RESUMEN
Gene therapy has become an important modality for a wide range of therapeutic indications with a rapid increase in the number of therapeutic candidates being developed in this field. Understanding the molecular biology underlying the gene therapy is often critical to develop appropriate safety assessment strategies. We aimed to discuss some of the commonly used gene therapy modalities and common preclinical toxicology testing considerations when developing gene therapies. Non-viral gene delivery methods such as electroporation, microinjection, peptide nanoparticles and lipid nanoparticles are deployed as innovative molecular molecular construct which are included in the design of novel gene therapies and the associated molecular biology mechanisms have become relevant knowledge to non-clinical toxicology. Viral gene delivery methodologies including Adenovirus vectors, Adeno-Associated virus vectors and Lentivirus gene therapy vectors have also advanced considerably across numerous therapeutic areas, raising unique non-clinical toxicology and immunological considerations. General toxicology, biodistribution and tumorigenicity are the pillars of non-clinical safety testing in gene therapies. Evaluating the tumorigenicity potential of a gene editing therapy often leverages molecular pathology while some translational challenges remain. Toxicology study design is entering a new era where science-driven customized approaches and program specific considerations have become the norm.
Asunto(s)
Edición Génica , Terapia Genética , Distribución Tisular , Terapia Genética/métodos , Técnicas de Transferencia de Gen , Vectores GenéticosRESUMEN
Pigs are considered one of the relevant animal models for ocular research as they share several histological and anatomical similarities with the human eye. With the increasing interest in juvenile animal models, this study aimed to describe the postnatal development of ocular structures in 16 Göttingen minipigs and 25 F2 domestic pigs, between birth and 6 months of age, using histopathology and immunohistochemistry against Ki-67, caspase-3, calbindin, glial fibrillary acidic protein, rhodopsin, and synaptophysin. All ocular structures in both pig breeds were incompletely developed at birth and for variable periods postnatally. Noteworthy histological features of immaturity included vascularization in the corneal stroma in neonatal Göttingen minipigs, increased cellularity in different substructures, remnants of the hyaloid vasculature, short and poorly ramified ciliary body processes, and a poorly developed cone inner segment. Increased cellular proliferation, highlighted by abundant Ki-67 immunolabeling, was observed in almost all developing structures of the pig eye for variable periods postnatally. Apoptosis, highlighted with caspase-3 immunolabeling, was observed in the retinal inner nuclear layer at birth and in the regressing hyaloid vasculature remnants. Immunohistochemistry against rhodopsin, synaptophysin, and calbindin demonstrated the short size of the developing photoreceptors and the immature cone inner segment morphology. Calbindin labeling revealed significant differences in the amount of positively labeled cone nuclei between the retinal area centralis and the non-area centralis regions. The elongation of Müller cell processes in the developing retina was shown with glial fibrillary acidic protein. In both pig breeds, the eyes reached histomorphological and immunohistochemical maturity at 6 months of age.
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Cuerpo Ciliar , Retina , Porcinos Enanos , Animales , Calbindinas , Inmunohistoquímica , Retina/crecimiento & desarrollo , Porcinos , Porcinos Enanos/crecimiento & desarrolloRESUMEN
The novel self-amplifying mRNA (SAM) technology for vaccines consists of an engineered replication-deficient alphavirus genome encoding an RNA-dependent RNA polymerase and the gene of the target antigen. To validate the concept, the rabies glycoprotein G was chosen as antigen. The delivery system for this vaccine was a cationic nanoemulsion. To characterize the local tolerance, potential systemic toxicity and biodistribution of this vaccine, two nonclinical studies were performed. In the repeated dose toxicity study, the SAM vaccine was administered intramuscularly to rats on four occasions at two-week intervals followed by a four-week recovery period. SAM-related changes consisted of a transient increase in neutrophil count, alpha-2-macroglobulin and fibrinogen levels. Transient aspartate aminotransferase and alanine aminotransferase increases were also noted in females only. At necropsy, observations related to the elicited inflammatory reaction, such as enlargement of the draining lymph nodes were observed that were almost fully reversible by the end of the recovery period. In the biodistribution study, rats received a single intramuscular injection of SAM vaccine and then were followed until Day 60. Rabies RNA was found at the injection sites and in the draining lymph nodes one day after administration, then generally decreased in these tissues but remained detectable up to Day 60. Rabies RNA was also transiently found in blood, lungs, spleen and liver. No microscopic changes in the brain and spinal cord were recorded. In conclusion, these results showed that the rabies SAM vaccine was well-tolerated by the animals and supported the clinical development program.
