RESUMEN
Messenger RNA (mRNA) vaccines were pivotal in reducing severe acute respiratory syndrome 2 (SARS-CoV-2) infection burden, yet they have not demonstrated robust durability, especially in older adults. Here, we describe a molecular adjuvant comprising a lipid nanoparticle (LNP)-encapsulated mRNA encoding interleukin-12p70 (IL-12p70). The bioactive adjuvant was engineered with a multiorgan protection (MOP) sequence to restrict transcript expression to the intramuscular injection site. Admixing IL-12-MOP (CTX-1796) with the BNT162b2 SARS-CoV-2 vaccine increased spike protein-specific immune responses in mice. Specifically, the benefits of IL-12-MOP adjuvantation included amplified humoral and cellular immunity and increased immune durability for 1 year after vaccination in mice. An additional benefit included the restoration of immunity in aged mice to amounts comparable to those achieved in young adult animals, alongside amplification with a single immunization. Associated enhanced dendritic cell and germinal center responses were observed. Together, these data demonstrate that an LNP-encapsulated IL-12-MOP mRNA-encoded adjuvant can amplify immunogenicity independent of age, demonstrating translational potential to benefit vulnerable populations.
Asunto(s)
Adyuvantes Inmunológicos , Vacunas contra la COVID-19 , Interleucina-12 , ARN Mensajero , SARS-CoV-2 , Vacunas de ARNm , Animales , Interleucina-12/metabolismo , SARS-CoV-2/inmunología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Vacunas contra la COVID-19/inmunología , Ratones , Nanopartículas/química , Femenino , COVID-19/prevención & control , COVID-19/inmunología , Vacuna BNT162 , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Ratones Endogámicos C57BL , Adyuvantes de Vacunas , Humanos , Lípidos/química , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad Celular , Inmunidad Humoral , LiposomasRESUMEN
The majority of newly discovered oral drugs are poorly water soluble, and co-administration with lipids has proven effective in significantly enhancing bioavailability of some compounds with low aqueous solubility. Yet, lipid-based delivery technologies have not been widely employed in commercial oral products. Lipids can impact drug transport and fate in the gastrointestinal (GI) tract through multiple mechanisms including enhancement of solubility and dissolution kinetics, enhancement of permeation through the intestinal mucosa, and triggering drug precipitation upon lipid emulsion depletion (e.g., by digestion). The effect of lipids on drug absorption is currently not quantitatively predictable, in part due to the multiple complex dynamic processes that can be impacted by lipids. Quantitative mechanistic analysis of the processes significant to lipid system function and overall impact on drug absorption can aid in the understanding of drug-lipid interactions in the GI tract and exploitation of such interactions to achieve optimal lipid-based drug delivery. In this review, we discuss the impact of co-delivered lipids and lipid digestion on drug dissolution, partitioning, and absorption in the context of the experimental tools and associated kinetic expressions used to study and model these processes. The potential benefit of a systems-based consideration of the concurrent multiple dynamic processes occurring upon co-dosing lipids and drugs to predict the impact of lipids on drug absorption and enable rational design of lipid-based delivery systems is presented.
Asunto(s)
Portadores de Fármacos/química , Lípidos/química , Modelos Biológicos , Absorción por la Mucosa Oral , Preparaciones Farmacéuticas/administración & dosificación , Administración Oral , Disponibilidad Biológica , Digestión/fisiología , Liberación de Fármacos , Humanos , Absorción por la Mucosa Oral/fisiología , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , SolubilidadRESUMEN
Orally delivered drugs and nutrients must diffuse through mucus to enter the circulatory system, but the barrier properties of mucus and their modulation by physiological factors are generally poorly characterized. The main objective of this study was to examine the impact of physicochemical changes occurring upon food ingestion on gastrointestinal (GI) mucus barrier properties. Lipids representative of postprandial intestinal contents enhanced mucus barriers, as indicated by a 10-142-fold reduction in the transport rate of 200 nm microspheres through mucus, depending on surface chemistry. Physiologically relevant increases in [Ca(2+)] resulted in a 2-4-fold reduction of transport rates, likely due to enhanced cross-linking of the mucus gel network. Reduction of pH from 6.5 to 3.5 also affected mucus viscoelasticity, reducing particle transport rates approximately 5-10-fold. Macroscopic visual observation and micro-scale lectin staining revealed mucus gel structural changes, including clumping into regions into which particles did not penetrate. Histological examination indicated food ingestion can prevent microsphere contact with and endocytosis by intestinal epithelium. Taken together, these results demonstrate that GI mucus barriers are significantly altered by stimuli associated with eating and potentially dosing of lipid-based delivery systems; these stimuli represent broadly relevant variables to consider upon designing oral therapies.
Asunto(s)
Calcio/metabolismo , Contenido Digestivo/química , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos/fisiología , Moco/química , Moco/metabolismo , Animales , Digestión/fisiología , Módulo de Elasticidad/fisiología , Alimentos , Concentración de Iones de Hidrógeno , Mucosa Intestinal/química , Masculino , Ratas , Ratas Sprague-Dawley , Porcinos , ViscosidadRESUMEN
Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.
Asunto(s)
Células Endoteliales/metabolismo , Nanopartículas/química , Polímeros/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular , Humanos , Ratones , Nanopartículas/ultraestructura , Neoplasias/genética , Neoplasias/terapia , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Complejos Multiproteicos/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Canales de Calcio/genética , Señalización del Calcio , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Respiración de la Célula/genética , Células HEK293 , Células HeLa , Humanos , Hígado/metabolismo , Potencial de la Membrana Mitocondrial/genética , Ratones , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Familia de Multigenes , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Interferencia de ARN , Alineación de SecuenciaRESUMEN
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
RESUMEN
Metalloproteins require soluble metal ions such as zinc to properly fold into their native and active state to maintain stability and biological activity. When protein products are produced during microbial fermentations, metals are made available to the metalloproteins via nutrient supplements. During the production at the manufacturing-scale of a recombinant product that required zinc as a cofactor, an insoluble precipitate formed in the preparation tank after steam sterilization of the nutrient feed containing methionine, glycerophosphate, and zinc sulfate (MGZ). The precipitated nutrient feed was believed to be the cause for not enough zinc delivered to the production fermentor, leading to poor product assembly and stabilization. This article explores several analytical techniques such as capillary zone electrophoresis, inductively coupled plasma and phosphate molybdate assays to identify and quantify the composition of the precipitate. Our results show that the glycerophosphate component of the combined MGZ nutrient feed contains inorganic phosphate, which precipitates zinc from the feed media.