Design of novel delivery systems to probe alternative routes of administration for a self-amplifying RNA rabies vaccine

Current vaccine design aims to develop safer vaccines based on one or few selected antigens. RNA-based vaccines can be engineered to encode any antigen of interest and have the potential for rapid, inexpensive and scalable manufacturing and have an acceptable safety profile. Moreover, they enable in situ antigen expression, mimicking a real viral infection hence eliciting robust humoral and cellular-mediated immune responses. RNA vaccines therefore represent a versatile tool to fight infectious diseases and emerging pathogens effectively and rapidly. Furthermore, the antigen can be designed in a self-amplifying RNA (SAM) to enhance the immunogenicity and to reduce the therapeutic dose compared to conventional non-amplifying mRNA vaccines.;RNAs can be encapsulated in delivery systems to protect them against degradation upon injection and to facilitate their delivery in host cells. Among them, lipid-based delivery systems and, more specifically, lipid nanoparticles (LNPs) are efficient non-viral delivery systems for RNA and SAM vaccines. Within this thesis, a panel of cationic LNPs (cLNPs), based on existing cationic lipids (e.g. DOTAP and DDA), was designed to deliver a SAM vaccine. The rabies virus was used as a model, as there is an established correlate of protection (neutralising antibodies) and there exist efficacious vaccines in the market (e.g. Rabipur) to be used as comparators. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG), the only target for neutralising antibodies, was used;Microfluidics-based methods for producing cLNPs of desired physicochemical properties were developed and optimal operating parameters (e.g. total flow rate and flow rate ratio) were established. Most promising SAM-cLNP candidates were chosen according to their physicochemical attributes, their ability to protect SAM from enzymatic degradation and their capacity to associate with cells and to induce antigen expression. These formulations were well retained at the injection site when administered intramuscularly or intradermally, while they were rapidly cleared following intranasal administration.;On the other hand, SAM-cLNPs induced protective levels of anti-RVG antibodies following intramuscular injection in mice and RVG-specific polyfunctional T cell responses even with a dose as low as 0.15 μg RVG-SAM. Remarkably, the immune responses elicited by SAM-cLNPs were comparable to Rabipur, a commercial vaccine based on an inactivated rabies virus, and a cationic nanoemulsion, a safe and well-established SAM delivery system which is currently being investigated in a phase I clinical trial in humans (as of September 2019). Intradermal administration of SAM-cLNPs resulted in similar humoral and cell-mediated immune responses, while significantly weaker immune responses were achieved when administered intranasally.Current vaccine design aims to develop safer vaccines based on one or few selected antigens. RNA-based vaccines can be engineered to encode any antigen of interest and have the potential for rapid, inexpensive and scalable manufacturing and have an acceptable safety profile. Moreover, they enable in situ antigen expression, mimicking a real viral infection hence eliciting robust humoral and cellular-mediated immune responses. RNA vaccines therefore represent a versatile tool to fight infectious diseases and emerging pathogens effectively and rapidly. Furthermore, the antigen can be designed in a self-amplifying RNA (SAM) to enhance the immunogenicity and to reduce the therapeutic dose compared to conventional non-amplifying mRNA vaccines.;RNAs can be encapsulated in delivery systems to protect them against degradation upon injection and to facilitate their delivery in host cells. Among them, lipid-based delivery systems and, more specifically, lipid nanoparticles (LNPs) are efficient non-viral delivery systems for RNA and SAM vaccines. Within this thesis, a panel of cationic LNPs (cLNPs), based on existing cationic lipids (e.g. DOTAP and DDA), was designed to deliver a SAM vaccine. The rabies virus was used as a model, as there is an established correlate of protection (neutralising antibodies) and there exist efficacious vaccines in the market (e.g. Rabipur) to be used as comparators. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG), the only target for neutralising antibodies, was used;Microfluidics-based methods for producing cLNPs of desired physicochemical properties were developed and optimal operating parameters (e.g. total flow rate and flow rate ratio) were established. Most promising SAM-cLNP candidates were chosen according to their physicochemical attributes, their ability to protect SAM from enzymatic degradation and their capacity to associate with cells and to induce antigen expression. These formulations were well retained at the injection site when administered intramuscularly or intradermally, while they were rapidly cleared following intranasal administration.;On the other hand, SAM-cLNPs induced protective levels of anti-RVG antibodies following intramuscular injection in mice and RVG-specific polyfunctional T cell responses even with a dose as low as 0.15 μg RVG-SAM. Remarkably, the immune responses elicited by SAM-cLNPs were comparable to Rabipur, a commercial vaccine based on an inactivated rabies virus, and a cationic nanoemulsion, a safe and well-established SAM delivery system which is currently being investigated in a phase I clinical trial in humans (as of September 2019). Intradermal administration of SAM-cLNPs resulted in similar humoral and cell-mediated immune responses, while significantly weaker immune responses were achieved when administered intranasally

