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

Hedlund, Hampus LU (2023) In Lund University, Faculty of Medicine Doctoral Dissertation Series
Abstract
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... (More)
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.
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Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor El Andaloussi, Samir, Karolinska Institutet
organization
publishing date
type
Thesis
publication status
published
subject
keywords
RNA Interference, RNA Therapeutics, Endosomal Escape, Lipid Mediated Delivery, Live-Cell Imaging
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
issue
2024:4
pages
78 pages
publisher
Lund University, Faculty of Medicine
defense location
Belfragesalen, BMC D15, Klinikgatan 32 i Lund. Join by Zoom: https://lu-se.zoom.us/j/69092710675
defense date
2024-01-12 09:15:00
ISSN
1652-8220
ISBN
978-91-8021-497-1
language
English
LU publication?
yes
id
72a2a70c-0d7d-4cf0-8d08-a562d8747b77
date added to LUP
2023-12-20 11:44:24
date last changed
2023-12-21 14:26:25
@phdthesis{72a2a70c-0d7d-4cf0-8d08-a562d8747b77,
  abstract     = {{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. <br/>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. <br/>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.<br/>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. <br/>}},
  author       = {{Hedlund, Hampus}},
  isbn         = {{978-91-8021-497-1}},
  issn         = {{1652-8220}},
  keywords     = {{RNA Interference; RNA Therapeutics; Endosomal Escape; Lipid Mediated Delivery; Live-Cell Imaging}},
  language     = {{eng}},
  number       = {{2024:4}},
  publisher    = {{Lund University, Faculty of Medicine}},
  school       = {{Lund University}},
  series       = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
  title        = {{Detection and quantification of lipid-mediated siRNA delivery to the cytosol}},
  url          = {{https://lup.lub.lu.se/search/files/167140268/E-Avhandling_Hampus_Hedlund.pdf}},
  year         = {{2023}},
}