RESUMEN
Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies.
Asunto(s)
Expresión Génica , Terapia Genética , ARN Mensajero/genética , Animales , Línea Celular , Índices de Eritrocitos , Eritropoyetina/sangre , Eritropoyetina/genética , Eritropoyetina/metabolismo , Genes Reporteros , Terapia Genética/métodos , Humanos , Lípidos/química , Macaca fascicularis , Ratones , Modelos Animales , Nanopartículas/química , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Mutación Silenciosa , Sus scrofaRESUMEN
Protein- and peptide-based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non-coding, long-chain RNA molecule, termed RNAdjuvant(®) , profoundly increased immunogenicity of both antigen formats. RNAdjuvant(®) induced balanced, long-lasting immune responses that resulted in a strong anti-tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen-specific multifunctional CD8(+) T-cell responses and mediate anti-tumor responses induced by peptide derived from HPV-16 E7 protein in the syngeneic TC-1 tumor, a murine model of human HPV-induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA-based adjuvant exhibited an excellent pre-clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up-regulation of pro-inflammatory and anti-viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA-based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.
Asunto(s)
Adyuvantes Inmunológicos , Vacunas contra el Cáncer/inmunología , ARN Largo no Codificante/inmunología , Neoplasias del Cuello Uterino/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/farmacología , Línea Celular Transformada , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Humanos , Memoria Inmunológica/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas E7 de Papillomavirus/inmunología , Péptidos/inmunología , Péptidos/farmacología , Poli I-C/inmunología , Poli I-C/farmacología , ARN Largo no Codificante/genética , Resultado del Tratamiento , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
Poxviruses are a large and complex family of viruses with members such as monkeypox virus and variola virus. The possibility of an outbreak of monkeypox virus (or a related poxvirus) or the misuse of variola virus justifies the development of countermeasures. Furthermore, poxviruses can be a useful surrogate for developing technology involving antibody therapies. In our experiments, we explored the feasibility of utilizing unmodified mRNA that encodes three previously described monoclonal antibodies, c8A, c6C, and c7D11, as countermeasures to smallpox in a relatively large (>3 kg) laboratory animal (rabbits). We confirmed in vitro translation, secretion, and biological activity of mRNA constructs and identified target monoclonal antibody levels from a murine vaccinia virus model that provided a clinical benefit. Individually, we were able to detect c7D11, c8A, and c6C in the serum of rabbits within 1 day of an intramuscular jet injection of lipid nanoparticle (LNP)-formulated mRNA. Injection of a combination of three LNP-formulated mRNA constructs encoding the three different antibodies produced near equivalent serum levels compared with each individual construct administered alone. These data are among the first demonstrating the feasibility of launching multiple antibodies using mRNA constructs in a large, nonrodent species. Based on empirically derived target serum level and the observed decay rate, the antibody levels attained were unlikely to provide protection.
RESUMEN
The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.
RESUMEN
Visualization of the spatio-temporal trafficking of vaccines after their delivery would help evaluate the efficacy of candidate formulations and aid their rational design for preclinical and translational studies. Here, we show that a dual radionuclide-near-infrared probe allows for quantitative, longitudinal and non-invasive monitoring, via positron emission tomography-computed tomography and near-infrared imaging of cynomolgus macaques, of the trafficking dynamics to draining lymph nodes of a model messenger RNA vaccine labelled with the probe. After intramuscular administration of the vaccine to the monkeys, we observed the dynamics of the mRNA vaccine at the injection site and in the draining lymph nodes, performed cellular analyses of the involved tissues using flow cytometry and identified through immunofluorescence that professional antigen-presenting cells are the primary cells containing the injected mRNA and encoding the antigen. This approach may reveal spatio-temporal determinants of vaccine efficacy in preclinical and translational studies employing large mammals.
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Técnicas de Transferencia de Gen , Tomografía Computarizada por Tomografía de Emisión de Positrones , ARN Mensajero/administración & dosificación , Espectroscopía Infrarroja Corta , Vacunas/administración & dosificación , Animales , Células Presentadoras de Antígenos/metabolismo , Radioisótopos de Cobre/química , Células HeLa , Humanos , Ganglios Linfáticos/diagnóstico por imagen , Macaca fascicularis , Masculino , Músculos/metabolismoRESUMEN
The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro, depending on the delivery vehicle, resulting in variations in protein expression levels. Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on â¼10â¯kPa hydrogels. This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines.
