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|Development of immunomodulatory RNA therapeutics for immune disorders
|Iyer, Vaishnavi Srinivasan
|Nanyang Technological University
|Iyer, V. S. (2022). Development of immunomodulatory RNA therapeutics for immune disorders. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/161897
|Autoimmune diseases such as Rheumatoid Arthritis (RA) are complex and involve many different cell types. Of these cell types, T cells are thought to be key drivers of the inflammation. The current treatment options available are restricted to small molecule drugs and Biologics, typically exerting its therapeutic activity by interfering with exact protein structures. Notably, only a fraction of the proteins in the body are predicted to be druggable by such drug modalities, greatly reducing the potential drug targets. Antisense Oligonucleotides (ASOs) are synthetic oligonucleotides that are designed to manipulate expression of their target genes at the post-transcriptional level. As ASOs have the potential to target any gene, thus greatly expanding the universe of potential drug targets, it represents a viable therapeutic approach to modulate aberrant pathways in diseases like RA. In paper I, we present a present a novel way of using CRISPR-mediated gene knock out to validate the role of target genes in particular cellular pathways of interest. We describe the RCC (rapid CRISPR competitive) assay, where we exploit the heterogeneity of the CRISPR-Cas9 mediated genome editing together with sanger sequencing to study the role of the target gene based on both the enrichment or depletion of mutated sequences but also the type of mutation (frameshift or silent mutations). In particular, LCP2 was validated as good target to reduce the activation potential of T cells. Thus in paper II, ASOs targeting LCP2 were designed and evaluated for their knockdown potential and their subsequent ability to reduce T cell activation. We show that these ASOs can modulate T cell behavior in human cell lines and human primary cells, validating the potential of ASOs as a therapeutic approach for immune diseases. There has been a lot of research into the phenotype of T cells found in RA patients and how they differ from healthy controls. One of the hypotheses for the progression of the disease is that T cells reacting to specific peptides are thought to contribute to the disease. Thus, paper III and paper IV focus on studying TCR specificities to gain a better understanding of the particular T cells that could be the culprits. In particular, these papers focus on development of methods to identify interesting TCR sequences from patients and re-expression of these TCRs in vitro and in vivo to study them in detail with methods to recapitulate patient T cells in vitro. Though focused currently on limited gene targets, this paves the way to answer the overarching questions forming the basis of my PhD: what is a good drug target, and how can technological developments transform the field of drug development?
|School of Chemistry, Chemical Engineering and Biotechnology
|This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).
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Updated on Feb 28, 2024
Updated on Feb 28, 2024
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