Design and application of RNA therapeutics for splice site mutations

Abstract: Precursor messenger RNA splicing is one of the most fundamental and complex mechanisms in eukaryotes. Over 90% of the human genes undergo alternative splicing, which is essential for the regulation of gene expression. A dynamic RNA-protein complex called the spliceosome catalyzes splicing. The spliceosome recognizes core splice site signals, and its function is furthermore regulated by other sequence elements that can either silence or enhance splicing in a cell-specific manner. These regulatory elements are recognized by trans-acting protein factors which modulate the function of the spliceosome. However, the fact that splicing is one of the most regulated mechanisms in the cell also makes it prone to dysfunctions caused by mutations. Mis-splicing diseases account for up to 30% of the inherited genetic diseases. They can be caused by mutations in the core splice sites as well as in the regulatory elements. Mutations that disrupt the splicing mechanism may result in RNA degradation, non-functional protein products, or toxic proteins that might alter the cellular environment. The understanding of mis-splicing disease mechanisms has been subject to various studies aiming at finding the appropriate therapeutics. In parallel with the development of gene therapy, where the classical aim is to introduce the corrected gene to cure a disease, another field has emerged; antisense oligonucleotide therapeutics. These RNA-DNA-based oligonucleotides can be designed to alter gene expression as well as to manipulate the splicing mechanism. Since their emergence during 1970s, antisense oligonucleotides have been extensively studied within the mis-splicing disease field, with several clinical trials ongoing. In this thesis, in paper I, we report a novel solid-phase synthesis method with a biological proof-of-concept. This synthesis method allows the conjugation of therapeutic oligonucleotides via a cleavable disulfide linker. The developed method can be applied to target several transcripts or different parts within the same transcript by allowing delivery of equimolar amounts of therapeutics. In paper II, we explore the possibility of using splice- correction approach for restoring the aberrant splicing of the gene BTK. Lack of BTK causes a primary immunodeficiency disease called XLA, which is a B cell developmental disorder. For the first time, we show splice-correction in B cells by modified oligonucleotide therapeutics both ex vivo and in vivo. In paper III, we aim at developing methods to rescue the core splice site mutations in BTK by using bifunctional oligonucleotide therapeutics. These oligonucleotides have the ability to recruit splice factor proteins, improving the splicing of mutated sites. We show that rescuing of core splice site mutations is possible yet it has its own challenges, which need to be taken into account in the design process. The results in this thesis have provided new therapeutics for a genetic disease, and more generally explores new methods for improving the function and delivery of oligonucleotide therapeutics.