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The deployment of adenovirus‐containing gene activated matrices onto severed axons after central nervous system injury leads to transgene expression in target neuronal cell bodies
Author(s) -
Maria Gonzalez Ana,
Berlanga Oscar,
Leadbeater Wendy E.,
CooperCharles Lisa,
Sims Karen,
Logan Ann,
Eliceiri Brian,
Berry Martin,
Baird Andrew
Publication year - 2009
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1354
Subject(s) - transgene , central nervous system , microbiology and biotechnology , biology , nervous system , cell bodies , gene expression , genetic enhancement , neuroscience , gene , genetics
Background In previous studies, we showed that gene activated matrices (GAMs) containing nonviral vectors successfully deliver genes to neurons after optic nerve and spinal cord injury. In the present study, we evaluated whether adenoviral vectors delivered within a GAM increase the efficiency of local gene delivery to injured CNS neurons. Lyophilized GAMs containing collagen and adenoviral vectors were assessed in vitro and in vivo . Methods We evaluated viral vector stability, release kinetics and efficiency of transduction for this GAM formulation in vitro using the quantitative polymerase chain reaction (qPCR), flow cytometry and fluorescence microscopy. Using PCR, reverse transcriptase‐PCR and confocal microscopy, we assessed viral DNA retrograde axonal transport, green fluorescent protein (GFP) expression in retinal ganglion cells (RGCs) after GAM implantation into the wound of the rat transected optic nerve. Results qPCR analyses demonstrated that 100% of viral particles were retained within the collagen after lyophilization. In vitro studies demonstrated that 60% of the particles within the GAM were infective and not released from the collagen matrix when placed in water. By 24 h, GFP expression was detected within cells that have invaded the GAM. In vivo studies demonstrated that adenoviral particles were retrogradely transported in axons from the GAM implanted at the lesion site to the RGC in the retina where the corresponding mRNA was expressed. Analysis of the efficiency of cell transduction indicated that 69% of RGC express GFP. Conclusions These studies demonstrate that lyophilized GAMs containing adenoviral particles within collagen are stable, retain a significant proportion of their infectivity and successfully and efficiently deliver genes to neurons after central nervous system injury. Copyright © 2009 John Wiley & Sons, Ltd.