Research Of Neuronal Regeneration Induced By Brain-Targeting Gene Delivery System

Traumatic brain injury(TBI)is not only an external force-induced acute event in the general concept,but also a chronic disease with complex pathophysiology.Primary brain injury and subsequent secondary brain injury both lead to massive neuronal death that further result in the long-lasting loss of neurological function and increase the incidence of disability,epilepsy,neurodegenerative diseases even non-neurological disorders.After injury,astrocytes activate into reactive astrocytes(RAs)and form glial scar that limit injury area.In the chronic phase of TBI,glial scar as physical barrier to restrain axonal regeneration and secret toxic factor,therefore inhibit the recovery of neurological function.Unfortunately,due to adult neurogenesis generally limit only in rare areas,TBI-induced massive neuronal loss is hard to replenish.The outcome is irreversible.To replenish lost neurons,neuronal replacement therapy such as stem cells transplantation and neuronal reprogramming thrive.Stem cell transplantation therapy can effectively produce new-born neurons.However,the unlimited stem cell proliferation may lead to tumor formation.Neuronal reprogramming is an emerging idea that aims to convert non-neuronal cells to neurons,such as astrocyte-to-neuron(AtN)conversion.Unlike stem cell transplantation,neuronal reprogramming can directly generate neurons in vivo,therefore eliminate the risk of tumorigenesis and have promising clinical prospect.For example,multiple research reports that the downregulation of polypyrimidine tract-binding protein 1(PTBP1)can convert astrocytes into neurons.However,there are also views that neuronal reprogramming in vivo is an artifact due to the off-target effect of targeting delivery system.The astrocyte-targeting vectors mislabeled neurons were mistook for induced neurons(iNs).Therefore,the development of high-targeting gene delivery system not only clarify the controversy of basic scientific research,but pave the way for future clinical translation.Especially the strategy that develop neuronal reprogramming-based and glial scar-targeting delivery system not only replenish neurons,but also eliminate glial scar,improve microenvironment surrounding neurons and promote the survival and development of new-born neurons.To across blood brain barrier(BBB),the present research constructed an adeno-associated virus(AAV)type AAV/BBB to delivery short hairpin RNA(sh RNA)that downregulate PTBP1 gene expression via RNA interference(RNAi).AAV/BBB can be administrated via intravenous injection and transfect central nervous system(CNS).Glial fibrillary acidic protein(GFAP)is specific marker of astrocytes.After TBI,the expression of GFAP significantly upregulate in RAs.Therefore,this research target astrocytes and RAs via GFAP specific promoter.However,the off-target effect of GFAP promoter may result in the neuronal leaky-expression of AAV/BBB.To improve the targeting specificity to astrocytes and glial scar,this study used rational design strategy to insert astrocyte-targeting P1 peptide onto capsid surface of AAV9 that have appropriate insert site.The obtained AAV9P1 variants have astrocyte targeting ability.The P1 peptide recognizes specific receptors on the surface of astrocytes and induce internalization of AAV9P1.The GFAP promoter then initiates the expression of the target gene in the astrocytes.Since the target cells must simultaneously satisfy both P1 peptide-mediated internalization and GFAP promoter-mediated target gene expression,the delivery system has high-targeting ability to astrocytes and RAs.In comparison with AAV9 and AAV/BBB,AAVP1 significantly improve the transfection efficiency to astrocytes and RAs in vitro.Controlled cortical impact(CCI)model was firstly constructed for in vivo experiments.AAV9P1 and AAV/BBB vectors were injected via tail vein at one week post injury.AAV started expressing transgene at two weeks post injection.The immunofluorescence(IF)showed that AAV9P1 and AAV/BBB mainly accumulate in the peri-injury area.The western blot(WB)and quantitative real-time polymerase chain reaction(qRT-PCR)experiments displayed the AAV9P1 and AAV/BBB-mediated effective mRNA and protein downregulation of PTBP1 gene at four weeks post injection.At eight weeks post injection,AAV/BBB treatment group were observed more iNs than AAV/BBB control group.However,the control group also existed the iNs that shouldn’t exist in theory,demonstrating the off-target effect of GFAP promoter to neurons.In comparison with AAV/BBB,the AAV9P1 control group only have very few neurons and the AAV9P1 treatment group significantly increased the number of iNs.The results preliminarily demonstrated that the downregulation of PTBP1 induce neuronal reprogramming post TBI.In conclusion,this study designed an AAV9P1-GFAP dual-targeting delivery system that can realize non-invasive administration to CNS by intravenous injection and have high-targeting to astrocytes and RAs,providing a promising tool for in vivo cell fate trace and targeting delivery of gene drugs.The present study preliminarily confirmed the generation of iNs.In future study,AAV9P1 delivery system that combined with more stringent lineage-tracing technique will be redound to the absolute clarification of controversy in the neuronal reprogramming field.