Pedro Miguel Duarte Moreno - I3S/INEB, University of Porto
Title: Gene silencing in a spinal cord injury model by local application of LNAgapmer antisense oligonucleotides
Abstract:
In order to achieve nerve regeneration in the context of spinal cord injuries new molecular therapeutic strategies that block the growth inhibitory signals coming from the extracellular environment at the injury site and increase the intrinsic regenerative capacity of neurons are warranted(1).
We have thus developed a strategy to down-regulate local gene expression of important molecules participating in the inhibition of nerve regeneration in a spinal cord injury setting. LNA-based antisense oligonucleotides(2) were designed against two targets of interest, RhoA(3) and GSK3 (4), and loaded in a fibrin gel matrix as an integrated system for their local release in vivo in a spinal cord injury setting.
Fibrin gels prepared with AONs were found to contain the AONs co-localized with the fibrin fibers and a decreased fiber density suggesting some level of direct interaction between the fiber components and the AONs.
An in vitro DRG explant culture system with the DRGs embedded in AON-containing fibrin gels was set-up to mimic a 3D culture condition with cells preserving the natural extracellular matrix components. Using a Cy5-AON containing gel we verified that the AONs were able to distribute throughout the whole DRG, penetrating to its core, and be uptaken by cells.
An efficient reduction of targeted gene expression (>60% at RNA and protein level) was achieved when culturing DRG explants for 7 days in LNA-AON containing gels confirming that the AONs, besides being efficiently distributed throughout the DRG explant were also active inside the cells.
In vivo experiments were conducted in a rat model of spinal cord injury (hemisection). The lesion was filled with the AON-containing fibrin gel and covered with an additional gel patch. Five days post lesion, an extensive distribution of the AON throughout the lesion site, but also traveling rostral and caudal to the lesion, was observed. When using functional LNA-AONs, we observed approximately 80 % gene downregulation in vivo.
AON-loaded fibrin gels could be a promising approach to modulate local cellular gene expression and activity lead toing enhanced regeneration in a spinal cord injury setting.
Title: Gene silencing in a spinal cord injury model by local application of LNAgapmer antisense oligonucleotides
Abstract:
In order to achieve nerve regeneration in the context of spinal cord injuries new molecular therapeutic strategies that block the growth inhibitory signals coming from the extracellular environment at the injury site and increase the intrinsic regenerative capacity of neurons are warranted(1).
We have thus developed a strategy to down-regulate local gene expression of important molecules participating in the inhibition of nerve regeneration in a spinal cord injury setting. LNA-based antisense oligonucleotides(2) were designed against two targets of interest, RhoA(3) and GSK3 (4), and loaded in a fibrin gel matrix as an integrated system for their local release in vivo in a spinal cord injury setting.
Fibrin gels prepared with AONs were found to contain the AONs co-localized with the fibrin fibers and a decreased fiber density suggesting some level of direct interaction between the fiber components and the AONs.
An in vitro DRG explant culture system with the DRGs embedded in AON-containing fibrin gels was set-up to mimic a 3D culture condition with cells preserving the natural extracellular matrix components. Using a Cy5-AON containing gel we verified that the AONs were able to distribute throughout the whole DRG, penetrating to its core, and be uptaken by cells.
An efficient reduction of targeted gene expression (>60% at RNA and protein level) was achieved when culturing DRG explants for 7 days in LNA-AON containing gels confirming that the AONs, besides being efficiently distributed throughout the DRG explant were also active inside the cells.
In vivo experiments were conducted in a rat model of spinal cord injury (hemisection). The lesion was filled with the AON-containing fibrin gel and covered with an additional gel patch. Five days post lesion, an extensive distribution of the AON throughout the lesion site, but also traveling rostral and caudal to the lesion, was observed. When using functional LNA-AONs, we observed approximately 80 % gene downregulation in vivo.
AON-loaded fibrin gels could be a promising approach to modulate local cellular gene expression and activity lead toing enhanced regeneration in a spinal cord injury setting.