Decoupling the adsorption mechanisms of arsenate at molecular level on modified cube-shaped sponge loaded superparamagnetic iron oxide nanoparticles

Altres ajuts: acord transformatiu CRUE-CSICIn this study, a commercial cube-shaped open-celled cellulose sponge adsorbent was modified by in-situ co-precipitation of superparamagnetic iron oxide nanoparticles (SPION) and used to remove As(V) from aqueous solutions. Fe K-edge X-ray absorption spectroscopy (XAS) and TEM identified maghemite as the main iron phase of the SPION nanoparticles with an average size 13 nm. Batch adsorption experiments at 800 mg/L showed a 63% increase of adsorption capacity when loading 2.6 wt.% mass fraction of SPION in the cube-sponge. Experimental determination of the adsorption thermodynamic parameters indicated that the As(V) adsorption on the composite material is a spontaneous and exothermic process. As K-edge XAS results confirmed that the adsorption enhancement on the composite can be attributed to the nanoparticles loaded. In addition, adsorbed As(V) did not get reduced to more toxic As(III) and formed a binuclear corner-sharing complex with SPION. The advantageous cube-shape of the sponge-loaded SPION composite together with its high affinity and good adsorption capacity for As(V), good regeneration capability and the enhanced-diffusion attributed to its open-celled structure make this adsorbent a good candidate for industrial applications

Manufacturing considerations for the development of lipid nanoparticles using microfluidics

In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic—DOTAP and DDAB; ionisable—MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics

Investigating the impact of delivery system design on the efficacy of self-amplifying RNA vaccines

Messenger RNA (mRNA)-based vaccines combine the positive attributes of both live-attenuated and subunit vaccines. In order for these to be applied for clinical use, they require to be formulated with delivery systems. However, there are limited in vivo studies which compare different delivery platforms. Therefore, we have compared four different cationic platforms: (1) liposomes, (2) solid lipid nanoparticles (SLNs), (3) polymeric nanoparticles (NPs) and (4) emulsions, to deliver a self-amplifying mRNA (SAM) vaccine. All formulations contained either the non-ionizable cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDA) and they were characterized in terms of physico-chemical attributes, in vitro transfection efficiency and in vivo vaccine potency. Our results showed that SAM encapsulating DOTAP polymeric nanoparticles, DOTAP liposomes and DDA liposomes induced the highest antigen expression in vitro and, from these, DOTAP polymeric nanoparticles were the most potent in triggering humoral and cellular immunity among candidates in vivo

Sensibilidad de células de carcinoma de colon al tratamiento con células NK alogénicas activadas

El carcinoma de colon es la segunda causa de muerte más común dentro de los diferentes tipos de cáncer. El diagnóstico precoz resulta clave para un tratamiento temprano y efectivo que, en muchos de los casos, requiere cirugía para eliminar las células tumorales. Si el cáncer progresa y se detecta en estadios más avanzados, el tratamiento se basa en quimioterapia combinada con anticuerpos monoclonales como trastuzumab, cetuximab y panitumumab. En ambos casos, en un alto porcentaje de ocasiones, la efectividad es baja y los efectos secundarios abundantes, por lo que el cáncer reaparece. El descubrimiento de que las células NK se activan frente a las células cancerosas en pacientes con cánceres hematológicos trasplantados de médula ósea, ha supuesto una revolución en el tratamiento de leucemias de mal pronóstico. Se ha observado que las células NK alogénicas son capaces de eliminar células de diferentes tipos de leucemia y mielomas resistentes a tratamientos de quimioterapia. Sin embargo, estos resultados no están tan claros en cánceres sólidos. Por este motivo, en este trabajo nos hemos planteado estudiar la sensibilidad de diferentes líneas celulares de cáncer de colon humanas como HT29 y HCT116 frente células NK activadas in vitro mediante diferentes estímulos. Para ello, hemos analizado la muerte celular inducida por células NK y estudiado la expresión de las proteínas citotóxicas y receptores involucrados en tal proceso

Structural Reinforcement through Liquid Encapsulation

The liquid inside a solid material is one of the most common composite materials in nature. The interface between solid–liquid plays an important role in unique deformation. Here, model systems of two polymers (polydimethylsiloxane–polyvinylidenefluoride) are used to make sphere of solid with liquid inside it

Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP : a case study using protein-loaded liposomes

