title: Localized viral vector delivery to enhance in situ regenerative gene therapy.
abstract: A lyophilization method was developed to locally release adenoviral vectors directly from biomaterials for in situ regenerative gene therapy. Adenovirus expressing a beta-galactosidase reporter gene (AdLacZ) was mixed with different excipient formulations and lyophilized on hydroxyapatite (HA) disks followed by fibroblasts culturing and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, suggesting 1 M sucrose in phosphate-buffered saline had best viability. Adenovirus release studies showed that greater than 30% virus remained on the material surface up to 16 h. Lyophilized adenovirus could be precisely localized in defined patterns and the transduction efficiency was also improved. To determine if the lyophilization formulations could preserve viral bioactivity, the lyophilized AdLacZ was tested after being stored at varying temperatures. Bioactivity of adenovirus lyophilized on HA was maintained for greater than 6 months when stored at -80 degrees C. In vivo studies were performed using an adenovirus encoding BMP-2 (AdBMP-2). AdBMP-2 was lyophilized in gelatin sponges and placed into rat critical-size calvarial defects for 5 weeks. Micro-computed tomography (micro-CT) analysis demonstrated that free-form delivery of AdBMP-2 had only modest effects on bone formation. In contrast, AdBMP-2 lyophilized in gelatin sponges led to more than 80% regeneration of critical-size calvarial defects.
authors.affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109-1078, USA.
authors.forename: W-W
authors.initials: WW
authors.lastname: Hu
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: S J
authors.initials: SJ
authors.lastname: Hollister
authors.forename: P H
authors.initials: PH
authors.lastname: Krebsbach
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D000257:Adenoviridae Infections / Q000378:metabolism; D000818:Animals; D001672:Biocompatible Materials; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D001861:Bone Regeneration; D001842:Bone and Bones / Q000378:metabolism / Q000473:pathology; D017886:Durapatite; D050723:Fractures, Bone / Q000000981:diagnostic imaging / Q000628:therapy* / Q000821:virology; D005612:Freeze Drying / Q000379:methods; D005781:Gelatin Sponge, Absorbable; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D020007:Implants, Experimental; D007267:Injections; D051381:Rats; D011916:Rats, Inbred F344; D014057:Tomography, X-Ray Computed; D014161:Transduction, Genetic / Q000379:methods; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001672:Biocompatible Materials; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta; D017886:Durapatite; D001616:beta-Galactosidase