Closed loop aptameric directed evolution selection of therapeutic aptamers for chronic myeloid leukaemia

Abstract

Introduction: A class of aptamers comprising short single strands of nucleic acids that form three-dimensional structures can bind specified targets with high affinity. As strong competitors or alternatives to antibodies, aptamers have been widely applied in research and diagnostics as binding partners. However, examples of aptamers serving as drug candidates are very limited, and there is limited know-how available on the criteria for selecting therapeutic aptamers. By incorporating microarray technology, in silico approaches and sequence-fitness landscape modelling, a comprehensive sequence-fitness mapping on-chip aptamer selection approach, termed Closed Loop Aptameric Directed Evolution (CLADE) [1] was developed to identify aptamers with both diversity and breadth of binding affinities. My intention was to join this group and determine if CLADE offers exceptional value over SELEX for the discovery of high therapeutic index aptamers. BCR-ABL is a t(9;22) chimeric non-receptor tyrosine kinase, which is the most critical molecule responsible for pathogenesis and uncontrolled proliferation of Chronic Myeloid Leukaemia (CML). Three generations of ABL tyrosine kinase inhibitors (TKIs) have been developed to treat CML. However, various DNA sequence mutations in abl gene can adversely affect TKI binding, and therapy, especially the T315i mutant. Aim: This study aims to produce processes based on protein synthesis and CLADE to identify therapeutic aptamers as ABL kinase inhibitors for the treatment of CML by CLADE. 16 Methods: The ABL kinase domain was selected as the target for aptamers selection to improve the chances of therapeutic hits. The chimeric co-synthesis of ABL-eGFP and ABL(T315i)-eGFP served both as targets for functional binding of selected aptamers and to assess the localisation of ABL on chip. In CLADE, iterative cycles of on-chip ssDNA library synthesis, in vitro target-ssDNA interaction assay and genetic algorithm were performed to screen high binding sequences. Aptamer sequences potentially binding human mRNAs were eliminated to improve the therapeutic index by preventing potential anti-sense effects. Results: Prokaryotic and eukaryotic expression systems including bacterial, mammalian and insect-baculovirus hosts and vectors were developed for expressing and purifying the two recombinant proteins. CLADE was performed against ABL and T315i. No definitive aptamers have been found to date. The study has paused due to limited resources and time. Future studies: Synthesis and use of microarrays and the ensuing transformation of the data require attention, and this will be followed by further iterations of CLADE. Comparison of aptamers binding fingerprints to wild or mutated forms of ABL protein will be used to identify pertinent aptamer binding sites, and these will be tested in conjunction with TKIs on CML cell lines and primary CML cancer cells in the first instance. NMR studies will be employed to aid computational prediction of optimal binding events of aptamers to ABL variants. Indeed, in a longer-term prediction of CLADE, it is possible that AI will be able to use information from established protein- aptamer bindings and use these as a training set to accelerate the aptamer discovery process.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Joshua Knowles (Supervisor) & Philip Day (Supervisor)