Development of small interfering RNAs to treat fungal disease

UoM administered thesis: Phd

  • Authors:
  • Mireille Van Der Torre

Abstract

Aspergillus fumigatus is an ubiquitous saprophytic fungus that causes life-threatening disease in immunocompromised individuals. The increasing resistance to current antifungals makes the development of new antifungal drugs an important goal. Small RNA (sRNA) mediated gene silencing, whereby activation of the RNA interference (RNAi) pathway by dsRNA results in degradation of target mRNA, has shown to be promising in its ability to control a range of diseases. This pathway can potentially be exploited to target essential fungal genes leading to their down-regulation and resulting in growth inhibition or death. Major barriers for the use of small interfering RNA (siRNA) as therapeutics are the stability of the siRNA molecule and its delivery into the cell. If these obstacles can be overcome a new class of antifungals based on siRNA could be developed. This thesis focuses on the development of siRNAs to treat fungal disease. We show that the RNAi pathway is conserved and functional in A. fumigatus. Sequencing the small RNA transcriptome of wildtype and RNAi pathway mutants revealed siRNA-like sequences that are candidate therapeutics. We assessed the uptake and efficacy of siRNA treatment in A. fumigatus and the model filamentous fungus Neurospora crassa. We show that siRNAs had no significant impact on growth or target mRNA levels. Chemical modification of siRNAs (PNA, 2-fluoro, 2-O-methyl and/or phosphorothioate linkages) did not improve efficacy. Attempts to improve uptake of siRNAs using transfection reagents, cell-penetrating peptides and extracellular vesicles (EVs) were unsuccessful. However, lipid-based formulations, more specifically liposomes, were demonstrated to be a promising delivery method that should be tested in more detail in future.

Details

Original languageEnglish
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Supervisors/Advisors
Award date1 Aug 2019