Hydrogels-based viral vectors delivery: a self-assembly strategy for targeted gene therapy
Creators: name: Cembran, Arianna
Publisher: name: The Australian National University
Year: 2021

description: Over the past few decades, gene therapy has gained more and more success in finding new strategies and therapies to treat diseases that are hopeless with traditional approaches. Technological advances and a better understanding of pathogenic mechanisms have enabled identification on new therapeutic targets and novel vectors. The introduction of new genetic material into host cells to substitute, replace or silence specific genes is especially appealing for the central nervous system (CNS) due to its limited capacity to self-repair. Neurodegeneration as a result of neurodegenerative diseases or brain injuries is represented by progressive neural loss of function of neurons, including neural death. Moreover, the CNS's chronic inflammatory response represents a barrier to regeneration and functional recovery. Therefore, regenerative medicine is progressing to find promising alternatives to encourage neuroregeneration. On this spot, tissue engineering emerges to bring alternatives rather than traditional therapies combining principles of engineering and biology to advance functional constructs in order to restore, preserve and ameliorate damaged tissues. Mainly, the outcomes corresponds to biomaterials, an attractive solution candidate that can mimic the extracellular matrix (ECM) of the brain, presenting biologically relevant cues and facilitate cell growth and organization. Through investigation of how biomaterials can enhance brain tissue regeneration, we propose to improve brain tissue reparative outcomes merging our selected novel scaffolds with gene therapy. In this thesis, the development and functionalization of nanofibrous three-dimensional scaffolds with encapsulation of designed viral vector payloads are described. Different peptide sequences were incorporated into self-assembling peptides (Fmoc-SAPs) to form an hydrogel networks. All peptides presented IKVAV laminin-derived epitope to better mimic the brain ECM. Following materials advancement and characterisation, we assessed identification of specific viral vectors (adeno-associated viral vectors, AAVs) that efficiently transduced targeted cell types in the CNS. Initial evaluation was carried out in vitro. We selected three AAV serotypes and three cell types and we found that a specific chimera serotype showed high and consistent tropism in all cell types. We also evaluated the transduction efficiency in vivo and we notably demonstrated that all vectors were able to precisely express GFP (gene reporter) after 12 days. The work was then expanded on by investigation of our SAPs to immobilize and deliver the AAV vectors. We demonstrated that with tuning the bulk properties of our peptides without changing the assembly mechanisms of the nanostructure, it is possible to diversify release profiles in accordance to the specific approach required to the nature of the disease. Thus, these SAP systems are promising as 3D biomaterial for improving gene therapy. Also, we designed AAV vectors encoding specific genes in order to convert reactive astrocytes into functional neurons to permit functional recovery in response to the CNS's inflammatory response. We observed that a great percentage of astrocytes already after 7 days post-infection expressed a neural marker and after 14 days post-infection, they started to express a protein for synaptic signalling vesicle. The described engineering of functionalized nanofibrous scaffolds that can be tailored to impact viral vector delivery highlights the research and development process required for improving neurodegeneration. Indeed, we provide a podium for further expansion not just in designing biomaterials for tissue engineering but for future application in gene therapy.