Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems


Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 × 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.

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Figure 1: Engineered AAV capsids for efficient transduction across the peripheral and central nervous systems.
Figure 2: AAV-PHP.eB transduces several CNS regions more efficiently than AAV-PHP.B.
Figure 3: AAV-PHP.S efficiently transduces peripheral neurons.
Figure 4: AAV-PHP.eB and S transfer multiple genomes per cell and enable tunable multicolor labeling.
Figure 5: AAV-PHP.eB can be used with gene regulatory elements to achieve cell-type-restricted gene expression throughout the brain.

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We thank E. Mackey and K. Beadle for assistance with cloning and viral production, P. Anguiano for administrative assistance, and the entire Gradinaru group for discussions. We thank G. Stevens and V. Anand for their efforts in image analysis; P. Rajendran and S. Kalyanam at University of California, Los Angeles and C. Fowlkes at the University of California, Irvine, for discussions, the University of Pennsylvania vector core for the AAV2/9 Rep-Cap plasmid, and M. Brenner at the University of Alabama for the GfABC1D promoter. pAAV-Ef1a-DIO EYFP, pAAV-EF1a-Cre and pAAV-Ef1a-fDIO EYFP were gifts from K. Deisseroth (Addgene plasmids 27056, 55636 and 55641). pEMS2113 and pEMS2115 were gifts from E. Simpson (Addgene plasmids 49138 and 49140). This work was primarily supported by the National Institutes of Health (NIH) through grants to V.G.: Director's New Innovator DP2NS087949 and PECASE; SPARC OT2OD023848-01; National Institute on Aging R01AG047664; BRAIN U01NS090577; and National Institute of Mental Health (NIMH) R21MH103824. Additional funding included the Gordon and Betty Moore Foundation through grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative (to V.G.), the Curci Foundation (to V.G.), the Hereditary Disease Foundation (to V.G. and B.E.D.), the Beckman Institute (to V.G. and B.E.D.) and Rosen Center (to C.L. and V.G.) at Caltech, NIH U01 MH109147 02S1 (to C.L. and V.G.), NIH NS085910 (to S.K.M. and V.G.), the Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO; to V.G. and B.E.D.) and the Friedreich's Ataxia Research Alliance (FARA) and FARA Australasia (to B.E.D.). S.K.M and V.G. are Heritage Principal Investigators supported by the Heritage Medical Research Institute.

Author information




K.Y.C. and B.E.D. designed and performed experiments, imaged samples and analyzed data. K.Y.C. prepared figures with input from B.E.D. and V.G. M.J.J. analyzed data, prepared figures and assisted with experiments and in manuscript preparation. B.B.Y. assisted with tissue processing, imaging and virus production. A.G. helped with image analysis. N.R. assisted with molecular cloning. W.-L.W. and L.S.-G. assisted in tissue processing. C.L. and S.K.M. assisted in experimental designs. K.Y.C., B.E.D. and V.G. wrote the manuscript with support from all authors. B.E.D. and V.G. conceived the project. V.G. supervised all aspects of the work.

Corresponding authors

Correspondence to Benjamin E Deverman or Viviana Gradinaru.

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Competing interests

The California Institute of Technology has filed patent applications related to this work with B.E.D., K.Y.C. and V.G. listed as inventors. B.E.D. and V.G. receive research support from Voyager Therapeutics; this support was not used in preparation of this manuscript or for the studies described therein.

Integrated supplementary information

Supplementary Figure 1 AAV-PHP.eB transduces several brain regions more efficiently than AAV-PHP.B.

(a-c) The cumulative distribution plots show native GFP fluorescence, measured by the median NLS-GFP intensity within individual nuclei in the cortex (a), striatum (b) and within calbindin+ Purkinje cell bodies (c). In (a and b), ROIs were drawn manually around each DAPI+ nuclei. In (c), the ROI detection was automated (see Online Methods and Supplementary Fig. 2). (a-c) The dotted lines represent the 50% intersect, where 50% of the cells have the corresponding median intensity or lower. (c) The solid black vertical line displays the cutoff point used during the automated detection to classify whether a cell had been transduced with ssAAV-CAG-NLS-GFP. Cells with a median intensity below or equal to 0.1 were classified as non-transduced while those above 0.1 were classified as transduced. (a-c) n = 4 individual animals per group. P ≤ 0.0001 for each region, Kolmogorov-Smirnov test.

