mRNA Modification and delivery strategies towards the establishment of a platform for safe and effective gene therapy
(2015)
- Author
- Oliwia Andries (UGent)
- Promoter
- Niek Sanders (UGent) , Stefaan De Smedt (UGent) and Tasuku Kitada
- Organization
- Abstract
- For many years, the instability of RNA had raised doubts as to whether it was possible to effectively use mRNA for gene therapy. However, rapid advances in messenger RNA-based technologies in the last decade have transformed mRNA into an increasingly popular therapeutic modality, especially in the field of vaccination against cancer and viral infections. Today, mRNA is considered a safer alternative to pDNA-based therapeutics, as it does not pose the risk of genomic integration, unlike DNA. Furthermore, mRNA-based approaches offer immediate expression of a protein of interest even in non-dividing cells. In Chapter 2 of the dissertation we reviewed the general properties and advantages of RNA as a therapeutic modality. Moreover, we discussed specific attributes, limitations and benefits of unmodified, modified and self-replicating mRNA platforms. Additionally, we also provide insights into the instability of the mRNA molecule and strategies to improve the efficiency of the transfection of in vitro transcribed (IVT) mRNA. In Chapter 3, we compared DNA and RNAbased strategies for heterologous gene expression using cationic liposomes as delivery system. We showed that transfection of human lung adenocarcinoma cells with mRNA complexes results in a much faster expression compared to pDNA complexes. While the efficacy of mRNA complexes is independent of the cell cycle, pDNA complexes result in weak expression in nondividing cells. Thus, these data demonstrate that the nuclear barrier is a crucial obstacle for pDNA but not for mRNA. However, when mRNA and pDNA complexes encoding luciferase were administered intranasally to the lungs of mice, only the pDNA complexes gave rise to a detectable bioluminescent signal. This is likely due to the differences in the stability of the complexes as we showed that mRNA complexes are less stable in biological fluids compared to DNA complexes. However, as described in Chapter 4, the innate immune response of the cells in the mouse lung is also likely to be a major cause of the reduced expression from mRNA. Regardless, these results demonstrated the functional limitations of the traditional unmodified mRNA platform and encouraged us to develop a more stable and efficient RNA platform as we described in Chapter 5. In Chapter 4, we showed that carriermediated delivery of mRNA may activate TLR3 signaling in respiratory cells. Carrier-mediated delivery of mRNA caused activation of the innate immune system accompanied by a massive production of immunostimulatory cytokines, such as IL-6 or TNFα in vitro as well as in mice following intranasal instillation. Furthermore, the presented data demonstrate that the recognition of mRNA by the innate immune system is also associated with cell death, which proceeds in human respiratory cells via pyroptosis, a form of programmed cell death mediated by overexpression of caspase-1. Finally, we showed that recognition of the delivered unmodified mRNA by the innate immune system had a negative effect on mRNA translation by comparing the unmodified mRNA with innate immuneevading double modified 5-methylcytidine (m5C) and pseudouridine (Ψ) mRNA. Finally, in Chapter 5, with the lessons learned in the previous two chapters in mind, we advanced the state-of-the-art modified RNA expression platform by discovering that incorporation of N1-methylpseudouridine (mΨ) into mRNA enables stronger and more sustained gene expression compared to pseudouridine (Ψ)-modified mRNA. The impact of this modification on the level and duration of gene expression, cellular viability, and the innate immune response was evaluated in vitro in different cell types as well as in vivo in mice. While endocytosisdependent delivery (lipofection) of unmodified mRNA caused overexpression of TLR3 in respiratory cells, electroporation of the RNA into the same cell types resulted in a reduced innate immune response and less in vitro cytotoxicity. Nevertheless, future research is still required to address the numerous outstanding limitations of mRNA therapeutics. Possible technologies to solve these problems are discussed in Chapter 7 (Appendix A). This chapter reviews the latest advances in synthetic biology including RNA devices to control protein expression and discusses the possibilities of applying them to mRNA-based vaccination.
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-5914818
- MLA
- Andries, Oliwia. MRNA Modification and Delivery Strategies towards the Establishment of a Platform for Safe and Effective Gene Therapy. Ghent University. Faculty of Veterinary Medicine, 2015.
- APA
- Andries, O. (2015). mRNA Modification and delivery strategies towards the establishment of a platform for safe and effective gene therapy. Ghent University. Faculty of Veterinary Medicine, Merelbeke, Belgium.
- Chicago author-date
- Andries, Oliwia. 2015. “MRNA Modification and Delivery Strategies towards the Establishment of a Platform for Safe and Effective Gene Therapy.” Merelbeke, Belgium: Ghent University. Faculty of Veterinary Medicine.
