RESUMO
While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per µg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 1011 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.
Assuntos
DNA , Vetores Genéticos , Adenoviridae/genética , Animais , Vetores Genéticos/genética , Genômica , Injeções Intravenosas , Fígado/metabolismo , CamundongosRESUMO
Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases, with approximately half of the HPV-infected people being adolescents and young adults. A recently developed quadrivalent HPV vaccine, GARDASIL((R)), has been shown to be highly effective in the prevention of a number of HPV-mediated diseases. The objective of the present study was to evaluate the potential effects of the vaccine on female fertility and F1 development, growth, behavior, and reproductive performance. In addition, anti-HPV antibodies in the F0 females and F1 offspring were measured during the study. Two groups of 65 virgin Sprague-Dawley rats were administered two or four intramuscular injections of the vaccine (full human dose of 0.5 mL at 5 and 2 weeks prior to mating, on Gestation Day [GD] 6, and Lactation Day [LD] 7; or GD 6 and LD 7 only). Additional groups of rats were administered phosphate-buffered saline or Merck Aluminum Adjuvant (MAA) at the same four times. All females were mated to males of the same stock. Cesarean sections were performed on 22/group on GD 21, 22/group were allowed to deliver, and remaining females used for blood collections or replacements. F0 female fertility parameters were evaluated. An extensive number of prenatal, perinatal, and postnatal parameters were evaluated in the F1 generation. There were no unscheduled deaths during the study. There was no evidence of toxicity in the F0 females given either MAA or vaccine. There were no effects on the fertility or reproductive performance of the F0 females. There was no evidence of developmental toxicity to the F1 generation, including fetal body weight and morphology, postnatal growth and development, behavior, and reproductive performance. The quadrivalent vaccine induced a specific antibody response to the four HPV types in the F0 female rats following one or multiple injections. Antibodies against all four HPV types were transferred to the F1 generation during gestation and/or lactation, likely via the placenta and milk, respectively. The passively transferred antibodies persisted up to Postnatal Day 77 when they were last measured. These results demonstrate that this quadrivalent HPV vaccine had no detectable adverse effects in either the treated F0 female rats or the F1 generation.
Assuntos
Alphapapillomavirus/patogenicidade , Fertilidade/efeitos dos fármacos , Desenvolvimento Fetal/efeitos dos fármacos , Feto/efeitos dos fármacos , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/toxicidade , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Feminino , Feto/imunologia , Vacina Quadrivalente Recombinante contra HPV tipos 6, 11, 16, 18 , Humanos , Lactação/efeitos dos fármacos , Masculino , Nível de Efeito Adverso não Observado , Tamanho do Órgão/efeitos dos fármacos , Infecções por Papillomavirus/imunologia , Gravidez , Ratos , Ratos Sprague-Dawley , Reprodução/efeitos dos fármacosRESUMO
Peroxisome proliferators (PPs) induce liver tumors in rodents through an unknown mechanism requiring PP-activated receptor (PPAR) alpha. Since PPs possess growth modulatory activities that may be important to their hepatocarcinogenicity, we aimed at dissociating the activation of growth signaling pathways from the PPARalpha-mediated response induced by PPs in cultured rat primary hepatocytes. Pretreatment with the differentiation-promoting agent dimethylsulfoxide (DMSO) increased PPARalpha mRNA/protein and enhanced the expression of PPARalpha-regulated genes [fatty acyl Co-A oxidase (FACO), cytochrome P450 4A1 (CYP4A1)] induced by PPs. In contrast, DMSO reduced the expression of immediate early genes (IEG) expression (c-myc, c-jun, c-fos, junB, egr-1) and inhibited mitogen-activated protein kinase (MEK) kinase/extracellular signal-regulated kinases (ERKs) and p38 phosphorylation. Furthermore, the inhibitors Tyrphostin and PD98059 dowregulated IEG/ERKs induction and slightly enhanced the FACO/CYP4A1 response induced by the PP WY-14,643. The stimulation of signal transduction pathways by PPs can be dissociated from PPARalpha activation, thus suggesting that PPs could activate growth regulatory pathways largely via PPARalpha-independent mechanisms.
Assuntos
Hepatócitos/efeitos dos fármacos , Proliferadores de Peroxissomos/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Células Cultivadas , Dimetil Sulfóxido/farmacologia , Inibidores Enzimáticos/farmacologia , Hepatócitos/metabolismo , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Ativação Transcricional/efeitos dos fármacosRESUMO
There is an abundance of vaccines currently in development, with most of them exploring novel mechanisms, adjuvants and/or delivery systems not only for traditional prophylactic use, but also for therapeutic uses. As vaccines are generally administered to healthy individuals, ensuring their quality, potency and safety becomes crucial, especially prior to evaluation in humans. To ensure these key attributes, vaccine developers need to incorporate them as early in the development program as possible, starting in basic research and continuing through preclinical, clinical and postmarketing development. Fortunately for vaccine developers, ample guidance is available from various regulatory agencies to enlighten the long and arduous path of vaccine development. This review will highlight these regulatory expectations, and provide some clarity as to why they are in place.