ABSTRACT
The environment within the spinal cord after injury, which changes in the progression from the acute to chronic stages, limits the extent of regeneration. The delivery of inductive factors to promote regeneration following spinal cord injury has been promising, yet, few strategies are versatile to allow delivery during acute or chronic injury that would facilitate screening of candidate therapies. This report investigates the intrathecal delivery of lentiviruses for long-term expression of regenerative factors. Lentivirus-filled sponges were inserted into the intrathecal space surrounding the spinal cord, with transgene expression observed within multiple cell types that persists for 12 weeks for both intact and injured spinal cord, without any apparent damage to the spinal cord tissue. Sponges loaded with lentivirus encoding for Sonic hedgehog (Shh) were investigated for acute (delivered at 0 weeks) and chronic (at 4 weeks) injuries, and for multiple locations relative to the injury. In an acute model, sponges placed directly above the injury increased oligodendrocyte and decreased astrocyte presence. Sponges placed caudal to the injury had reduced impact on oligodendrocytes and astrocytes in the injury. In a chronic model, sponges increased oligodendrocyte and decreased astrocyte presence. Furthermore, the effect of Shh was shown to be mediated in part by reduction of Bmp signaling, monitored with an Msx2-sensitive reporter vector. The implantation of lentivirus-loaded biomaterials intrathecally provides the opportunity to induce the expression of a factor at a specified time without entering the spinal cord, and has the potential to promote gene delivery within the spinal cord, which can influence the extent of regeneration.
Subject(s)
Gelatin Sponge, Absorbable , Gene Transfer Techniques , Genetic Vectors , Hedgehog Proteins/genetics , Lentivirus/genetics , Spinal Cord Injuries/therapy , Acute Disease , Animals , Astrocytes/cytology , Astrocytes/metabolism , Chronic Disease , Gelatin Sponge, Absorbable/chemistry , Genetic Therapy/methods , HEK293 Cells , Humans , Hydrogels/chemistry , Injections, Spinal , Luciferases/genetics , Mice , Oligodendroglia/cytology , Oligodendroglia/metabolism , Polyethylene Glycols/chemistry , Porosity , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Cord Injuries/genetics , Spinal Cord Injuries/pathology , Spinal Cord Regeneration/genetics , TransfectionABSTRACT
Hydrogels have been extensively used for regenerative medicine strategies given their tailorable mechanical and chemical properties. Gene delivery represents a promising strategy by which to enhance the bioactivity of the hydrogels, though the efficiency and localization of gene transfer have been challenging. Here, we functionalized porous poly(ethylene glycol) hydrogels with heparin-chitosan nanoparticles to retain the vectors locally and enhance lentivirus delivery while minimizing changes to hydrogel architecture and mechanical properties. The immobilization of nanoparticles, as compared to homogeneous heparin and/or chitosan, is essential to lentivirus immobilization and retention of activity. Using this gene-delivering platform, we over-expressed the angiogenic factors sonic hedgehog (Shh) and vascular endothelial growth factor (Vegf) to promote blood vessel recruitment to the implant site. Shh enhanced endothelial recruitment and blood vessel formation around the hydrogel compared to both Vegf-delivering and control hydrogels. The nanoparticle-modified porous hydrogels for delivering gene therapy vectors can provide a platform for numerous regenerative medicine applications.