603. Novel DNA-Shuffled AAV Capsids Show Robust and Unique Gene Delivery and Expression Profiles in the CNS Via Intrathecal Injection

Abstract

Aim: Adeno-associated virus (AAV) has great potential for treatment of central nervous system (CNS) diseases. Currently, a few AAV serotypes including AAV9 effectively deliver genes in the CNS. We aimed to screen more AAV serotypes for expression in CNS and compare them to AAV9.Material and methods: Using an existing library containing shuffled capsid genes of AAV serotypes 1 through 9, we characterized a few novel capsids for high expression in the CNS in vivo. Three novel serotypes, AAVH43, AAVH48, and AAVHH67 containing the reporter gene lacZ driven by a CMV promoter were injected in C57BL/6 mice and AAV9 was included as a positive control group (n=4 per group). Prior to vector injection, 25% mannitol solution 200μl was given by tail vein to deduce intracranial pressure. Twenty minutes later, 10μl of virus vectors (1×1011 vg) were slowly injected into the cerebrospinal fluid (CSF) at the intervertenral space between lumbar vertebrae L3 and L4 by a 33-gauge needle connected to a 50μl Hamilton syringe with PE10 tubing. Two weeks later, tissue samples were harvested for lacZ staining and immunostaining.Results: LacZ gene delivered by AAVH43, AAVH48, and AAVHH67 vectors expressed efficiently and similarly to AAV9 in the spinal cord by intrathecal injection. The entire spinal cord from the lumbar up to cervical region showed gene expression. The infected cells were mainly distributed in the posterior funiculus and gray matter. In addition, all 4 vectors infected dorsal root ganglia neurons very efficiently. Immunostaining showed AAVH43, AAVH48 and AAVHH67 vectors with a CMV promoter infected mainly neurons but not glial cells, similar to AAV9. In addition, AAVH43 and AAVH48 had some common characteristics and infected cerebellar cortex much more efficiently than AAV9. Those neurons showed distinctive morphology as Purkinje cells. Further confirmation with cell-type specific antibodies is needed.Conclusion: The AAVH43, AAVH48 and AAVHH67 vectors have similar ability to infect the spinal cord and dorsal root ganglia, equivalent to AAV9, when delivered by intrathecal injection. Moreover, the cerebellar cortex is more susceptible to AAVH43 and AAVH48 infection. With further investigation, the three novel AAV serotypes could be suitable alternative candidates for CNS gene delivery and therapy.