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ARN Mensajero/farmacocinética , Vacunas Antirrábicas/farmacocinética , Animales , Femenino , Inyecciones Intramusculares , Masculino , ARN Mensajero/administración & dosificación , Vacunas Antirrábicas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Medición de Riesgo , Distribución TisularRESUMEN
Histological examination of the rat placenta and fetus is uncommon. Toxicological studies mainly rely on gross examination of the fetus and on fetal and placental weights. These are often insufficient to assess the fetal and placental toxicity of xenobiotics. The small size of the fetus makes its dissection labor-intensive. Thus, our objective was to develop a simple and accurate technique to evaluate the rat fetus and placenta. Sprague-Dawley rat fetuses at gestational day 19.5 ( n = 18) and their placentas ( n = 32) were fixed in formalin. Placentas were cut transversally in the center. Fetuses were cut following a freehand whole-body serial sectioning diagram adapted from Wilson's method. Sections were stained with hematoxylin-eosin-phloxine-saffron, and histomorphometry was used to measure the area of the fetal placental region (27.2 ± 1.7 mm2), including the labyrinth (22.2 ± 1.0 mm2) and the basal zone (4.8 ± 0.8 mm2). Our whole-fetus serial sectioning technique resulted in 12 precise cutting planes that fit on 3 histological slides, enabling the examination of most organs without labor-intensive dissection. Quantitative analysis of placental areas improves the understanding of the pathogenesis of treatment-related changes. This technique provides a standardized method for future research in pertinent fields such as developmental biology and toxicology.
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Feto/anatomía & histología , Técnicas de Preparación Histocitológica , Placenta/anatomía & histología , Animales , Femenino , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
As in many altricial species, rats are born with fused eyelids and markedly underdeveloped eyes. While the normal histology of the eyes of mature rats is known, the histomorphological changes occurring during postnatal eye development in this species remain incompletely characterized. This study was conducted to describe the postnatal development of ocular structures in Sprague-Dawley (SD) rats during the first month of age using histology and immunohistochemistry (IHC). Both eyes were collected from 51 SD rats at 13 time points between postnatal day (PND)1 and PND30. Histologic examination of hematoxylin and eosin-stained sections was performed, as well as IHC for cleaved-caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to evaluate apoptosis, and IHC for Ki-67 and phospho-histone-H3 to evaluate cell proliferation. Extensive ocular tissue remodeling occurred prior to the eyelid opening around PND14 and reflected the interplay between apoptosis and cell proliferation. Apoptosis was particularly remarkable in the maturing subcapsular anterior epithelium of the lens, the inner nuclear and ganglion cell layers of the developing retina, and the Harderian gland, and was involved in the regression of the hyaloid vasculature. Nuclear degradation in the newly formed secondary lens fibers was noteworthy after birth and was associated with TUNEL-positive nuclear remnants lining the lens organelle-free zone. Cell proliferation was marked in the developing retina, cornea, iris, ciliary body and Harderian gland. The rat eye reached histomorphological maturity at PND21 after a rapid phase of morphological changes characterized by the coexistence of cell death and proliferation.
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Ojo/crecimiento & desarrollo , Ratas Sprague-Dawley/crecimiento & desarrollo , Animales , Animales Recién Nacidos/anatomía & histología , Animales Recién Nacidos/crecimiento & desarrollo , Apoptosis , Proliferación Celular , Cuerpo Ciliar/anatomía & histología , Cuerpo Ciliar/crecimiento & desarrollo , Córnea/anatomía & histología , Córnea/crecimiento & desarrollo , Ojo/anatomía & histología , Femenino , Glándula de Harder/anatomía & histología , Glándula de Harder/crecimiento & desarrollo , Histonas/metabolismo , Iris/anatomía & histología , Iris/crecimiento & desarrollo , Antígeno Ki-67/metabolismo , Cristalino/anatomía & histología , Cristalino/crecimiento & desarrollo , Masculino , Ratas , Ratas Sprague-Dawley/anatomía & histología , Retina/anatomía & histología , Retina/crecimiento & desarrolloRESUMEN
Both a rodent and a nonrodent species are required for evaluation in nonclinical safety studies conducted to support human clinical trials. Historically, dogs and nonhuman primates have been the nonrodent species of choice. Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety as an alternate nonrodent species. The pig is an appropriate option for these toxicology studies based on metabolic pathways utilized in xenobiotic biotransformation. Both similarities and differences exist in phase I and phase II biotransformation pathways between humans and pigs. There are numerous breeds of pigs, yet only a few of these breeds are characterized with regard to both xenobiotic-metabolizing enzymes and background pathology findings. Some specific differences in these enzymes based on breed and sex are known. Although swine have been used extensively in biomedical research, there is also a paucity of information in the current literature detailing the incidence of background lesions and differences between commonly used breeds. Here, the xenobiotic-metabolizing enzymes are compared between humans and pigs, and minipig background pathology changes are reviewed with emphasis on breed differences.
