Using CRISPR-X for Optimization of Antibodies Towards A30P α-synuclein Oligomers in Immunotherapy of Parkinson’s Disease

Introduction: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive cell death in the substantia nigra portion of the midbrain, which results in severe dopamine imbalance and a subsequent loss of refined motor control. Pathogenicity arises from mutations driving the misfolding of alpha synuclein (ɑ-syn), thus leading to oligomerization and formation of Lewy body aggregates in neurons. Previous research has identified the A30P mutated oligomers, which are connected to familial early-onset PD, as particularly toxic intermediates. However, there are insufficient methods of targeting them in isolation of other forms of the ɑ-syn protein. We aim to modify existing approaches to immunotherapy of PD, by use of CRISPR-X and phage display, to design antibodies that are highly specific to the A30P oligomer. Methods: We will use CRISPR-X to produce a diverse library of antibodies by introducing point mutations in the sequences encoding their variable light (VL) and heavy (VH) chains. Then, via insertion into bacteriophages for multiple rounds of phage display selection, we should obtain a polyclonal selection of antibodies that exclusively recognize epitopes on A30P ɑ-syn. Each variant then produces a distinct monoclonal phage colony to be incubated with relevant antigens for affinity testing via enzyme-linked immunosorbent assay (ELISA). Expected Results: We expect that measurements from ELISA would indicate low dissociation constants (KD) within the low micromolar (10-6) to nanomolar (10-7 to 10-9) range, when incubated with A30P oligomers. We predict that the resultant antibodies would have a higher affinity for the A30P oligomeric ɑ-syn than other forms of the protein. Furthermore, the novel antibodies are expected to have a higher specificity to this target antigen than the original antibodies that were modified. Discussion: By determining binding affinity, we verify the effectiveness of this method for improving specificity of in vitro antibody design. Upon further investigation using mice models, we would anticipate these novel antibodies could produce a specific reversal of the toxic effects of A30P in neuronal tissue. Conclusion: This study aims to establish a potential for CRISPR-X and phage selection to be used in antibody optimization for developing an immunotherapeutic treatment for PD.

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