RESUMEN
Although there is a WHO guidance for a limit on residual DNA for parenterally administered vaccines produced on continuous cell lines, there is no corresponding guidance for oral vaccines. To help determine an oral limit, we performed a study of Vero cell DNA uptake in rats, in which the relative uptake and persistence of Vero cell DNA administered orally was compared to its uptake when delivered intramuscularly (IM). The results of this study allowed the generation of an empirically derived IM versus oral factor (10(6)) representing the relative inefficiency of DNA uptake by oral administration. This factor was then applied to the WHO recommended parenteral limit of 10 ng/dose to determine a corresponding upper limit on the level of residual Vero cell DNA for an oral vaccine of 10 mg. As a conservative approach, this empirically determined limit was reduced 100-fold to 100 microg. Thus, the results of this animal study, together with additional evidence in the literature, support a residual DNA safety limit of 100 microg per dose for an oral vaccine produced on a continuous cell line.
Asunto(s)
ADN/administración & dosificación , ADN/efectos adversos , Vacunas/normas , Administración Oral , Animales , Línea Celular , Chlorocebus aethiops , ADN/farmacocinética , Desoxirribonucleasas , Endocitosis , Endosomas/fisiología , Femenino , Humanos , Masculino , Guías de Práctica Clínica como Asunto , Vacunas/administración & dosificación , Células Vero , Organización Mundial de la SaludRESUMEN
Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six- to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay - referred to as repeat-anchored integration capture (RAIC) PCR - that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.
Asunto(s)
ADN/metabolismo , Eritropoyetina/genética , Terapia Genética/métodos , Genoma , Animales , ADN/administración & dosificación , Electroporación , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa/métodos , Transfección/métodosRESUMEN
Peroxisome proliferators (PPs) act as nongenotoxic tumor promoters in rodents. Their hepatocarcinogenicity requires the presence of the PP-activated receptor alpha (PPARalpha); however, the exact role played by this transcription factor in the liver, more precisely in liver cell growth and differentiation, is not known. The aim of this study was to investigate the role of PPARalpha in oval cells, which are considered to be closely related to liver stem cells, act as bipotential progenitors for the two main hepatic lineages, and have been implicated as playing a role in several models of liver carcinogenesis. We studied the PPARalpha-mediated response of primary oval cells isolated from rats fed a choline-deficient ethionine-supplemented diet (CDE diet, a regimen commonly used for the induction of oval cell proliferation in rodents) with or without cotreatment with WY14,643, a prototype PPARalpha-activator. PPARalpha was expressed at relatively low levels in primary oval cells from rats fed the CDE diet alone. In vivo treatment with WY14,643 for 2-6 weeks induced, in the oval cells, the expression of PPARalpha as well as that of the PPARalpha-responsive genes encoding fatty acyl-CoA oxidase and cytochrome P450 4A1. Moreover, the oval cell response to WY14,643 was accompanied by an overall phenotypic modulation toward the hepatocyte lineage. In addition, the PPARalpha activator induced, among the oval cells, a subpopulation of transitional cells showing features of maturing hepatocytes expressing the oncofetal marker, alpha-fetoprotein. These results show that oval cells are responsive to PPs and strongly argue for a role of PPARalpha in the differentiation/maturation of rat oval cells. In the absence of the CDE diet regimen, 9-week treatment with WY14,643 lead to the appearance of a population of large-sized cells somewhat similar to the transitional cells. However, these cells showed little expression of markers of mature hepatocytes, consistent with a block during their maturation process, i.e., they are resistant to PPARalpha-mediated differentiation. Interestingly, the phenotype of these cells resembled that of the cells usually found in neoplastic foci induced by PPs. Our results, together with previous reports, suggest the involvement of oval cells in the hepatocarcinogenicity of PPs.
Asunto(s)
Neoplasias Hepáticas/inducido químicamente , Hígado/efectos de los fármacos , Proliferadores de Peroxisomas/toxicidad , Pirimidinas/toxicidad , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Citocromo P-450 CYP4A , Sistema Enzimático del Citocromo P-450/genética , Masculino , Oxigenasas de Función Mixta/genética , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiologíaRESUMEN
The primary safety concern for DNA vaccines is their potential to integrate into host cellular DNA. We describe a sensitive and quantitative assay for investigating the tissue distribution and integration of plasmid DNA vaccines. By including gonadal tissues in the analysis, the potential for germline transmission is also assessed. At various time points after injection, total DNA is isolated from a variety of tissues and assayed by PCR for the presence of plasmid. To test for integration, genomic DNA is first purified away from free plasmid using a series of different gel electrophoresis procedures. The gel-purified genomic DNA is then assayed for integrated plasmid using PCR. Stringent methods are used to prevent contamination. The assay, validated using a variety of positive and negative controls, is capable of detecting one copy of plasmid per ug DNA (approximately 150,000 diploid cells). Using this assay, we have carried out intramuscular studies in mice or guinea pigs for four different DNA vaccine plasmids. There was no evidence of integration to a sensitivity of about one copy/microg DNA, which is at least three orders of magnitude below the spontaneous mutation frequency.