Cell-penetrating Peptides Enhance The Transduction Of Adeno-associated Virus Serotype 9 In The Central Nervous System

Objective: Recombinant adeno-associated viruses(r AAVs)have been widely used in the gene therapy field for decades.However,because of the challenge of effectively delivering r AAV vectors through the blood-brain barrier(BBB),their applications for treatment of central nervous system(CNS)diseases are quite limited.Our study will help to improve the applications of AAV gene delivery vectors for the treatment of CNS diseases.Methods: 1.Firstly,AAV9 with GFP were constructed,and AAV9 was used to interact with different cell penetrating peptides(CPP)and infect HEK293T?h CMEC/D3?HAs cell lines with different concentrations.The GFP expression of AAV9 gene was detected by fluorescence microscope,RT-q PCR and Western blot.2.The direct interaction between AAV9 and CPP was studied by immunofluorescence co-localization.3.By changing the interaction time and temperature of AAV9 and CPP,the transduction efficiency of the complex was detected.4.To test whether AAV9-CPP complex can improve the ability of AAV9 to penetrate the blood-brain barrier by in vitro blood-brain barrier model.5.The expression of GFP and inflammatory factors in vivo were detected by immunohistochemistry and RT-q PCR.Results: In vitro experiments,Through the detection of molecular and protein levels.We found that several CPP can significantly enhance the transduction efficiency of AAV9 in vitro,and the best one is LAH4.The enhancement of AAV9 transduction by LAH4 depends on the binding of AAV9 capsid and peptide.In vivo,we demonstrated that AAV9-LAH4 complex increased the transduction of AAV9 in CNS after systemic administration by immunohistochemistry.Our results indicate that LAH4 may interact with AAV9 to increase the ability of AAV vector to cross BBB,thus further inducing high expression of target genes in brain.Conclusion: 1.The complex of AAV9 and CPP can enhance the transduction of AAV9 in HEK293T?h CMEC/D3?HAs cells,and the best one is LAH4.2.The complex of AAV9 and LAH4 can improve the ability of AAV9 to penetrate the blood-brain barrier in vivo,and the complex does not cause inflammatory reaction.