Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration

Abstract

We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse 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 eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing 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.

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

“…AAVs were produced and purified as previously described 61 . Briefly, AAVs were generated by triple transient transfection of HEK293T cells (ATTC, CRL-3216) using polyethylenimine (Polysciences, 23966-2) 62 .…”

“…Intravenous administration of AAV vectors was performed by retro-orbital injection with a 31gauge needle in 6-8 week old mice as previously described 61 . Following injection, 1-2 drops of proparacaine (Akorn Pharmaceuticals, 17478-263-12) were applied to the cornea to provide local analgesia.…”

Tapping into the brain’s star power

“…To that end, Viviana Gradinaru's lab at Caltech has engineered vectors based on adenoassociated viruses (AAVs) that can deliver genes to the mouse brain after intravenous injection. These vectors allow researchers to dial up or down the number of cells that get labelled while maintaining high expression 9 .…”

Whole-Brain Analysis of Cells and Circuits by Tissue Clearing and Light-Sheet Microscopy

“…Advancing modern neuroscience endeavors is the development of techniques for modifying chemically-distinct and/or genetically-specified cells and circuits for high-resolution imaging at depth. Among these techniques are as follows: (A) HTC-based biomolecule stabilization and tissue-clearing (images adapted from Treweek et al, 2015;Greenbaum et al, 2017a;, (B) viral vector engineering [left, Cre recombinase-based AAV targeted evolution (CREATE); right, vector-assisted spectral tracing (VAST); Chan et al, 2017;Bedbrook et al, 2018;Challis et al, 2018], and (C, E) methodologies for cell-profiling in vivo and in situ. Recent advances in modern microscopy [e.g., LSFM and ultramicroscopy (Dodt et al, 2007); CLARITY-optimized light-sheet microscopy (Tomer et al, 2014); two-photon microscopy and microendoscopy (Jung et al, 2004;Barretto et al, 2011;Marshall et al, 2016)], allow researchers to visualize deep tissue structures in real-time (C), and to image large tissue volumes rapidly and with subcellular resolution HCR (Choi et al, 2014;Greenbaum et al, 2017b)].…”

“…Recent advances in modern microscopy [e.g., LSFM and ultramicroscopy (Dodt et al, 2007); CLARITY-optimized light-sheet microscopy (Tomer et al, 2014); two-photon microscopy and microendoscopy (Jung et al, 2004;Barretto et al, 2011;Marshall et al, 2016)], allow researchers to visualize deep tissue structures in real-time (C), and to image large tissue volumes rapidly and with subcellular resolution HCR (Choi et al, 2014;Greenbaum et al, 2017b)]. Complementing these technologies are improved strategies for targeting individual cell populations and neuronal circuits, such as through the use of genetically engineered animal models (e.g., rodent fluorescent reporter and Cre driver lines; D, E) or through the systemic delivery (e.g., via retro-orbital (RO) injection) of viral vectors with unique cell tropism Chan et al, 2017); B, Top left, For CNS targeting, AAV-PHP.eB was used to package single-stranded (ss) rAAV genomes expressing nuclear localized (NLS) fluorescent reporters (XFP) from cell type-specific promoters for neurons (hSyn1, green), oligodendrocytes (MBP, red), or astrocytes (GFAP, blue), resulting in brain-wide gene expression upon RO delivery; bottom left, for PNS targeting, AAV-PHP.S was used to package ss-rAAV genomes expressing XFPs from either neuron-specific (hSyn1) or tyrosine hydroxylase (rTH)-specific promoters, with RO injection of ssAAVPHP.S:rTH-GFP and ssAAV-PHP.S:hSyn1-tdTomato-f (farnesylated) resulting in gene expression throughout THϩ-containing cell bodies (green) and nerve bundles (red) of the myenteric and submucosal plexus of the duodenum; images adapted from Challis et al, 2018) and/or multicolor labeling capabilities (Chan et al, 2017; e.g., VAST; B, right; E, left), the latter of which promotes cell-sorting and long-range projection mapping endeavors (Bedbrook et al, 2018). D, E, Likewise, methodologies for labeling RNA transcripts and protein epitopes have been adapted for use alongside tissue-clearing protocols (E; Shah et al, 2016; Greenbaum et al, 2017b).…”