Cellular reprogramming for brain repair

Abstract

The inability of the central nervous system (CNS) to regenerate effectively is a barrier to the successful treatment of traumatic injuries and neurodegenerative diseases. As with other cells in the body, neurons are sensitive to injury and degenerative/inflammatory conditions that often result in cell death. While neurons are frequently lost in response to injury or degeneration, astrocytes on the other hand become activated, proliferative, and assemble to enclose a necrotic region and form glial scars. The astrocyte response to injury presents a valuable therapeutic target as they perform both cytotrophic and cytotoxic functions, sometimes concomitantly, after injury. An adeno-associated virus (AAV) vector encoded with specific transcription factors (TFs) that targets astrocytes and potentially converts/reprograms them to functional neurons is of interest in developing novel therapeutic solutions for traumatic brain injury (TBI). Viral vectors such as lentivirus and retrovirus integrate with host-cell genome and pose a series of risks including insertional mutagenesis, transgene integration, and strong immunogenicity[1,2]. However, AAV does not have the problem associated with the integration of host-cell genome as it forms circular concatemers that persist as episomes in the nucleus of transduced cells and does not integrate into host genomes[3]. This project aims to reprogram reactive astrocytes to neurons using TFs both in vitro and in vivo. Furthermore, a bio-inspired self-assembling peptide (SAP) hydrogel is used to provide spatiotemporal release of AAVs in vivo to address multiple difficulties with viral vector delivery by shielding and confining the vectors to the site of therapeutic need. Additionally, these SAP hydrogels have been functionalised to provide relevant biologically active motifs. These motifs can mimic the native cellular microenvironment of the brain. To further drive the survival, differentiation, and maturation of reprogrammed cells, we have developed a hybrid composite biomaterial, incorporating electrospun short fibres (SFs) loaded with valproic acid (VPA) small molecule within our novel SAP hydrogel matrix. We found that NeuroD1/ and SOX2 TFs can trans-differentiate and dedifferentiate reactive astrocytes to neurons in vitro, respectively. More importantly, both SOX2 and NeuroD1-mediated reprogramming resulted in neural replenishment in vivo. Furthermore, NeuroD1 encoded AAV could significantly reduce the glial scar intensity 28 days post-implantation in vivo in a brain injury model. The presentation of SAP+AAV-NeuroD1 also altered the morphology of astrocytes far away from the lesion site due to the alleviation of inflammatory cells (astrocytes and microglia) in the injury site. This demonstrates the ability of NeuroD1 to reprogram reactive astrocytes to functional neurons and potentially alter the phenotype of reactive astrocytes near the injury site. Thus, this SAP hydrogel is promising as a 3D biomimetic cell culture environment (high water content, stiffness within the range of soft tissue, presenting laminin-derived IKVAV peptide as a bio-functional epitope to encourage cell infiltration into the scaffold for neural tissue engineering) and payload delivery platform for future studies of tissue engineering strategies that can manipulate the inflammatory response to improve functional recovery outcomes. To summarise, the research reported here demonstrates i) functionalisation and materials characterisation, ii) the effect of AAV encoded with SOX2 and NeuroD1 TFs on reprogramming reactive astrocytes to functional neurons, iii) the impact of VPA in neural differentiation and maturation, iv) the effect of SAP hydrogels in AAV distribution in brain injury model in vivo. Show more