Exploring novel splice modulating antisense oligonucleotides for tackling FGF23- related disorders

Arpitha Chikkanna

Antisense oligonucleotides (ASOs) are a class of custom-made short single-stranded nucleic acid molecules that are designed to specifically bind to a target RNA through Watson-crick base pairing. This targeted approach can be utilised to modulate gene expression by either blocking the translation of RNA into protein or promoting its degradation. Since the 90s, ASOs have been widely recognised as potent therapeutic tools for addressing a multitude of disease conditions. Typically, naturally occurring RNA is prone to nuclease degradation that affects its stability and binding affinity. To address these limitations, various chemical modifications are introduced to enhance the pharmacological properties of ASOs. Among the ASO approaches, splice modulating ASOs are crafted to bind to a specific site in the pre-messenger RNA (premRNA) to alter the splicing process and potentially inducing splice modulation. This thesis explores the potential of novel chemically modified splice modulating ASOs for tackling fibroblast growth factor 23 (FGF23) related disorders. Chapter 1 provides a comprehensive introduction to nucleic acid technologies, emphasizing the importance of their chemical modifications and different drug delivery approaches for therapeutic applications. Furthermore, this chapter provides an overview of FGF23, detailing its mechanism of action and its involvement in vitamin D metabolism. Chapter 2 specifies the materials and methods used in this study. Chapter 3 is focused on the design and evaluation of novel splice modulating ASOs targeting human FGF23 in chronic kidney disease (CKD). ASOs incorporating 2’O-methyl phosphorothioate (2’OMe-PS)-modifications and phosphorodiamidate morpholino oligomers (PMO) modifications were designed, synthesized, and tested for their ability to induce exon- 2 skipping. The ASOs PNAT669 and PNAT670 efficiently induced exon-2 skipping in FGF23, leading to premature termination codons in exon 3, thereby downregulating FGF23 protein expression in human embryonic kidney 293 cells (HEK293). Moreover, the ASOs PNAT669 and PNAT-PMO 669 demonstrated significant impact on the downstream enzymes, with both significantly increasing 1α-hydroxylase (1α(OH)ase) and reducing 24-hydroxylase (24(OH)ase). The focus of chapter 4 was to design and evaluate novel splice modulating antisense oligonucleotides targeting human FGF23 in tumour induced osteomalacia (TIO). The ASOs PNAT669 and PNAT670 effectively induced exon-2 skipping and caused protein knockdown in osteosarcoma cells (Saos- 2). Furthermore, PNAT-PMO 669 and PNAT-PMO 670 induced exon-2 skipping and knockdown of FGF23 protein, while also affecting the expression of the downstream enzymes. The focus of Chapter 5 was to design and evaluate splice modulating ASOs targeting human and mouse FGF23 for the treatment of X-linked hypophosphatemia (XLH). To evaluate the efficacy of human ASOs, we have obtained B-lymphocytes (derived from LCL) from a female adult and her son, both with XLH. However, due to the hard-to-transfect nature and extremely low expression of FGF23 of these cells, we were not able to generate any meaningful data with the human ASOs. On the other hand, mouse variants of the FGF23-targeting ASOs were evaluated in alpha mouse liver 12 (AML-12) cells, where we observed efficient exon-2 skipping induced by ASO4. Additionally, in AML-12 cells, ASO3 and ASO4 both demonstrated significant reduction in the expression of mouse FGF23 protein. This positive outcome encourages further testing of ASO3 and 4 in animal models. Overall, the research outcomes presented in this thesis validates the scope of using splice modulating antisense oligonucleotides towards the development of potential therapeutic molecules for the treatment of FGF23 related disorders.

Exploring novel splice modulating antisense oligonucleotides for tackling FGF23- related disorders

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