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Modelos Animales , Porcinos/anatomía & histología , Porcinos/metabolismo , Toxicología/métodos , Animales , Humanos , Pruebas de Toxicidad/métodosRESUMEN
Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety assessment of small molecules, biopharmaceutical agents, and medical devices as an alternate nonrodent species. Although swine have been used extensively in biomedical research, there is a paucity of information in the current literature detailing the incidence of background lesions and differences in incidence between commonly used breeds. This article is a collaborative effort between multiple organizations to define and document lesions found in the common breeds of minipigs used for toxicological risk assessment in North America (NA) and the European Union (EU). We retrospectively assessed 10 years of historical control data from several institutions located in NA and EU, covering the period of 2004-2015. Here we report the background lesions with consideration of breed and geographical location. To our knowledge, this is the first report documenting spontaneous background lesions in commonly used breeds of swine in both NA and EU. This report serves as a resource to pathologists and will aid in interpretation of findings and differentiation of background from test article-related changes.
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Investigación Biomédica , Enfermedades de los Porcinos , Porcinos Enanos , Animales , Investigación Biomédica/organización & administración , Investigación Biomédica/normas , Bases de Datos Factuales , Incidencia , Porcinos , Enfermedades de los Porcinos/clasificación , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/patología , Pruebas de Toxicidad/normasRESUMEN
Purpose: Characterization of a novel partial-body irradiation (PBI) shielding strategy in nonhuman primates (NHP; rhesus macaques), aimed at protecting the oral cavity, with respect to various gastrointestinal acute radiation syndrome (GI-ARS) syndrome parameters as well as buccal ulceration development.Materials and methods: NHPs were irradiated using a Cobalt-60 gamma source, in a single uniform dose, ranging from 9-13 Gy and delivered at 0.60-0.80 Gy min-1. Animals were either partially shielded via oral cavity shielding (PBIOS) or underwent total-body irradiation (TBI).Results: Clinical manifestations of GI-ARS, and also radiation-induced hematology and clinical chemistry changes, following PBIOS were comparable to the PBI NHP GI-ARS model utilizing shielding of the distal pelvic limbs and were significantly milder than TBI at similar radiation doses. Nadir citrulline levels were comparable between PBIOS and TBI but signs of recovery appeared earlier in PBIOS-treated animals. The PBIOS model prevented oral mucositis, whereas the TBI model presented buccal ulcerations at all tested radiation dose levels.Conclusions: Taken together, these results suggest that the PBIOS model is a suitable alternative to traditional PBI. For GI-ARS investigations requiring orally administered medical countermeasures, PBIOS confers added value due to the prevention of oral mucositis over traditional PBI.
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Boca/efectos de la radiación , Protección Radiológica/métodos , Síndrome de Radiación Aguda/sangre , Síndrome de Radiación Aguda/etiología , Síndrome de Radiación Aguda/patología , Animales , Citrulina/sangre , Radioisótopos de Cobalto/efectos adversos , Rayos gamma/efectos adversos , Macaca mulatta , Masculino , Análisis de Supervivencia , Úlcera/sangre , Úlcera/etiología , Úlcera/patologíaRESUMEN
Bovine viral diarrhea (BVD) viruses are pestiviruses that have been isolated from domestic and wild ruminants. There is serologic evidence of pestiviral infection in more than 40 species of free-range and captive mammals. Vertical transmission can produce persistently infected animals that are immunotolerant to the infecting strain of Bovine viral diarrhea virus (BVDV) and shed virus throughout their lives. Seven species (white-tailed deer, mouse deer, eland, domestic cattle, alpaca, sheep, and pigs) have been definitively identified as persistently infected with BVDV. This study provides serological, molecular, immunohistochemical, and histological evidence for BVDV infection in 2 captive mountain goats from a zoological park in Idaho. The study was triggered by isolation of BVDV from tissues and immunohistochemical identification of viral antigen within lesions of a 7-month-old male mountain goat (goat 1). Blood was collected from other mountain goats and white-tailed and mule deer on the premises for BVDV serum neutralization, viral isolation, and reverse transcription polymerase chain reaction. One 3-month-old mountain goat (goat 2) was antibody negative and BVDV positive in serum samples collected 3 months apart. This goat subsequently died, and though still antibody negative, BVDV was isolated from tissues and identified by immunohistochemistry within lesions. Sequencing and phylogenetic analysis identified the isolates as BVDV-2. These findings provide evidence of persistent infection in a mountain goat, underscoring the need for pestivirus control strategies for wild ruminants in zoological collections.
