477. Three-Dimensional Imaging of Multiple AAV Serotype/Mutant Vector Transductions in the Non-Human Primate Brain By Massively Parallel Sequencing

Abstract

Global therapeutic gene delivery to the central nervous system (CNS) offers a promising approach to treat CNS diseases and preclinical studies using rodents have shown AAV9 as a potential candidate to achieve this goal. However, recent work has highlighted the existence of nonhuman primate (NHP)-rodent differences in AAV vector biology and transduction profiles in the CNS after gene delivery, making imperative a more rigorous evaluation of various AAV serotype and capsid mutant vectors in NHPs. It is of particular importance to understand how each AAV strain (serotype or mutant) is distributed and transduces cells in the brain of a primate species closely related to humans when injected into the bloodstream or cerebrospinal fluids (CSF). Here we report a novel approach to generate three-dimensional (3D) vector transduction heat maps of more than one hundred of AAV strains in the rhesus monkey brain when vectors are delivered via the cisterna magna. In this approach, we integrate the vector transduction data obtained by AAV Barcode-Seq (an Illumina sequencing-based high-throughput vector transduction analysis) into an MRI image. To show proof-of-principle of the method, a barcoded-AAV library containing 145 different AAV strains was directly injected into CSF via the cisterna magna of three rhesus macaques (4×1012 vg/kg) and their brains were subjected to MRI analysis before collection of the organ 6 weeks post-injection. Six mm-thick coronal sections of the brain were made and photographed, and the right half of each section slice was diced into 6 × 7 × 7 mm3 cubes using a matrix, generating more than 100 cubes from a cerebral hemisphere. Total DNA was then extracted from each cube using an automated magnetic particle processor and subjected to AAV Barcode-Seq analysis to determine transduction efficiency of each AAV strain in each cube by multiplexed Illumina sequencing. The coordinates of each cube were determined by aligning the photo images of each coronal section to the corresponding MRI images using an image alignment algorithm. This strategy allowed us to create 3D heat maps showing distribution and transduction of each AAV strain in the entire NHP brain. A preliminary analysis using one of the three monkeys revealed that AAV1 vector spreads globally and transduces all brain regions including the cerebral cortex while the distribution of AAV2 was limited locally. Unexpectedly, we found that many other serotypes and mutants transduced the NHP brain better than AAV9 when delivered into CSF. Thus, imaging of AAV vector distribution and transduction throughout the NHP brain by AAV Barcode-Seq combined with MRI provides a powerful approach to comprehensively study AAV vector-mediated CNS gene delivery in clinically relevant animal models. Our results emphasize the large difference in AAV distribution and transduction efficiency that exists in the brain of higher primates compared to rodent models.