Capsid Library Screening And Functional Analysis Of Retrograde AAV

Recombinant adeno-associated virus(r AAV)is one of the most widely used vectors in neuroscience and gene therapy.However,the application of r AAV still has many limitations,such as the deficiency of retrograde tracing capability and cell typespecific transduction,and the limited packaging capacity.It's important to further analyze and modify the AAV to promote its application.In this thesis,the key sites of retrograde transport for r AAV2-retro and AAVXR had been mutated,and we evaluated the contribution of each mutation to retrograde transport in prefrontal cortex-striatum and amygdala-striatum pathways,respectively.Results showed that the LADQDYTKTA inserted peptide is critical in the process of retrograde transport,mutating even one amino acid almost completely abolishes the retrograde access to neurons projecting to striatum.Surprisingly,we also found that the mutant that restored V708 I had a stronger retrograde tracing ability in the amygdala-striatum circuit than r AAV2-retro.Compared with AAVXR,the mutant that restored N385 D showed improved retrograde tracing capability in the prefrontal cortex-striatum circuit.In this thesis,an AAV capsid library with random heptapeptide insertion was constructed,and we screened the capsid variants for retrograde transport in the striatum-substantia nigra neural circuit.Here,one new capsid variants AAV9lib-R1 was successfully identified and verified.In the future,this AAV library will be improved and used for retrograde tracing and cell type-specific capsid screening.I also analyzed and shortened the GFAP promoter,and packaged into the capsid of AAV which could infect the whole brain of mice to verify its expression pattern.Finally,a short promoter with good performance named GFAP-M6 was obtained.At the same time,the function of each fragment of the GFAP promoter in expression regulation was analyzed.In this thesis,I functionally analyzed the mutations endowing AAV-retro with retrograde tracing capacity,and obtained the new variants of retrograde tracing AAV through capsid library screening.Besides,I screened and analyzed the GFAP promoter in the framework of AAV and obtained a shorter astrocyte-specific promoter.These studies are beneficial to promote the application of r AAV vector in neuroscience research.