RESUMEN
Idiosyncratic drug reactions (IDRs) are associated with significant patient morbidity/mortality and lead to considerable drug candidate attrition in drug development. Their idiosyncratic nature makes the study of IDRs difficult. In particular, nevirapine is associated with a relatively high risk of serious skin rash and liver injury. We previously found that nevirapine causes a similar skin rash in female Brown Norway rats, but these animals do not develop significant liver injury. Programmed cell death protein-1 (PD-1) is an immune checkpoint involved in immune tolerance, and anti-PD-1 antibodies have been used to treat cancer. However, they increase the risk of liver injury caused by co-administered drugs. We found that PD-1-/- mice are more susceptible to drug-induced liver injury, but PD-1-/- mice are not a good model for all drugs. In particular, they do not develop a skin rash when treated with nevirapine, at least in part because they lack the sulfotransferase in their skin that forms the reactive metabolite responsible for the rash. Therefore, we developed a PD-1 mutant (PD-1m/m) rat, with an excision in the ligand-binding domain of PD-1, to test whether nevirapine would cause a more serious skin rash in these animals. The PD-1m/m rat was based on a Sprague Dawley background, which has a lower incidence of skin rash than Brown Norway rats. The treated PD-1m/m rats developed more severe liver injury than PD-1-/- mice, but in contrast to expectations, they did not develop a skin rash. Functional knockouts provide a unique tool to study the mechanisms of IDRs.
Asunto(s)
Nevirapina , Receptor de Muerte Celular Programada 1 , Ratas Endogámicas BN , Animales , Nevirapina/toxicidad , Femenino , Receptor de Muerte Celular Programada 1/genética , Ratas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Técnicas de Inactivación de Genes , MasculinoRESUMEN
Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Nevirapina , Humanos , Ratas , Ratones , Animales , Nevirapina/toxicidad , Nevirapina/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Sistemas CRISPR-Cas , Modelos Animales , Hígado/metabolismo , Mamíferos/metabolismoRESUMEN
Of all methods exercised in modern molecular biology, modification of cellular properties through the introduction or removal of nucleic acids is one of the most fundamental. As such, several methods have arisen to promote this process; these include the condensation of nucleic acids with calcium, polyethylenimine or modified lipids, electroporation, viral production, biolistics, and microinjection. An ideal transfection method would be (1) low cost, (2) exhibit high levels of biological safety, (3) offer improved efficacy over existing methods, (4) lack requirements for ongoing consumables, (5) work efficiently at any scale, (6) work efficiently on cells that are difficult to transfect by other methods, and (7) be capable of utilizing the widest array of existing genetic resources to facilitate its utility in research, biotechnical and clinical settings. To address such issues, we describe here Pressure-jump-poration (PJP), a method using rapid depressurization to transfect even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner. Finally, PJP-induced transfection in primary versus transformed cells reveals a surprising dichotomy between these classes which may provide further insight into the process of cellular transformation.
Asunto(s)
Electroporación , Ácidos Nucleicos , Presión Hidrostática , Transfección , Electroporación/métodos , Células CultivadasRESUMEN
Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1-10 µl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections.
Asunto(s)
Electroporación , Transfección , Transfección/métodos , Electroporación/métodos , Animales , Ratones , Línea Celular , Humanos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismoRESUMEN
A novel brain-targeted and reactive oxygen species-activatable manganese dioxide containing nanoparticle system functionalized with anti-amyloid-ß antibody (named aAß-BTRA-NC) developed by our group has shown great promise as a highly selective magnetic resonance imaging (MRI) contrast agent for early detection and multitargeted disease-modifying treatment of Alzheimer's disease (AD). To further evaluate the suitability of the formulation for future clinical application, we investigated the safety, biodistribution, and pharmacokinetic profile of aAß-BTRA-NC in a transgenic TgCRND8 mouse AD model, wild type (WT) littermate, and CD-1 mice. Dose-ascending studies demonstrated that aAß-BTRA-NC was well-tolerated by the animals up to 300 µmol Mn/kg body weight [b.w.], 3 times the efficacious dose for early AD detection without apparent adverse effects; Histopathological, hematological, and biochemical analyses indicated that a single dose of aAß-BTRA-NC did not cause any toxicity in major organs. Immunotoxicity data showed that aAß-BTRA-NC was safer than commercially available gadolinium-based MRI contrast agents at an equivalent dose of 100 µmol/kg b.w. of metal ions. Intravenously administered aAß-BTRA-NC was taken up by main organs with the order of liver, kidneys, intestines, spleen, followed by other organs, and cleared after one day to one week post injection. Pharmacokinetic analysis indicated that the plasma concentration profile of aAß-BTRA-NC followed a 2-compartmental model with faster clearance in the AD mice than in the WT mice. The results suggest that aAß-BTRA-NC exhibits a strong safety profile as a nanotheranostic agent which warrants more robust preclinical development for future clinical applications.