TOWARDS A COMBINATION THERAPY FOR SPINAL CORD INJURY: PRNA-3WJ NANOTHERAPEUTICS AND TRANSPLANTATION OF INDUCED-NEURAL STEM CELLS

Spinal cord injury (SCI) is a debilitating pathology that has increased in prevalence over the last few decades. Despite improvements in modern medicine leading to a normal life expectancy, there are limited treatment options and still no fully restorative therapies. Several experimental therapies have been employed to ameliorate the hostile injured environment, amongst which stem cell transplantation is a standout. Neural stem cell (NSC) transplantation has shown promising results in promoting functional recovery in SCI models. However, major accessibility, ethical and immunocompatibility issues impede their clinical translation. These barriers can be overcome through the use of induced neural stem cells (iNSCs), obtained by direct reprogramming of autologous somatic cells, leading to the possibility of fully immunocompatible transplants. However, stem cell transplantations in pre-clinical models of SCI showed only limited CNS repair and protection, likely due to limited capacity of transplanted cells to robustly integrate in vivo. In this perspective, we envision that a combination of interventions aiming to first modulate the injured microenvironment will create a more hospitable context for the subsequent stem cell transplantation. Expression of Lipocalin 2 (Lcn2), a siderophore-binding protein implicated in modulation of inflammatory response in CNS diseases, is upregulated in reactive astrocytes that play a major role in inhibiting regeneration in SCI. We aim to ameliorate the deleterious injured environment through the downregulation of Lcn2 expression by delivering packaging RNA (pRNA) nanostructures to create a more amenable niche for improving the engraftment and differentiation of transplanted iNSCs. To achieve this, we first established a reproducible and reliable in vitro protocol for NPC- derived astrocyte differentiation, a more homogeneous population compared to primary astrocyte cultures, subsequently employed for safety and efficacy screening of pRNA nanostructures. NPC- derived astrocytes showed mature astrocytic phenotype after 15 days in vitro culture and gene expression changes upon activation, as observed in vivo. To silence Lcn2 expression we employed pRNA nanostructures, a bio-inspired construct and promising candidates amongst different nanotechnologies. Remarkably, pRNA transfection did not show any cytotoxicity on cultured astrocytes and we observed a specific and significant decrease in the expression of target mRNA upon pRNA transfection, i.e. reduction of classical hallmarks of activated astrocytes (GFAP and vimentin upregulation) here assayed as a proof of concept, and significant reduction in Lcn2 expression as a new therapeutic target. Finally, before combining pRNA transfection and iNSC transplantation in vivo, we first aimed to characterize and compare the therapeutic efficacy of transplanted iNSCs with the well-described NPCs in a murine contusion model of SCI. Preliminary data showed a similar survival rate after transplantation between iNSCs and NPCs and interestingly a similar trend in improvements of fine locomotor recovery 7 weeks after transplantation. Therefore, transplantation of fully immunocompatible iNSCs represents an innovative and promising therapeutic approach for SCI and in vitro results obtained from Lcn2 silencing showed encouraging results for a future promising combinatorial approach that aims at healing the injured environment and promoting functional recovery in SCI.