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
PER.C6, a cell line derived from human embryonic retinal cells transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes, is used to produce E1-deleted Ad5 vectors such as the MRKAd5 HIV-1 gag vaccine. While whole, live PER.C6 cells are capable of growing as tumours when transplanted subcutaneously into immunodeficient nude mice at a high dosage, the process for vaccine production includes filtration steps and other methods which effectively preclude contamination by intact viable substrate cells. However, because of the neoplastic nature of this cell line, we carried out a series of investigations to assess the tumorigenic risk posed by residuals from the cell substrate in a vaccine. To address concerns about transmission of oncogenic DNA, we demonstrated that purified PER.C6 cellular DNA does not induce tumours in newborn hamsters or nude mice. To address concerns about other potential residuals, including hypothetical adventitious tumour viruses, we demonstrated that a PER.C6 cell lysate and a MRKAd5 HIV-1 gag vaccine produced on PER.C6 cells do not induce tumours in newborn hamsters or newborn rats. These results, in conjunction with the wide panel of viral safety tests performed on these cells, support the safety of the PER.C6 as a cell substrate for vaccine production.
Asunto(s)
Vacunas contra el SIDA/biosíntesis , Adenovirus Humanos/genética , Vacunas contra el SIDA/normas , Animales , Animales Recién Nacidos , Secuencia de Bases , Pruebas de Carcinogenicidad , Línea Celular Transformada , Cricetinae , Cartilla de ADN , Vectores Genéticos , Células HeLa , Humanos , Ratones , Ratones Desnudos , Neoplasias/epidemiología , Neoplasias/etiología , Reacción en Cadena de la Polimerasa , Ratas , Retina/virologíaRESUMEN
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
p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein. The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain). The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Nucleares , Factores de Transcripción/química , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN Complementario/metabolismo , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Distribución Tisular , Factores de Transcripción/genéticaRESUMEN
A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro. Six weeks following intramuscular injection, the vast majority of the plasmid was detected in the muscle and skin near the injection site; lower levels of plasmid were also detected in the draining lymph nodes. At early time points (1-7 days) after injection, a low level of systemic exposure could be detected. Occasionally, plasmid was detected in gonads, but it dissipated rapidly and was extrachromosomal - indicating a low risk of germline transmission. Aluminum phosphate adjuvant had no effect on the tissue distribution and did not result in a detectable increase in integration frequency. Biojector delivery, compared with needle injection, greatly increased the uptake of plasmid (particularly in skin at the injection site), but did not result in a detectable increase in integration frequency. Finally, injection of a plasmid DNA vaccine containing the human papilloma virus type 16 E7 gene, known to increase integration in vitro, did not result in detectable integration in mice. These results suggest that the risk of integration following intramuscular injection of plasmid DNA is low under a variety of experimental conditions.
Asunto(s)
Plásmidos/genética , Plásmidos/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas Virales/genética , Adyuvantes Inmunológicos/farmacología , Compuestos de Aluminio/farmacología , Animales , Secuencia de Bases , ADN/análisis , Gónadas/química , Cobayas , Humanos , Ratones , Músculos/química , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Fosfatos/farmacología , Plásmidos/efectos adversos , Piel/química , Distribución Tisular , Vacunación , Vacunas Virales/administración & dosificación , Virosis/prevención & controlRESUMEN
p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex.