AAV production and purification v1

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing. *IMPORTANT* This protocol is based on Challis et al., Nat Protoc 14: 379, published in 2019. Since that publication, we have made a number of modifications to the protocol to increase throughput or yield. With these modifications, we routinely achieve yields of >5e12 vg per 150 cm dish, with a well packaging capsid (e.g. AAV9, AAV-PHP.eB, and AAV.CAP-B10). In Challis et al., Nat Protoc 14: 379, a Type 70 Ti rotor is used for ultracentrifugation of the AAV through a density gradient (steps 32-36). This rotor can hold 8 tubes, allowing for simultaneous purification of 8 separate AAV variants, at 1-10 plates (150 mm plates) per variant. To increase throughput, we also provide a smaller scale purification workflow with a Type 70.1 Ti rotor, which can allow for purification of 12 separate AAV variants, at 1-3 plates (150 mm plates) per variant. Significant modifications to the referenced protocol are indicated by *MODIFICATION*. Read and understand the original protocol, before following a modified protocol. Following the original protocol should also yield expected results.