Nanomedicines are well recognised for their ability to improve therapeutic outcomes. Yet, due to their complexity, nanomedicines are challenging and costly to produce using traditional manufacturing methods. For nanomedicines to be widely exploited, new manufacturing technologies must be adopted to reduce development costs and provide a consistent product. Within this study, we investigate microfluidic manufacture of nanomedicines. Using protein-loaded liposomes as a case study, we manufacture liposomes with tightly defined physico-chemical attributes (size, PDI, protein loading and release) from small-scale (1 mL) through to GMP volume production (200 mL/min). To achieve this, we investigate two different laminar flow microfluidic cartridge designs (based on a staggered herringbone design and a novel toroidal mixer design); for the first time we demonstrate the use of a new microfluidic cartridge design which delivers seamless scale-up production from bench-scale (12 mL/min) through GMP production requirements of over 20 L/h using the same standardised normal operating parameters. We also outline the application of tangential flow filtration for down-stream processing and high product yield. This work confirms that defined liposome products can be manufactured rapidly and reproducibly using a scale-independent production process, thereby de-risking the journey from bench to approved product

PEG-copolymer-coated iron oxide nanoparticles that avoid the reticuloendothelial system and act as kidney MRI contrast agents

In vitro experiments have shown the great potential of magnetic nanocarriers for multimodal imaging diagnosis and non-invasive therapies. However, their extensive clinical application is still jeopardized by a fast retention in the reticuloendothelial system (RES). The other issue that restrains their potential performance is slow degradation and excretion, which increases their risks of toxicity. We report a promising case in which multicore iron oxide nanoparticles coated with a poly(4-vinylpyridine) polyethylene glycol copolymer show low RES retention and high urinary excretion, as confirmed by single photon emission computerized tomography (SPECT), gamma counting, magnetic resonance imaging (MRI) and electron microscopy (EM) biodistribution studies. These iron oxide-copolymer nanoparticles have a high PEG density in their coating which may be responsible for this effect. Moreover, they show a clear negative contrast in the MR imaging of the kidneys. These nanoparticles with an average hydrodynamic diameter of approximately 20 nm were nevertheless able to cross the glomerulus wall which has an effective pore size of approximately 6 nm. A transmission electron microscopy inspection of kidney tissue revealed the presence of iron containing nanoparticle clusters in proximal tubule cells. This therefore makes them exceptionally useful as magnetic nanocarriers and as new MRI contrast agents for the kidneys.Financial support by the Spanish Ministry of Science and Innovation (MAT2014-52069-R) (SAF2014-53413-R) (PC2015-1-05 (53-80)) is gratefully acknowledged.Peer reviewe

Silencing COI1 in Rice Increases Susceptibility to Chewing Insects and Impairs Inducible Defense

The jasmonic acid (JA) pathway plays a key role in plant defense responses against herbivorous insects. CORONATINE INSENSITIVE1 (COI1) is an F-box protein essential for all jasmonate responses. However, the precise defense function of COI1 in monocotyledonous plants, especially in rice (Oryza sativa L.) is largely unknown. We silenced OsCOI1 in rice plants via RNA interference (RNAi) to determine the role of OsCOI1 in rice defense against rice leaf folder (LF) Cnaphalocrocis medinalis, a chewing insect, and brown planthopper (BPH) Nilaparvata lugens, a phloem-feeding insect. In wild-type rice plants (WT), the transcripts of OsCOI1 were strongly and continuously up-regulated by LF infestation and methyl jasmonate (MeJA) treatment, but not by BPH infestation. The abundance of trypsin protease inhibitor (TrypPI), and the enzymatic activities of polyphenol oxidase (PPO) and peroxidase (POD) were enhanced in response to both LF and BPH infestation, but the activity of lipoxygenase (LOX) was only induced by LF. The RNAi lines with repressed expression of OsCOI1 showed reduced resistance against LF, but no change against BPH. Silencing OsCOI1 did not alter LF-induced LOX activity and JA content, but it led to a reduction in the TrypPI content, POD and PPO activity by 62.3%, 48.5% and 27.2%, respectively. In addition, MeJA-induced TrypPI and POD activity were reduced by 57.2% and 48.2% in OsCOI1 RNAi plants. These results suggest that OsCOI1 is an indispensable signaling component, controlling JA-regulated defense against chewing insect (LF) in rice plants, and COI1 is also required for induction of TrypPI, POD and PPO in rice defense response to LF infestation

Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences

The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & Nemésio 2007; Donegan 2008, 2009; Nemésio 2009a–b; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on 18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based researchers who signed it in the short time span from 20 September to 6 October 2016