Development of novel antisense oligonucleotide molecules towards tackling rare and acquired diseases

Prithi Raguraman

Antisense oligonucleotides (ASOs) are a class of short synthetic molecules that can bind to RNA targets through Watson and Crick base pairing rules and regulate gene expression. ASO-based therapy has been well-studied in the past decade for tackling various genetic conditions. Antisense oligo-mediated modulation RNA splicing has been well-established as a therapeutic strategy for conditions like Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). This thesis focuses on developing novel chemically modified splice-modulating ASOs for therapeutic applications including DMD, cancer and skin inflammation including psoriasis. Chapter 1 of the thesis provides a general overview of oligonucleotide therapeutics. It also highlights the mechanism of different classes of therapeutic oligonucleotides including their potential therapeutic applications. Chapter 2 focuses on the evaluation of different chemically modified ASOs in a DMD model system in vitro. In this study, the potential of three distinct chemically modified ASOs with modifications in the sugar, phosphate group and nucleobase namely, alpha-L-Locked nucleic acid, 2’-O-methyl- 5-(1-phenyl-1,2,3-triazol-4-yl) uridine and 4-(trimethylammonio)butylsulfonyl or tosyl sulfonyl phosphoramidates were tested for their efficacy to induce exon-23 skipping in the mouse dystrophin transcripts in vitro. Results indicated that compared to the already established 2O- methyl phosphorothioate ASOs, the newly studied ASOs showed better efficacy to induce exon skipping. Chapter 3 of the thesis detailed the design and development of ASOs targeting IL-36G, IL-23A, C10orf99, IGFL-1 and S100A7 to find a therapeutic ASO for Psoriasis. Psoriasis is an auto-immune/auto-inflammatory disease that is characterised by the abnormal differentiation and proliferation of keratinocytes. Genes that are overexpressed in psoriasis were selected for this study. The results indicated that the ASOs developed were efficient to reduce the expression of IL-36G, IL-23A, C10orf99 and IGFL-1. Chapter 4 of the thesis focused on the design and evaluation of the ASOs targeting Required for Meiotic Nuclear Divison homolog 1 (RMND1) in human cancer cells. RMND1 belongs to the family sif2 proteins and has been implicated as a minor pan-cancer site suggesting its involvement in cancer progression. ASO targeting RMND1 was developed and its efficiency to induce exon 3 skipping and subsequently its efficacy to reduce the protein expression was studied. In summary, novel ASO candidates were developed and evaluated against diseases including DMD, psoriasis and cancer, and the efficient ASO identified in this study hold significant potential for future studies and therapeutic development.

Development of novel antisense oligonucleotide molecules towards tackling rare and acquired diseases

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