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Diarrea Mucosa Bovina Viral/diagnóstico , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Enfermedades de las Cabras/diagnóstico , Animales , Animales Salvajes , Anticuerpos Antivirales/análisis , Bovinos , Ciervos/virología , Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Virus de la Diarrea Viral Bovina Tipo 2/aislamiento & purificación , Cabras/virología , Hepatitis Viral Animal/patología , MasculinoRESUMEN
Healthy ruminants carry intestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stx has antiviral activities in vitro and STEC numbers correlate with reduced early viremia in sheep experimentally infected with bovine leukemia virus (BLV). This study assessed the impact of intestinal STEC on BLV-induced disease for one year post-BLV-challenge. High STEC scores (CFU/g feces x frequency of STEC-positive samples) correlated with good health, whereas poor weight gain, distress, and tumor development occurred only among animals with low STEC scores. STEC carriage was associated with increased percentages of B cells in peripheral blood.
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Infecciones por Deltaretrovirus/veterinaria , Intestinos/microbiología , Virus de la Leucemia Bovina/fisiología , Enfermedades de las Ovejas/microbiología , Escherichia coli Shiga-Toxigénica/fisiología , Animales , Infecciones por Deltaretrovirus/microbiología , Masculino , OvinosRESUMEN
In this review paper, literature data on pre- and postnatal eye development are compared between humans and nonclinical species that are commonly used for human safety assessment, namely, mouse, rat, rabbit, dog, minipig, and nonhuman primates. Some new data on rat and minipig ocular development are also included. This compiled information can be helpful for species selection in juvenile toxicity studies or assist in the interpretation of (non)clinical data during pediatric drug development. Despite some differences in developmental windows and anatomical peculiarities, such as the lack of a fovea centralis in nonprimate species or the presence of a nictitating membrane in some nonclinical species, the functioning and development of the eye is strikingly similar between humans and other mammals. As such, all commonly used nonclinical species appear to be relatively good models for human eye development, although some practical constraints such as size may be a limiting factor. Birth Defects Research 109:1540-1567, 2017. © 2017 Wiley Periodicals, Inc.
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Ojo/embriología , Ojo/crecimiento & desarrollo , Especificidad de la Especie , Animales , Perros , Humanos , Ratones , Primates , Conejos , Ratas , Porcinos , Porcinos EnanosRESUMEN
We evaluated the effects of immunostimulation in the development of postweaning multisystemic wasting syndrome (PMWS) in 930 pigs 53 to 54 d old in a grower/finisher barn with a history of PMWS. The pigs were allocated to 5 treatment groups: 4 groups received a single intramuscular injection of RespiSure-ONE (a commercial Mycoplasma hyopneumoniae vaccine; n = 197), Emulsigen (an oil-based adjuvant; n = 172), Alhydrogel (an aluminum-hydroxide-based adjuvant; n = 172), or physiologic saline (n = 218); 1 group received no treatment (n = 171). Pigs affected by PMWS were found in all the groups. Antigen to Porcine circovirus type 2 (PCV-2) was detected by immunohistochemical testing within lesions of mesenteric lymph node, spleen, Peyer's patch, and lung of affected pigs. There was no significant difference in the incidence of PMWS among the groups. The findings indicate that immunostimulation did not influence the expression of PMWS in this study. Thus, routine vaccination against swine diseases may not significantly contribute to the occurrence of PMWS under field conditions.
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Adyuvantes Inmunológicos/farmacología , Vacunas Bacterianas/farmacología , Infecciones por Circoviridae/veterinaria , Enfermedades de los Porcinos/epidemiología , Vacunas Virales/farmacología , Síndrome Debilitante/veterinaria , Hidróxido de Aluminio , Animales , Animales Recién Nacidos , Antígenos Virales/análisis , Infecciones por Circoviridae/epidemiología , Infecciones por Circoviridae/inmunología , Infecciones por Circoviridae/patología , Circovirus , Inmunohistoquímica/veterinaria , Infecciones por Mycoplasma/epidemiología , Infecciones por Mycoplasma/inmunología , Infecciones por Mycoplasma/veterinaria , Distribución Aleatoria , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/patología , Síndrome Debilitante/epidemiología , Síndrome Debilitante/inmunología , Síndrome Debilitante/patologíaRESUMEN
Small intestinal obstruction caused by 2 fibrous bands was found in a steer. Distended small intestine was palpable per rectum. Each band was located bilaterally between the caudal abdominal wall and the pelvic inlet. The compromised portion of intestine was considered nonviable and the animal was euthanized.