mRNA Modification and delivery strategies towards the establishment of a platform for safe and effective gene therapy

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

T Cell epitope mapping of the E-protein of West Nile virus in BALB/c mice

West Nile virus (WNV) is a zoonotic virus, which is transmitted by mosquitoes. It is the causative agent of the disease syndrome called West Nile fever. In some human cases, a WNV infection can be associated with severe neurological symptoms. The immune response to WNV is multifactorial and includes both humoral and cellular immunity. T-cell epitope mapping of the WNV envelope (E) protein has been performed in C57BL/6 mice, but not in BALB/c mice. Therefore, we performed in BALB/c mice a T-cell epitope mapping using a series of peptides spanning the WNV envelope (E) protein. To this end, the WNV-E specific T cell repertoire was first expanded by vaccinating BALB/c mice with a DNA vaccine that generates subviral particles that resemble West Nile virus. Furthermore, the WNV structural protein was expressed in Escherichia coli as a series of overlapping 20-mer peptides fused to a carrier-protein. Cytokine-based ELISPOT assays using these purified peptides revealed positive WNV-specific T cell responses to peptides within the different domains of the E-protein

Synthetic biology devices and circuits for RNA-based 'smart vaccines': a propositional review

Nucleic acid vaccines have been gaining attention as an alternative to the standard attenuated pathogen or protein based vaccine. However, an unrealized advantage of using such DNA or RNA based vaccination modalities is the ability to program within these nucleic acids regulatory devices that would provide an immunologist with the power to control the production of antigens and adjuvants in a desirable manner by administering small molecule drugs as chemical triggers. Advances in synthetic biology have resulted in the creation of highly predictable and modular genetic parts and devices that can be composed into synthetic gene circuits with complex behaviors. With the recent advent of modified RNA gene delivery methods and developments in the RNA replicon platform, we foresee a future in which mammalian synthetic biologists will create genetic circuits encoded exclusively on RNA. Here, we review the current repertoire of devices used in RNA synthetic biology and propose how programmable 'smart vaccines' will revolutionize the field of RNA vaccination

Pulmonary delivery of mRNA: in vitro and in vivo evaluation

C

Detection of cellular and humoral immune response following pDNA-based vaccination.

<p>IFN-γ production by (a) CD4-depleted and (c) CD8-depleted splenocytes after stimulation with purified recombinant GST tagged E-protein derived peptides. The WNV E-protein specific T-cell repertoire in BALB/c mice was expanded by two DNA vaccinations. Splenocytes obtained two weeks after the boost were stimulated with different recombinant GST tagged E-protein derived peptides and the numbers of cells producing IFN-γ were determined via ELISPOT. (b) Detection of serum IgG1 and IgG2a titers to the WNV E-protein two weeks after the boost via ELISA.</p

Location of the peptide sequences in the E protein that, based on our <i>in vivo</i> experiments, contain strong CD4+ (underlined) and CD8+ (bold) T cell epitopes.

<p>The shown amino acid sequence is that of the E protein of lineage 1 WNV strain Ita09. Sequences that are in bold and underlined contain strong CD4+ as well as CD8+ T cell epitopes.</p