Supplementary Figure 2 An automated cell detection pipeline used for counting Purkinje cells in the cerebellum.

The pipeline used for automated cell body and nucleus detection. (a) To detect cell bodies from the original image, a morphological filter with a disk-shaped structural element is first used to blur thin fibrous processes (morphological filtering). A circular Hough transform was subsequently applied to the filtered image (circular object detection). Red circles indicate the objects detected, each of which corresponds to a single cell body. (b) In Fig. 2c, we further analyzed the GFP channel to detect the nucleus of each cell. First, the same area at each cell body region was cropped from the GFP image (cropped image of each cell body). The background of each cropped image, estimated by 2D Gaussian smoothing with a standard deviation of 5-fold the cell body size, was subtracted (background subtraction). After thresholding the image to make it binary (binarization), the 2-D convex hull of the object (convex hull) was obtained as the final nuclear region (NLS).

Supplementary Figure 3 Representative immunohistochemistry in the cortex and striatum after transduction with AAV-PHP.B or AAV-PHP.eB.

Single-plane confocal images of native GFP fluorescence (green) after immunohistochemistry (IHC) with DAPI (DNA labeling, blue), NeuN (neuronal marker, magenta) and S100 (glial marker, red) in (a) cortex or (b) striatum. These data are presented in support of Fig. 2. All scale bars are 50 μm. All imaging and display conditions for the GFP channel are matched across panel pairs to allow for comparisons. All images are from tissue extracted at three weeks post intravenous administration of ssAAV-CAG-NLS-GFP at 1 × 1011 vg to adult mice.

Supplementary Figure 4 AAV-PHP.S transduces peripheral organs through intravenous delivery in adult mice.

(a) Representative images of the liver, lungs and cardiac muscle transduced by AAV9 (top) or AAV-PHP.S (bottom). (b) Representative images of several layers of the stomach (longitudinal muscle, myenteric plexus, and circular muscle) transduced by AAV9 (top) or AAV-PHP.S (bottom). The myenteric plexus also contains muscle cells due to the uneven topography of the stomach. (c) Representative images of the submusocal plexus stained with neuronal marker PGP9.5 (red) and astrocyte marker S100b (blue) in the duodenum, jejunum, ileum, and proximal colon. (a-c) All images show native GFP fluorescence taken with confocal microscopy. Images in (a) are 20 μm maximum intensity projections (MIPs) and images in (b and c) are single-plane images. All imaging and display conditions for the GFP channel are matched across panel pairs to allow for comparisons. All scale bars are 50 μm. All images are from tissue extracted three weeks post intravenous administration of ssAAV-CAG-NLS-GFP at 1 × 1012 vg to adult mice. All experiments and imaging for direct comparisons were performed in parallel.

Supplementary Figure 5 The FLPo-FRT two-component system allows sparse gene expression.

(a) Schematic of the two viral vectors used for sparse gene expression with FlpO recombinase encoded in one viral vector and a transgene flanked by FRT sites on a second viral vector. In the basal state, the XFP is expressed in a double inverted orientation (fDIO) and upon the presence of FLPo is inverted into the orientation in line with the CAG promoter for expression. (b) Representative images of native mNeonGreen fluorescence two weeks after intravenous administration of ssAAV-PHP.B:CAG-fDIO-mNeonGreen at 1 × 1012 vg/mouse together with the indicated dose of ssAAV-PHP.B:EF1α-FLPo. (c) Magnified images of the cortex, striatum, and cerebellum. Scale bars are 1 mm (b) and 50 μm (c).

