542. Novel Barcode-Based In Vivo Screening Method for Generating De Novo AAV Serotypes for CNS-Directed Gene Therapy

Abstract

Capsid modification is a useful strategy to create adeno-associated virus (AAV) vectors with subtype specific neuronal targeting and enhanced retrograde transport. Incorporating known cell-specific binding ligands is a rational method, but the creation of vectors without prior knowledge has the potential to reveal novel targets. Directed evolution and phage display are broadly utilized high-throughput methods, but are inefficient due to displaying random peptides wherein the vast majority will be non-functional.Here, we have developed a novel AAV library in which each virus particle display a peptide derived from known neuron-related proteins on the surface of an AAV2 capsid. The packaged viral genome encodes a unique barcode sequence to facilitate capsid identification. 92398 unique oligos encoding 14-amino-acid peptides derived from 135 proteins were synthesized using microarray. Four-fragment Gibson assembly and novel emulsion PCR was then used to generate a plasmid library by inserting oligos into the capsid gene and barcodes between the inverted terminal repeats. This plasmid library was then used to assemble a diverse library of AAV capsids, such that particles were composed of only peptide-modified capsid proteins which package an expression cassette containing RNA expressed barcodes providing post hoc identification of the capsid structure.In parallel, the plasmid library was sequences using Illumina paired-end sequencing to link the RNA expressed barcodes to the de novo capsid structures. The successfully generated AAV library efficiently infected neurons and astrocytes in vitro and displayed a subset of peptides that had efficient retrograde transport ability in neurons in vivo (e.g., transported from striatum to substantia nigra). Functional peptides, which successfully promoted neuronal infectivity or retrograde transport, were identified by Illumina sequencing of RNA expressed barcodes both in vitro and in vivo and efficacy modeled through barcode counting. In conclusion, we developed a high-throughput combinatorial method to generate peptide-modified AAV libraries that are valuable for evaluation of receptor expression of neuronal populations and have the potential to generate novel vectors with unique properties for in vivo gene transfer in the CNS.Figure 1Polar plot showing the number of unique peptides found in custom array and plasmid library followed by the number found in infective AAV particles, at injection site and for transported AAVs.View Large Image | Download PowerPoint Slide