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Mecanotransducción Celular/fisiología , Músculo Esquelético/metabolismo , ARN Mensajero/metabolismo , Animales , Línea Celular , Endosomas/química , Matriz Extracelular/química , Femenino , Citometría de Flujo , Células HeLa , Humanos , Hidrogeles/química , Ratones , Ratones Endogámicos BALB C , Nanopartículas/químicaRESUMEN
mRNA represents a promising new vaccine technology platform with high flexibility in regard to development and production. Here, we demonstrate that vaccines based on sequence optimized, chemically unmodified mRNA formulated in optimized lipid nanoparticles (LNPs) are highly immunogenic and well tolerated in non-human primates (NHPs). Single intramuscular vaccination of NHPs with LNP-formulated mRNAs encoding rabies or influenza antigens induced protective antibody titers, which could be boosted and remained stable during an observation period of up to 1 year. First mechanistic insights into the mode of action of the LNP-formulated mRNA vaccines demonstrated a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that the mRNA technology is a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases.
RESUMEN
Rabies is a zoonotic infectious disease of the central nervous system (CNS). In unvaccinated or untreated subjects, rabies virus infection causes severe neurological symptoms and is invariably fatal. Despite the long-standing existence of effective vaccines, vaccine availability remains insufficient, with high numbers of fatal infections mostly in developing countries. Nucleic acid based vaccines have proven convincingly as a new technology for the fast development of vaccines against newly emerging pathogens, diseases where no vaccine exists or for replacing already existing vaccines. We used an optimized non-replicating rabies virus glycoprotein (RABV-G) encoding messenger RNA (mRNA) to induce potent neutralizing antibodies (VN titers) in mice and domestic pigs. Functional antibody titers were followed in mice for up to one year and titers remained stable for the entire observation period in all dose groups. T cell analysis revealed the induction of both, specific CD4+ as well as CD8+ T cells by RABV-G mRNA, with the induced CD4+ T cells being higher than those induced by a licensed vaccine. Notably, RABV-G mRNA vaccinated mice were protected against lethal intracerebral challenge infection. Inhibition of viral replication by vaccination was verified by qRT-PCR. Furthermore, we demonstrate that CD4+ T cells are crucial for the generation of neutralizing antibodies. In domestic pigs we were able to induce VN titers that correlate with protection in adult and newborn pigs. This study demonstrates the feasibility of a non-replicating mRNA rabies vaccine in small and large animals and highlights the promises of mRNA vaccines for the prevention of infectious diseases.
Asunto(s)
Glicoproteínas/inmunología , Fragmentos de Péptidos/inmunología , ARN Mensajero/inmunología , Vacunas Antirrábicas , Rabia/veterinaria , Enfermedades de los Porcinos/prevención & control , Proteínas Virales/inmunología , Animales , Animales Recién Nacidos , Secuencia de Bases , Relación Dosis-Respuesta Inmunológica , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Embarazo , ARN Mensajero/química , Rabia/mortalidad , Rabia/prevención & control , Vacunas Antirrábicas/genética , Vacunas Antirrábicas/inmunología , Porcinos , Linfocitos T , Vacunas Sintéticas/inmunologíaRESUMEN
mRNA represents a new platform for the development of therapeutic and prophylactic vaccines with high flexibility with respect to production and application. We have previously shown that our two component self-adjuvanted mRNA-based vaccines (termed RNActive® vaccines) induce balanced immune responses comprising both humoral and cellular effector as well as memory responses. Here, we evaluated the early events upon intradermal application to gain more detailed insights into the underlying mode of action of our mRNA-based vaccine. We showed that the vaccine is taken up in the skin by both non-leukocytic and leukocytic cells, the latter being mostly represented by antigen presenting cells (APCs). mRNA was then transported to the draining lymph nodes (dLNs) by migratory dendritic cells. Moreover, the encoded protein was expressed and efficiently presented by APCs within the dLNs as shown by T cell proliferation and immune cell activation, followed by the induction of the adaptive immunity. Importantly, the immunostimulation was limited to the injection site and lymphoid organs as no proinflammatory cytokines were detected in the sera of the immunized mice indicating a favorable safety profile of the mRNA-based vaccines. Notably, a substantial boostability of the immune responses was observed, indicating that mRNA can be used effectively in repetitive immunization schedules. The evaluation of the immunostimulation following prime and boost vaccination revealed no signs of exhaustion as demonstrated by comparable levels of cytokine production at the injection site and immune cell activation within dLNs. In summary, our data provide mechanistic insight into the mode of action and a rational for the use of mRNA-based vaccines as a promising immunization platform.
