Developing gene modification technology based therapies for ankylosing spondylitis. a proof of principle study

Rasiukienė, Olga (2024) Developing gene modification technology based therapies for ankylosing spondylitis. a proof of principle study. Doctoral thesis, Northumbria University.

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Abstract

Ankylosing Spondylitis (AS) is an autoimmune inflammatory arthritis strongly associated with Major Histocompatibility Complex (MHC) Class I molecule – Human Leukocyte Antigen (HLA)-B*27. There are differential associations with AS and HLA-B*27 subtypes. The subtypes which exhibit strong associations with AS include HLA-B*27:05, 04 and 02 subtypes, whilst 06 and 09 have weak or no association with disease. HLA-B*27:05 represents the most common subtype and exhibits few amino acid differences when compared to the 06 and 09 subtypes.

The role of HLA-B*27 in AS disease development remains undetermined. It has been proposed that HLA-B*27 either presents peptides that trigger an autoimmune response or that the molecule itself, via its propensity to misfold, participates in the inflammatory process. As the key amino acid differences between 05 and 06 are located in the peptide binding groove, which can potentially influence peptide presentation and protein folding, in this thesis I propose to use the differences between the disease and non-disease associated subtypes to develop a potential therapeutic approach using gene editing.

In this thesis the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system will be utilised to modify HLA-B*27:05 HC cDNA converting it into the 06 subtype targeting the following changes D77S, V152E and H114D/D116Y. To evaluate the effect of gene editing on protein expression, the gene edited cell lines will be compared biochemically to cell lines expressing identical mutants generated utilising Site Directed Mutagenesis (SDM) PCR approach and expressed at physiological copy numbers.

CRISPR-Cas9 system was successfully applied to generate the V152E mutation within HLA-B*27:05 HC cDNA, however, this was not the case for D77S. An alternative strategy was employed to modify p114/116 due to an unwanted Protospacer Adjacent Motif (PAM) site at p120, which could not be removed by introducing silent mutations. Therefore, a conserved glycine encoded by p120 was altered to either a valine (G120V) or alanine (G120A) alongside H114D/D116Y. Sequencing revealed successful introduction of the G120V edit but the H114D/D116Y modification was lacking.

This was the first study utilising the CRISPR-Cas9 system to introduce subtle nucleotides substitutions within a highly polymorphic MHC class I molecule. My study provides a proof of principle demonstration that Homologous Direct Repair (HDR) can be accomplished in such a challenging target, therefore, it could be explored further as a potential therapeutic approach against AS and other similar diseases where only subtle genetic changes might be the key to treatment or even a cure.

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