Abstract
Purpose :
Antisense oligonucleotides (AONs) are a versatile tool to treat inherited retinal diseases (IRDs). Amongst other functions, AONs can modulate splicing, and for example correct disease-causing aberrantly spliced transcripts. However, despite the therapeutic potential of these molecules, little is known on how different chemical modifications of AONs can affect their biodistribution, toxicity and uptake in the retina. We addressed this question by extensively comparing splice-switching AONs with three different chemical modifications commonly used in a clinical setting (2’O-methyl-phosphorothioate (2-OMe/PS), 2’O-methoxyethyl-phosphoriate (2-MOE/PS), and phosphorodiamidite morpholino oligomers (PMO)).
Methods :
Eight different genes exclusively expressed in specific retinal cell types were selected. We focused on rods, cones, bipolar and retinal pigment epithelium cells, which are frequently affected in IRDs. Several AONs for each target gene were designed and evaluated in vitro by employing splice reporter vectors in human and mouse-derived cell lines. The best performing AON for each target was then assessed in vivo by intravitreal delivery to C57BL/6J wild-type mice. Animals were examined for one week and eyes were collected for molecular and morphological studies. The animal research was conducted according to the regulations of the ARVO statement for the use of animals in ophthalmic and vision research. All procedures were conducted in the Netherlands.
Results :
Our studies in vitro and in vivo showed that 2-OMe/PS and 2-MOE/PS AONs have comparable efficacy and safety profiles. In contrast, in vivo PMO-oligonucleotides (ivPMO), conjugated to an octa-guanidine dendrimer caused a severe toxic effect observed already after 24 hours. This was evidenced by externally visible ocular phenotypes in 88.5% of all ivPMO-treated animals, accompanied by severe alterations at the morphological level. However, delivery of unmodified PMO-AONs did not cause any toxicity, although it significantly reduced the efficacy.
Conclusions :
These results showed that the same AON sequence with different chemical modifications displayed different splicing modulation efficacies, being the 2’MOE/PS the most efficacious molecule both in vitro and in vivo. This is the first systematic comparison of different chemical modifications of AONs in the retina, providing important insights for clinical applications.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.