with small molecule ligands for ocular surface diseases. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3372.
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© ARVO (1962-2015); The Authors (2016-present)
Precision medicine strategies using locked nucleic acids (LNA) are an attractive approach to treat corneal diseases. LNAs induce sequence specific degredation of mRNA via RNAse H. LNAs could target molecular mediators of disease in the cornea. However, attempts to use LNAs in the cornea have been hindered by pharmacokinetics (PK), necessitating delivery vehicles using lipids or polymers.Small molecule ligand (SML) conjugates to LNA (LNA-SML) are an alternative to transfection reagent formulations. SML are hypothesized to enhance local PK by binding to cells and nearby matrix to promote cell uptake and retention. This technology would allow any gene or miRNA to be inhibited, enabling a new generation of personalized corneal therapeutics. In this study, we developed and characterized LNA-SML conjugates for enhancing corneal uptake and LNA bioactivity.
Phosphorothioated LNA were terminated with a 5’ hexylamino linker. SMLs including 1,2-dithiolane-4-carboxylic acid (Fig 1A, AspA) were conjugated to the LNA amine via EDC/NHS coupling and purified molecular weight cut off spin filtration precipitation and HPLC to yield LNA-SML (Fig 1B). LNA-SML was confirmed by LC/MS. LNA against MALAT1 was used as a tool sequence. The LNA-SML was applied to ARPE-19 and HCE-T cells for 30 minutes in PBS then washed with media with 10% FBS, simulating instillation via eye drops and challenge by tear proteins.
Treatment with 40 nM LNA-AspA results in significant reduction (-41%) of the model gene MALAT1, while untargeted LNA results in +8% MALAT1 levels (Fig 1C). AspA targeting induces ~10-fold increase of LNA uptake relative to control (Fig 1D). Microscopy shows that treatment with LNA or LNA-AspA does not affect monolayer morphology, suggesting favorable tolerability profiles for LNA-SML at therapeutic doses.
We synthesized LNA-SMLs and showed proof-of-concept data that SMLs enable intracellular LNA delivery and significantly reduce the model gene MALAT1 relative to untargeted LNA. The facile synthesis and superior bioactivity suggest LNA-SMLs are preferable to lipid or polymer formulations. These data support further inquiry into this class of molecules that enable LNA delivery to corneal cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
A) SML structure: 1,2-dithiolane-4-carboxylic acid B) AspA attached to LNA via 5' amide C) LNA-SML significantly improves MALAT1 knockdown (-48%, p < 0.05) D) LNA-SML enhances LNA uptake by ~10-fold
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