title: Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.
abstract: Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture.
authors.affiliation: Instituto de Biología Molecular en Medicina y Terapia Génica, Departamento de Biologìa Molecular y Genómica, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada 950, Guadalajara, Jalisco 44340, Mexico.
authors.forename: Juan
authors.initials: J
authors.lastname: Armendáriz-Borunda
authors.forename: Blanca Estela
authors.initials: BE
authors.lastname: Bastidas-Ramírez
authors.forename: Ana
authors.initials: A
authors.lastname: Sandoval-Rodríguez
authors.forename: Jaime
authors.initials: J
authors.lastname: González-Cuevas
authors.forename: Belinda
authors.initials: B
authors.lastname: Gómez-Meda
authors.forename: Jesús
authors.initials: J
authors.lastname: García-Bañuelos
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002586:Cesium; D002712:Chlorides; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000302:isolation & purification*; D008297:Male; D051381:Rats; D013053:Spectrophotometry
chemical_list: D002712:Chlorides; D002586:Cesium; C028019:cesium chloride