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Title: Evaluating the genotoxic potential of oligonucleotide pharmaceuticals
Author: Reshat, Reshat
ISNI:       0000 0004 2732 1293
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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According to regulatory guidelines, routine genotoxicity tests are not appropriate for biotechnology derived pharmaceuticals, including oligonucleotide based therapeutics, as they are not expected to interact with genomic DNA. However, reports of oligonucleotides capable of binding duplex DNA in a sequence specific manner to form triple-helix (triplex) or displacement-loop (D-loop) structures that in turn cause mutation have raised concern. The European Medicines Agency (EMA) has questioned the capability of antisense oligonucleotide (ASO) therapeutics to form such structures at genomic DNA. Additionally, concern has been expressed regarding the fate of chemically modified ASO degradation products (nucleotide analogues). It is well established that non-canonical antiretroviral nucleoside analogues, employed in antiretroviral therapy, result in gross chromosome aberrations following incorporation into genomic DNA. This study has addressed these concerns by evaluating the genotoxic potential of a triplex forming oligonucleotide (TFO) and a D-loop forming ASO targeting genomic DNA. Furthermore, the incorporation efficiency and genotoxicity of nucleotide analogues derived from ASO degradation was investigated. Data presented here demonstrate a TFO targeting genomic DNA was not capable of inducing mutation above the detection limit of this assay. However, a biologically active ASO molecule induced sequence specific mutation ~4.4 fold above control in a system where RAD51 protein expression was induced. Additionally, DNA polymerase was capable of incorporating various ASO derived nucleotide analogues into a primed DNA template with reduced efficiency. Treatment with phosphorothioate nucleotide analogue, one of the most common chemical modifications used in ASO design, induced mutation ~100 fold above control. To conclude, ASO and their putative degradation products appeared to be capable of off-target mutagenesis providing favourable conditions were met. However, as genotoxicity data has been presented for a single ASO and nucleotide analogue, it seems plausible to suggest this work can provide a foundation to test future ASO therapeutics and putative degradation products.
Supervisor: Gooderham, Nigel Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral