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1.
Nano Lett ; 15(11): 7300-6, 2015 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-26469188

RESUMEN

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.


Asunto(s)
Técnicas de Transferencia de Gen , Lípidos/química , Nanopartículas/química , ARN Mensajero/administración & dosificación , Línea Celular Tumoral , Humanos , Lípidos/administración & dosificación , Liposomas/administración & dosificación , Liposomas/química , Hígado/efectos de los fármacos , Nanopartículas/administración & dosificación , Fosfatidiletanolaminas/administración & dosificación , Fosfatidiletanolaminas/química , Polietilenglicoles/química , ARN Mensajero/química , Transfección
2.
Adv Mater ; 29(33)2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28681930

RESUMEN

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.


Asunto(s)
Lípidos/química , Linfocitos B , Hígado , Nanopartículas , ARN Mensajero
3.
Biomaterials ; 109: 78-87, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27680591

RESUMEN

mRNA has broad potential for treating diseases requiring protein expression. However, mRNA can also induce an immune response with associated toxicity. Replacement of uridine bases with pseudouridine has been postulated to modulate both mRNA immunogenicity and potency. Here, we explore the immune response and activity of lipid nanoparticle-formulated unmodified and pseudouridine-modified mRNAs administered systemically in vivo. Pseudouridine modification to mRNA had no significant effect on lipid nanoparticle physical properties, protein expression in vivo, or mRNA immunogenicity compared to unmodified mRNA when delivered systemically with liver-targeting lipid nanoparticles, but reduced in vitro transfection levels. Indicators of a transient, extracellular innate immune response to mRNA were observed, including neutrophilia, myeloid cell activation, and up-regulation of four serum cytokines. This study provides insight into the immune responses to mRNA lipid nanoparticles, and suggests that pseudouridine modifications may be unnecessary for therapeutic application of mRNA in the liver.


Asunto(s)
Lípidos/química , Nanopartículas/química , Seudouridina/química , ARN Mensajero/química , Animales , Citocinas/metabolismo , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Inmunidad Innata , Hígado/metabolismo , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Nanopartículas/administración & dosificación , Tamaño de la Partícula , ARN Mensajero/administración & dosificación , ARN Mensajero/biosíntesis , ARN Mensajero/inmunología , Propiedades de Superficie , Transfección
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