Expression kinetics and innate immune response after electroporation and LNP-mediated delivery of a self-amplifying mRNA in the skin

In this work, we studied the expression kinetics and innate immune response of a self-amplifying mRNA (sa-RNA) after electroporation and lipid-nanoparticle (LNP)-mediated delivery in the skin of mice. Intradermal electroporation of the sa-RNA resulted in a plateau-shaped expression, with the plateau between day 3 and day 10. The overall protein expression of sa-RNA was significantly higher than that obtained after electroporation of plasmid DNA (pDNA) or non-replication mRNAs. Moreover, using IFN-beta reporter mice, we elucidated that intradermal electroporation of sa-RNA induced a short-lived moderate innate immune response, which did not affect the expression of the sa-RNA. A completely different expression profile and innate immune response were observed when LNPs were used. The expression peaked 24 h after intradermal injection of sa-RNA-LNPs and subsequently showed a sharp drop. This drop might be explained by a translational blockage caused by the strong innate immune response that we observed in IFN-beta reporter mice shortly (4 h) after intradermal injection of sa-RNA-LNPs. A final interesting observation was the capacity of sa-RNA-LNPs to transfect the draining lymph nodes after intradermal injection

Zika virus protection by a single low dose nucleoside modified mRNA vaccination

Zika virus (ZIKV) has recently emerged as an explosive pandemic associated with severe neuropathology in newborns and adults1. There are no ZIKV-specific treatments or preventatives; thus, development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins2,3. Here, we demonstrate that a single low-dose intradermal immunization with lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope (prM-E) glycoproteins of a 2013 ZIKV outbreak strain elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNPs protected mice from ZIKV challenges at 2 weeks or 5 months post-vaccination, and a single dose of 50 μg was sufficient to protect non-human primates from a challenge at 5 weeks post-vaccination. These data demonstrate that nucleoside-modified mRNA-LNPs elicit rapid and durable protective immunity and thus represent a new and promising vaccine candidate for the global fight against ZIKV

Influence of osmotic forces and transbilayer membrane area imbalances on the stability and morphology of model membrane vesicles

This thesis is focused on the effects of osmotic pressure and transbilayer area asymmetry on the morphology and stability of model lipid vesicle systems. These forces have been implicated in many diverse biological functions including membrane fusion, maintenance of cellular shape and membrane trafficking. The osmotic stability of these large unilamellar vesicles (LUVs) in the present of plasma has also been investigated. This is of practical interest as these vesicles are currently being used as in vivo drug carriers. The LUVs used in this thesis are made by the extrusion procedure which involves repeatedly passing an aqueous lipid dispersion through small pore sized filters. The morphology of the resulting egg phosphatidylcholine: cholesterol (55:45,mol:mol) LUVs formed by the extrusion technique (LUVETs) is found to be predominantly non-spherical, a property which has important effects on their osmotic properties. In particular, the initial influx of water that results from exposure of these vesicles to a hypo osmotic solution is first accomodated by the vesicles "rounding up" to maximize their volume to surface area ratio. Further studies show that osmotically induced vesicle lysis is a very rapid event with most of the solute release occurring within the first 30 seconds. However. lysis results in only partial release of solute such that a residual osmotic gradient results. This residual gradient is similar to the gradient required to initiate lysis. The maximum residual osmotic gradients were measured for LUVET systems with different mean diameters (90 to 340 nin). These results indicate that the osmotic properties of LUVETs obey Laplace's law for a spherical vesicle, relating the pressure difference across a close elastic membrane to the membrane tension. Osmotic lysis studies were also conducted in the presence of plasma with palmitoyloleoylphosphatidylcholine:cholesterol (55:45. mol:mol) LUVETs. Plasma was found to enhance solute release. However, both the residual and threshold osmotic gradients are reduced to the same extent. The plasma component responsible for the reduction in the membrane lysis tension is demonstrated to be the lipoproteins, with the high density lipoproteins exerting the greatest effect. The third area of investigation concerns the morphological consequences of imbalances between the surface areas of the vesicle's inner and outer monolayers as examined by cryo-electron microscopy techniques. Surface area imbalances are generated by inducing net transbilayer transport of DOPG indioleoylphosphatidylcholine: dioleoylphosphatidylglycerol (DOPC:DOPG. 9:1. mol:mol)vesicles in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner monolayer to the outer monolayer. initially invaginated LUVETs are transformed to long narrow tubular structures. or spherical structures with one or more tubular extensions. Conversely. when DOPG is transported from the outer monolayer to the inner monolayer of non-invaginated LUVETs, a reversion to invaginated structures is observed. These results are consistent with proposals that factors leading to imbalances in monolayer surface areas could play important roles in intracellular membrane transport processes.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

A multi-targeting, nucleoside-modified mRNA influenza virus vaccine provides broad protection in mice

Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50 ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates

Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

Dysfunction of the lymphatic system leads to secondary lymphedema and results in degradation of quality of life. Here, the authors show that delivery of nucleoside-modified Vascular Endothelial Growth Factor C (VEGFC) mRNA, encapsulated in lipid nanoparticles, induces organ-specific lymphatic growth and reverses experimental lymphedema

Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles

Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo

Characterization of HIV-1 Nucleoside-Modified mRNA Vaccines in Rabbits and Rhesus Macaques

Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability. Keywords: nucleoside modification, mRNA vaccine, HIV-1, rhesus macaque, neutralizing antibody, ADC

The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs

The regulatory approval of Onpattro, a lipid nanoparticle-based short interfering RNA drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis, paves the way for clinical development of many nucleic acid-based therapies enabled by nanoparticle delivery