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Inmunidad Adaptativa , Adyuvantes Inmunológicos/administración & dosificación , Inmunidad Innata , ARN Mensajero/inmunología , Vacunas/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Citocinas/inmunología , Femenino , Inyecciones Intradérmicas , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Angiogenesis is the sprouting of new blood capillaries from surrounding preexisting blood vessels. This process is fundamental for embryonic development, wound healing and inflammation. In healthy adults angiogenesis is of minor importance. However, aberrant angiogenesis is essentially involved in disorders as diabetic retinopathy, rheumatoid arthritis and tumor growth, and blocking angiogenesis has emerged as a promising target for antagonizing these diseases. Therefore the development of new anti-angiogenic drugs is of great interest in academic and industrial research. This review focuses on the employment of peptidomimetics in inhibiting pathologic angiogenesis. It will survey the individual aspects of angiogenesis where the usage of peptidomimetics is favored and will consider the current progresses on this field.
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Inhibidores de la Angiogénesis/farmacología , Neovascularización Patológica/tratamiento farmacológico , Péptidos/farmacología , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Animales , Humanos , Integrinas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Neovascularización Patológica/patología , Péptidos/síntesis química , Péptidos/químicaRESUMEN
Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive(®) vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive(®) vaccines. RNActive(®) vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive(®) vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive(®) could be successfully translated to humans.
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Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , ARN/administración & dosificación , ARN/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Humanos , Receptor Toll-Like 7/inmunología , Vacunación/métodosRESUMEN
Nucleic acid based therapies have so far mainly been focused on plasmid DNA (pDNA), small interfering RNA (siRNA), antisense and immunostimulatory oligonucleotides. Messenger RNA (mRNA) was the subject of only a few studies. The objective of this investigation was the preparation of new composite polyplexes with mRNA consisting of poly(ethylene imine) (PEI) and poly(ethylene imine)-poly(ethylene glycol)-copolymers (PEI-PEG) as blends. These complexes were designed to increase the stability of mRNA, to improve transfection efficiency and to reduce cytotoxicity. Hydrodynamic diameters of the polyplexes were measured by dynamic light scattering, polyplex stability was analyzed by gel retardation assay and transfection efficiency of luciferase (Luc) encoding mRNA was evaluated under in vitro conditions. Most of the polyplexes generated showed small particle sizes <200 nm and positive zeta-potentials of +20 mV to +30 mV. Stable complexes were formed even at low nitrogen to phosphate ratios. Polyplexes with mRNA Luc and blends of low molecular weight PEI(5 kDa) and PEI(25k Da)-PEG(20 kDa)1-block-copolymer showed protein expression as high as polyplexes with PEI(25 kDa). Moreover, luciferase expression was significantly higher than that obtained with one of the components alone. These results suggest that delivery systems for pulmonary application of mRNA merit further investigation under in vitro and in vivo conditions.
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Iminas/química , Polietilenglicoles/química , Polietileneimina/análogos & derivados , Polietilenos/química , ARN Mensajero/administración & dosificación , Transfección , Animales , Línea Celular , Supervivencia Celular , Fibroblastos/metabolismo , Ratones , Tamaño de la Partícula , Polietileneimina/química , ARN Mensajero/genética , Transfección/métodosAsunto(s)
Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Indometacina/síntesis química , Indometacina/farmacología , Fosfotransferasas/antagonistas & inhibidores , Inhibidores de la Angiogénesis/química , Inhibidores Enzimáticos/química , Indometacina/químicaRESUMEN
Oligonucleotides with a 3'-terminal 5-alkynyl-3'-amino-2',3'-dideoxyuridine residue were prepared, starting from 2'-deoxyuridine. The optimized route employs a 2',3'-dideoxy-3'-trifluoroacetamido-5-iodouridine 5'-phosphoramidite as building block for DNA synthesis and involves on-support Sonogashira coupling with N-tritylpropargylamine to generate oligonucleotides. The amino group of the propargylamine side chain was acylated to accelerate primer extension reactions involving the 3'-amino group. Three acyl groups were identified that decrease the half-life for DNA-templated extension steps with 7-azabenzotriazole esters of 2'-deoxynucleotides. The residue of 4-pyrenylbutyric acid was found to accelerate primer extension reactions and to render them more selective than those of the control primer. With this substituent, primer extension is also faster than previously measured for three-strand systems involving template, aminoprimer, and a downstream-binding helper oligonucleotide. Fast-reacting primers might become useful for genotyping single nucleotides.