Detection and quantification of lipid-mediated siRNA delivery to the cytosol

Forskningsoutput: AvhandlingDoktorsavhandling (sammanläggning)

117 Nedladdningar (Pure)

Sammanfattning

Cancer is one of the leading causes of death globally. Novel therapeutics is urgently needed to advance treatment, especially for generalized disease where current treatment options have a poor prognosis. In particular, targeted therapy that can address genetic changes would be of great value.
RNA interference is an evolutionary conserved gene regulatory mechanism that can be used by introducing exogenous synthetic double-stranded RNAs, so called small interfering RNA (siRNA). siRNAs are sequence-specific inhibitors that are easily designed and could in theory target any gene of interest, making siRNA a promising modality for targeted therapy. However, a key challenge in translating siRNA into the clinic is the inefficacy to deliver siRNA across the plasma membrane, but most importantly, to escape the endosomal system and reach the cytosol where they can interact with the RNA interference machinery. Multiple delivery strategies have been proposed to improve delivery to the cytosol, but because of a lack of methods to accurately quantify this step, the efficacy of current delivery strategies is unknown and the scope for improvement is thus unclear.
The aim of this thesis was to develop novel methods to study the process of endosomal escape and cytosolic delivery of RNA. In particular, advanced high resolution microscopy techniques have been used to in detail characterize and determine the efficacy of lipid mediated delivery of RNA. With these methods, single-cell knockdown kinetics of cytosol delivered siRNA is determined, and the dose-response correlation between knockdown and intracellular siRNA concentration is elucidated. In a second study, endosomal structures damaged by membrane-destabilizing substances are characterized and their potential improvements on cholesterol conjugated siRNA delivery. Lastly, several mechanistic barriers limiting the lipid nanoparticle delivery of siRNA and mRNA are identified.
This thesis advances our understanding on the limiting step of endosomal escape and cytosolic entry of RNA during lipid-based delivery. The tools and knowledge presented in this work will contribute to the development of future delivery strategies aimed at enhancing the effectiveness of siRNA and other nucleic acid-based therapeutics.
Originalspråkengelska
KvalifikationDoktor
Tilldelande institution
  • Institutionen för kliniska vetenskaper, Lund
Handledare
  • Wittrup, Anders, handledare
  • Nordenfelt, Pontus, Biträdande handledare
  • Swaminathan, Vinay, Biträdande handledare
Tilldelningsdatum2024 jan. 12
UtgivningsortLund
Förlag
ISBN (tryckt)978-91-8021-497-1
StatusPublished - 2023

Bibliografisk information

Defence details
Date: 2024-01-12
Time: 09:15
Place: Belfragesalen, BMC D15, Klinikgatan 32 i Lund. Join by Zoom: https://lu-se.zoom.us/j/69092710675
External reviewer(s)
Name: El Andaloussi, Samir
Title: professor
Affiliation: Karolinska Institutet

Ämnesklassifikation (UKÄ)

  • Biokemi och molekylärbiologi

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