SCIREGENASENSE · TARGETED DELIVERY OF NEW ANTISENSE MOLECULES FOR REGENERATION ENHANCEMENT AFTER SPINAL CORD INJURY

TARGETED DELIVERY OF NEW ANTISENSE MOLECULES FOR REGENERATION ENHANCEMENT AFTER SPINAL CORD INJURY

The reversal of paralysis after spinal cord injury (SCI) is among the most daunting challenges of neuroscience research. Current treatments involve surgery and physiotherapy often combined with functional electrical stimulation. There are, however, no fully restorative therapies at present. With the understanding of the molecular pathology involved in neural injury and regeneration, new molecular targets are now under investigation aiming at developing therapeutic approaches in order to achieve axon re-growth over the SCI site. We proposed an innovative therapeutic strategy centered on the use of novel Locked Nucleic Acid (LNA) based single-stranded antisense oligonucleotides (ssAONs) and a multifunctional chitosan delivery platform delivery system specific for neural cells in the spinal cord. The project focused on achieving the simultaneous down-regulation of different genes involved in axonal regeneration inhibition, through the development of LNA-based antisense oligonucleotides and specific in vivo delivery system through the development of a chitosan-based vector, a biomaterial known for its biocompatibility and biodegradability properties. An initial in vitro screening of different 2’-O-methyl RNA gapmer antisense oligonucleotide against RhoA and GSK3β genes was done resulting in two sequences per target, active in downregulation (> 70% downregulation efficiency). These 22-mer oligonucleotide sequences were then the basis for designing shorter LNA gapmer oligonucleotides (14-mers) hoping to achieve the same or higher downregulation efficiencies. Four LNA oligonucleotides per target were designed and one sequence per target was selected having > 80% downregulation efficiencies. Of importance, the antisense oligonucleotide work as resulted in the establishment of a new and ongoing collaboration between the host group and the group of Prof. Jesper Wengel (University of Southern Denmark). In parallel the chitosan-based vector system was developed. In order to efficiently create stable nanocomplexes between chitosan (a polycation) and the oligonucleotides (ssAONs, as mentioned above) we had to modify chitosan by quaternization (trimethyl-chitosan, TMC) followed by the introduction of an 18C long acyl lipid chain (stearic acid). Trimethyl-chitosan possesses permanent positive charges independent of pH (due to the trimethyl groups) thus improving electrostatic interactions with the nucleic acids at physiological conditions. The hydrophobic unit conferred to the chitosan backbone self-assembly properties while also contributing to the interaction with the ssAONs. In a proof-of-concept study we observed that the self-assembly nature of the stearylated-TMC was found essential for the formation of stable nanoparticles with around 150 nm average diameter size and -8 mV of surface charge (zeta potential) with capacity to mediate the successful cellular delivery of single-stranded oligonucleotides. Subsequently, we have focused on refining the vector system towards achieving an efficient oligonucleotide delivery in neuronal cells in vivo. This involved the use of a fibrin gel system where nanoparticles would be embedded for later local application in an injury site (spinal cord injury site) in vivo. To test our systems we developed an in vitro explant culture using dorsal root ganglion (DRG) extracted from rat spinal cords, in order to mimic nanoparticle delivery in a closer to in vivo condition with cells surrounded by their natural extracellular matrix environment. DRGs were cultured inside a fibrin gel matrix where nanoparticles or free oligonucleotides are embedded, and through this system we could preliminarily assess the efficiency of delivery to neuronal cells in a 3D culture environment. We have found that delivery was successful since we were able to obtain more than 70% donwregulation of the target genes in the DRG. To our knowledge this is the first time such system is used for development and assay of delivery vector systems and could have an important impact in the field. We are at the moment assessing the functional effect of this downregulation in terms of the capacity of neurons to extend axons in an inhibitory environment mimicking what happens in a spinal cord injury situation. For this, transfected DRGs are transferred after 5 days to incubation chambers coated with inhibitory molecules such as chondroitin sulphate proteoglycans (CSPGs). Axonal length measurements are then preformed over a period of 8-24h. This developed culture system will allow us to pre-screen different gene targeted therapies and their functional effects, reducing the amount of animals needed in subsequent in vivo experimentations. Finally both the LNA antisense oligonucleotides in free form or delivered in nanoparticles are being tested in an in vivo rat model system of spinal cord injury. Initial studies using fluorescently labeled oligonucleotides have provided evidence that the fibrin gel system is able to sustain release of the oligonucleotide after implantation at the lesion site. Diffusion of oligonucleotide occurred in both rostral and dorsal direction. This provided evidence that the oligonucleotides can reach a large number of cells around the lesion area. For nanoparticle testing, a step of fibrin gel loading needed to be developed. As nanoparticles are formed in very dilute solutions in order to maintain size and low polydispersity a concentration step needs to be employed in order to achieve volumes appropriate for local application. This was achieved by concentrating nanoparticles in the gel by forcing flow-through using centrifugal force. At the moment an approximately 25% of initial particles are possible to retain in the gel. This allowed us to proceed for in vivo experimentation while further optimizations of the loading system are being pursued. Functional assays with both free oligonucleotides and nanoparticles are underway to test the in vivo downregulation capacity of our antisense oligonucleotide systems. In conclusion we were able to design efficient LNA-based antisense oligonucleotides against relevant genes acting in the inhibitory signaling that stops nerve regeneration after a lesion in the CNS. Due to their inherent stability and small size the LNA oligos can already be used for cell delivery in vivo unassisted by vector systems (“naked” oligonucleotide delivery). Envisaging future therapeutic applications and in order to increase the delivery efficiencies and decrease possible side-effects associated to the use of high amounts of “naked” oligonucleotides, a biomaterial-based vector was developed. This consisted on an amphiphilic modified chitosan polymer (trimethylated and stearylated), which proved essential for the formation of oligonucleotide containing nanoparticles stable against serum protein destabilization and increased interaction with cell membranes leading to improved uptake. Finally a fibrin based gel system has been studied regarding incorporation of both naked LNA oligonucleotides and nanoparticles, with the purpose to apply it locally in a spinal cord injury site for sustained release over time. This system is being tested in vivo in a rat spinal cord injury model. We believe that new tools have been developed that will be of value to the nanomedicine field and which can be further developed into a therapeutically/clinically relevant strategy that could be used in the context of spinal cord injuries. Nevertheless, the implications for the development and demonstration of usage of the oligonucleotide and polymer based vector platform could already be exploited in different therapeutic settings where antisense oligonucleotide delivery could be advantageous (eg. Duchenne muscular dystrophy, spinal muscular atrophy, Ataxias, neurodegenerative disease, …). As concluding remark, the project has opened new avenues of research dealing with the application of new antisense therapies in the regenerative medicine field.

Data: CORDIS, © European Union