RESUMO
Adenoviral oncolytic virotherapy represents an attractive treatment modality for central nervous system (CNS) neoplasms. However, successful application of virotherapy in clinical trials has been hampered by inadequate distribution of oncolytic vectors. Neural stem cells (NSCs) have been shown as suitable vehicles for gene delivery because they track tumor foci. In this study, we evaluated the capability of NSCs to deliver a conditionally replicating adenovirus (CRAd) to glioma. We examined NSC specificity with respect to viral transduction, migration and capacity to deliver a CRAd to tumor cells. Fluorescence-activated cell sorter (FACS) analysis of NSC shows that these cells express a variety of surface receptors that make them amenable to entry by recombinant adenoviruses. Luciferase assays with replication-deficient vectors possessing a variety of transductional modifications targeted to these receptors confirm these results. Real-time PCR analysis of the replication profiles of different CRAds in NSCs and a representative glioma cell line, U87MG, identified the CRAd-Survivin (S)-pk7 virus as optimal vector for further delivery studies. Using in vitro and in vivo migration studies, we show that NSCs infected with CRAd-S-pk7 virus migrate and preferentially deliver CRAd to U87MG glioma. These results suggest that NSCs mediate an enhanced intratumoral distribution of an oncolytic vector in malignant glioma when compared with virus injection alone.
Assuntos
Neoplasias Encefálicas/terapia , Glioma/terapia , Neurônios/virologia , Terapia Viral Oncolítica/métodos , Transplante de Células-Tronco/métodos , Adenoviridae/genética , Adenoviridae/fisiologia , Animais , Neoplasias Encefálicas/virologia , Movimento Celular/fisiologia , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Glioma/virologia , Humanos , Proteínas Inibidoras de Apoptose , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Transplante de Neoplasias , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , RNA Mensageiro/genética , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , Células-Tronco/virologia , Survivina , Transcrição Gênica , Células Tumorais Cultivadas , Replicação ViralRESUMO
Turtle eggs, 24 h old, were infected with Arizona hinshawii and treated 48 h later with gentamicin sulfate (Garasol; Shering Corp., Allantown, N.J.) by pressure differential egg dip treatment to ascertain the concentration of this reagent required to eradicate this pathogen from eggs. Infected eggs treated with 1,000 or 1,500 micrograms of gentamicin per ml of dip solution eliminated detectable A. hinshawii from eggs as determined by testing shells and embryo-yolk homogenates of 12-day-old eggs and the gastrointestinal tracts, kidneys, livers and gall bladders, and yolks of 50-day-old embryos. Treated eggs produced hatchlings which did not excrete detectable A. hinshawii at 72 h or 30 days after hatching, nor was this organism recovered from the visceral organs of these hatchlings when necropsied 30 days after hatching. Bacteriological assays on infected nontreated eggs showed that greater than 70% of the eggs harbored A. hinshawii, and eggs in this group produced hatchlings which actively excreted and harbored A. hinshawii. Eggs not infected or treated also produced turtles which excreted and systemically carried A. hinshawii and Salmonella spp. though not at the same level as did the turtles produced from infected, nontreated eggs.