- Chicago author-date (all authors)
- Andries, Oliwia. 2015. “MRNA Modification and Delivery Strategies towards the Establishment of a Platform for Safe and Effective Gene Therapy.” Merelbeke, Belgium: Ghent University. Faculty of Veterinary Medicine.
- Vancouver
- 1.Andries O. mRNA Modification and delivery strategies towards the establishment of a platform for safe and effective gene therapy. [Merelbeke, Belgium]: Ghent University. Faculty of Veterinary Medicine; 2015.
- IEEE
- [1]O. Andries, “mRNA Modification and delivery strategies towards the establishment of a platform for safe and effective gene therapy,” Ghent University. Faculty of Veterinary Medicine, Merelbeke, Belgium, 2015.
@phdthesis{5914818, abstract = {{For many years, the instability of RNA had raised doubts as to whether it was possible to effectively use mRNA for gene therapy. However, rapid advances in messenger RNA-based technologies in the last decade have transformed mRNA into an increasingly popular therapeutic modality, especially in the field of vaccination against cancer and viral infections. Today, mRNA is considered a safer alternative to pDNA-based therapeutics, as it does not pose the risk of genomic integration, unlike DNA. Furthermore, mRNA-based approaches offer immediate expression of a protein of interest even in non-dividing cells. In Chapter 2 of the dissertation we reviewed the general properties and advantages of RNA as a therapeutic modality. Moreover, we discussed specific attributes, limitations and benefits of unmodified, modified and self-replicating mRNA platforms. Additionally, we also provide insights into the instability of the mRNA molecule and strategies to improve the efficiency of the transfection of in vitro transcribed (IVT) mRNA. In Chapter 3, we compared DNA and RNAbased strategies for heterologous gene expression using cationic liposomes as delivery system. We showed that transfection of human lung adenocarcinoma cells with mRNA complexes results in a much faster expression compared to pDNA complexes. While the efficacy of mRNA complexes is independent of the cell cycle, pDNA complexes result in weak expression in nondividing cells. Thus, these data demonstrate that the nuclear barrier is a crucial obstacle for pDNA but not for mRNA. However, when mRNA and pDNA complexes encoding luciferase were administered intranasally to the lungs of mice, only the pDNA complexes gave rise to a detectable bioluminescent signal. This is likely due to the differences in the stability of the complexes as we showed that mRNA complexes are less stable in biological fluids compared to DNA complexes. However, as described in Chapter 4, the innate immune response of the cells in the mouse lung is also likely to be a major cause of the reduced expression from mRNA. Regardless, these results demonstrated the functional limitations of the traditional unmodified mRNA platform and encouraged us to develop a more stable and efficient RNA platform as we described in Chapter 5. In Chapter 4, we showed that carriermediated delivery of mRNA may activate TLR3 signaling in respiratory cells. Carrier-mediated delivery of mRNA caused activation of the innate immune system accompanied by a massive production of immunostimulatory cytokines, such as IL-6 or TNFα in vitro as well as in mice following intranasal instillation. Furthermore, the presented data demonstrate that the recognition of mRNA by the innate immune system is also associated with cell death, which proceeds in human respiratory cells via pyroptosis, a form of programmed cell death mediated by overexpression of caspase-1. Finally, we showed that recognition of the delivered unmodified mRNA by the innate immune system had a negative effect on mRNA translation by comparing the unmodified mRNA with innate immuneevading double modified 5-methylcytidine (m5C) and pseudouridine (Ψ) mRNA. Finally, in Chapter 5, with the lessons learned in the previous two chapters in mind, we advanced the state-of-the-art modified RNA expression platform by discovering that incorporation of N1-methylpseudouridine (mΨ) into mRNA enables stronger and more sustained gene expression compared to pseudouridine (Ψ)-modified mRNA. The impact of this modification on the level and duration of gene expression, cellular viability, and the innate immune response was evaluated in vitro in different cell types as well as in vivo in mice. While endocytosisdependent delivery (lipofection) of unmodified mRNA caused overexpression of TLR3 in respiratory cells, electroporation of the RNA into the same cell types resulted in a reduced innate immune response and less in vitro cytotoxicity. Nevertheless, future research is still required to address the numerous outstanding limitations of mRNA therapeutics. Possible technologies to solve these problems are discussed in Chapter 7 (Appendix A). This chapter reviews the latest advances in synthetic biology including RNA devices to control protein expression and discusses the possibilities of applying them to mRNA-based vaccination.}}, author = {{Andries, Oliwia}}, isbn = {{9789058644169}}, language = {{eng}}, pages = {{211}}, publisher = {{Ghent University. Faculty of Veterinary Medicine}}, school = {{Ghent University}}, title = {{mRNA Modification and delivery strategies towards the establishment of a platform for safe and effective gene therapy}}, year = {{2015}}, }