Supplementary Figure 6 The use of the tetracycline-inducible system with viral vectors provides tunable dense to sparse gene expression.

(a) A two-component vector system consisting of one virus, ssAAV-PHP.B:TRE-mNeonGreen, delivered at 1 × 1012 vg into adult mice, and a second virus, ssAAV-PHP.B:CAG-tTA, the dose of which was varied across mice. As a control, ssAAV-PHP.B:TRE-mNeonGreen was delivered alone to observe any basal expression from the ssAAV-PHP.B:TRE-mNeonGreen. All experimental, imaging and display conditions are matched across images. (b) Representative 20 μm MIPs of native mNeonGreen fluorescence from the cortex, striatum, and cerebellum at high and low activation doses. (c and d) Measurements of the shortest cell-to-cell distance (c) and cell density (d) in the cortex, striatum and cerebellum. n = 3 sections per group, mean ± SEM,unpaired t-test (***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; n.s., p ≥ 0.05). (e) A MIP image of a coronal section taken from the olfactory bulb of a Tbx21-Cre mouse transduced with vector-assisted spectral tracing (VAST) transgenes. The VAST cocktail consist of ssAAV-PHP.eB:TRE-DIO-3XFP (1 × 1012 vg total dose) and sAAV-PHP.eB:ihSyn1-tTA (1 × 1010 vg – high dose) or (1 × 108 vg – low dose). In (e, right) the inset box in gray represents a high magnification of the region of interest outlined. Scale bars are 1 mm (a) and 50 μm (b and e). All images are native GFP fluorescence taken with confocal microscopy.

Supplementary Figure 7 A positive feedback loop within the tTA inducer vector enhances gene expression.

Schematic of the two-component viral vector system without (a) and with (b) an inducible positive feedback loop with the viral vector containing the tTA. The schematic shows tTA protein (magenta hexagons) binding onto TRE elements (blue lines) to activate expression of (a) mNeonGreen or (b) mNeonGreen and the tTA containing vector. (c) Representative confocal images of sagittal brain sections transduced by the no-feedback vector and (d) positive-feedback tTA vector at 1 × 109 vg. Both tTA inducers were co-administered with 1 × 1012 vg of the mNeonGreen viral vector. Scale bar in (c) is 1 mm.

Supplementary Figure 8 Cell-type-restricted expression and sparse labeling in the enteric nervous system using AAV-PHP.S together with neuron-specific gene regulatory elements or Cre transgenic mice.

Images show XFP expression within the myenteric plexus. (a) ssAAV-PHP.S:hSyn1-3XFP was administered intravenously at a total dose of 1 × 1012 vg. (b) ssAAV-PHP.S:CAG-DIO-3XFP was administered intravenously at a total dose of 1 × 1012 vg to ChAT-IRES-Cre transgenic mice to achieve cell type-specific expression in cholinergic neurons. (c) Sparse labeling with the VAST system in the proximal colon of a ChAT-IRE-Cre transgenic using ssAAV-PHP.S-TET-DIO-3XFP at 1 × 1012 vg and ssAAV-PHP.S-ihSyn-DIO-tTA at 1 × 1010 vg. Color-coded square boxes display neurons traced manually in Imaris v8.3. (d) Rectangular boxes show higher magnification views highlighting how sparse multicolor labeling facilitates tracing of closely aligned processes. Double arrows mark traced processes. Scale bars in (a and d) are set to 50 μm and in (c) to 200 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–3 (PDF 3888 kb)

Supplementary Methods Checklist (PDF 347 kb)

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Supplementary Software (ZIP 776 kb)

A video demonstrating the transduction of AAV-PHP.S across multiple layers of the small intestine after optical clearing with refractive index matching solution (RIMS).

Native GFP fluorescence (green) from ssAAV-PHP.S:CAG-NLS-GFP at 1 × 1012 vg/mouse is shown. S100b staining (magenta) is shown for contrast. (MOV 9284 kb)

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Chan, K., Jang, M., Yoo, B. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci 20, 1172–1179 (2017).

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