Desenvolvimento de terapias inovadoras para a lesão medular

ReConnect aims at TRANSLATING pre-clinical data on potential pro-regenerative genes into PROMISING SCI THERAPIES. At its core is a unique dataset of early regulated genes with a high probability of triggering intrinsic pro-regenerative genetic programs in neurons, that will be explored using clever strategies and cutting-edge techniques, aiming to establish new effective therapies for SCI. To accomplish this, the following specific objectives were devised: 1. TO IDENTIFY CANDIDATE GENE TARGETS OF HIGH THERAPEUTIC POTENTIAL This first aim is based on the hypothesis that within our previous list of ~100 regeDEG there is a smaller set of non-redundant genes able to trigger most of the other pro-regenerative genes or their downstream functions. Various bioinformatics tools will be used to identify these genes (expected to be 20-40 from a preliminary bioinformatics analysis). These genes will be at the intersection of biological processes and pathways enriched only in regenerating conditions and exhibit a high number of physical and functional interactions with other genes within the list. Subsequent high-content screens, in differentiating PNS neurons where this small set of genes will be down-regulated, will reveal their role in neuritogenesis, a key cellular process in neuroregeneration. 2. TO QUANTIFY THE IN VITRO REGENERATIVE POTENTIAL OF KEY CANDIDATE TARGETS The second aim is to quantify the regenerative potential of 5-10 selected candidate targets from Aim 1 in vitro. It will test how the genetic modulation of these genes, alone or combined with each other, will affect the regenerative capacity of mature injured CNS neurons, under basal and neurotrophic-supplemented conditions. Short- and long-term survival, and neuritogenesis, will be the primary readouts to monitor, and the modulated genes will be prioritized in accordance with their capacity to aid central neurons to survive and regrow their neurites. 3. TO DEMONSTRATE THE IN VIVO REGENERATIVE VALUE OF A CORE SET OF CANDIDATE TARGETS New therapies must be tested in vivo, in proof-of-function assays that are closer to the real SCI environment. Fulfillment of Aims 1 and 2 will deliver a core set of genes whose modulation improves neuritogenesis in vitro under differentiation and regeneration conditions, and thus with a high probability of triggering a considerable neuroregenerative response in vivo. The effect of genetically modulating these candidate genes in a mouse model of SCI will be monitored, particularly on the axonal regrowth at the spinal cord. Parallel studies on an in vivo PNI model will also occur. 4. TO CONFIRM THE REGENERATIVE CAPACITY OF THE DESIGNED THERAPY/IES IN HUMAN NEURONS Aims 1-3 will close the loop on the knowledge and work in rodents (rat PNI vs SCI-derived transcriptomic data –> in vitro functional tests in rodent cells -> in vivo regeneration tests in rodents). However, some therapies fail their translation from rodents to humans. We thus aim to test our gene therapies in an innovative human pre-clinical model based on reprogrammed cells from SCI patients. We will also start testing the combinatorial therapeutic efficacy of applying the developed gene therapies with other tested therapies, such as Schwann cells (cell therapy) and biomaterials. To achieve these aims, ReConnect will take advantage of the know-how and assets of an established international consortium of researchers and will be advised by its clinical partners.