Technical Report

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

Received:
Accepted:
Published online:

Abstract

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.

  • Subscribe to Nature Neuroscience for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Accessions

Primary accessions

NCBI Reference Sequence

References

  1. 1.

    & Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success—a personal perspective. Hum. Gene Ther. 26, 257–265 (2015).

  2. 2.

    et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

  3. 3.

    et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 34, 334–338 (2016).

  4. 4.

    , & Targeting neural circuits. Cell 165, 524–534 (2016).

  5. 5.

    & DREADDs (designer receptors exclusively activated by designer drugs): chemogenetic tools with therapeutic utility. Annu. Rev. Pharmacol. Toxicol. 55, 399–417 (2015).

  6. 6.

    et al. Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain. Nat. Methods 13, 325–328 (2016).

  7. 7.

    , & Viral vector-based models of Parkinson's disease. Curr. Top. Behav. Neurosci. 22, 271–301 (2015).

  8. 8.

    , , , & Adeno-associated virus-based gene therapy for CNS diseases. Hum. Gene Ther. 27, 478–496 (2016).

  9. 9.

    & AAV-mediated gene therapy for research and therapeutic purposes. Annu. Rev. Virol. 1, 427–451 (2014).

  10. 10.

    et al. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial. Lancet 388, 661–672 (2016).

  11. 11.

    et al. Virally mediated optogenetic excitation and inhibition of pain in freely moving nontransgenic mice. Nat. Biotechnol. 32, 274–278 (2014).

  12. 12.

    et al. Virus-mediated shRNA knockdown of Nav1.3 in rat dorsal root ganglion attenuates nerve injury-induced neuropathic pain. Mol. Ther. 21, 49–56 (2013).

  13. 13.

    , , , & Vagal sensory neuron subtypes that differentially control breathing. Cell 161, 622–633 (2015).

  14. 14.

    et al. Sensory neurons that detect stretch and nutrients in the digestive system. Cell 166, 209–221 (2016).

  15. 15.

    et al. Chemogenetic disconnection of monkey orbitofrontal and rhinal cortex reversibly disrupts reward value. Nat. Neurosci. 19, 37–39 (2016).

  16. 16.

    et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 59–65 (2009).

  17. 17.

    et al. Widespread central nervous system gene transfer and silencing after systemic delivery of novel AAV-AS vector. Mol. Ther. 24, 726–735 (2016).

  18. 18.

    et al. Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer. Mol. Ther. 19, 1070–1078 (2011).

  19. 19.

    , , & Brain endothelial cell-targeted gene therapy of neurovascular disorders. EMBO Mol. Med. 8, 592–594 (2016).

  20. 20.

    et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016).

  21. 21.

    et al. Global representations of goal-directed behavior in distinct cell types of mouse neocortex. Neuron 94, 891–907 (2017).

  22. 22.

    et al. Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex. Nat. Neurosci. (2017).

  23. 23.

    & Adeno-associated viral vectors for mapping, monitoring, and manipulating neural circuits. Hum. Gene Ther. 22, 669–677 (2011).

  24. 24.

    et al. Circuit architecture of VTA dopamine neurons revealed by systematic input-output mapping. Cell 162, 622–634 (2015).

  25. 25.

    , , , & Improved tools for the Brainbow toolbox. Nat. Methods 10, 540–547 (2013).

  26. 26.

    et al. Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice. Front. Mol. Neurosci. 7, 81 (2014).

  27. 27.

    et al. Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol. Ther. 14, 45–53 (2006).

  28. 28.

    & Neuronal morphology goes digital: a research hub for cellular and system neuroscience. Neuron 77, 1017–1038 (2013).

  29. 29.

    et al. Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness. J. Gene Med. 6, 817–828 (2004).

  30. 30.

    & High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS One 2, e162 (2007).

  31. 31.

    & Evaluation of an autoregulatory tetracycline regulated system. Oncogene 16, 1879–1884 (1998).

  32. 32.

    et al. rAAV-compatible MiniPromoters for restricted expression in the brain and eye. Mol. Brain 9, 52 (2016).

  33. 33.

    et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 19, 1743–1749 (2016).

  34. 34.

    , , , & Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion. Gene Ther. 21, 44–51 (2014).

  35. 35.

    & Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23–36 (2013).

  36. 36.

    et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92, 372–382 (2016).

  37. 37.

    et al. Short promoters in viral vectors drive selective expression in mammalian inhibitory neurons, but do not restrict activity to specific inhibitory cell-types. Front. Neural Circuits 3, 19 (2009).

  38. 38.

    , , & Better targeting, better efficiency for wide-scale neuronal transduction with the synapsin promoter and AAV-PHP.B. Front. Mol. Neurosci. 9, 116 (2016).

  39. 39.

    , , , & Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68, 639–653 (2010).

  40. 40.

    , , , & High-throughput, high-resolution mapping of protein localization in mammalian brain by in vivo genome editing. Cell 165, 1803–1817 (2016).

  41. 41.

    , , & Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562–574 (2010).

  42. 42.

    et al. Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules. Science 349, 70–74 (2015).

  43. 43.

    et al. Viral-genetic tracing of the input-output organization of a central noradrenaline circuit. Nature 524, 88–92 (2015).

  44. 44.

    et al. Multiplex cell and lineage tracking with combinatorial labels. Neuron 81, 505–520 (2014).

  45. 45.

    , , , & Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo. Nat. Methods 5, 61–67 (2008).

  46. 46.

    , , , & Single-cell electroporation for gene transfer in vivo. Neuron 29, 583–591 (2001).

  47. 47.

    et al. Single-neuron labeling with inducible Cre-mediated knockout in transgenic mice. Nat. Neurosci. 11, 721–728 (2008).

  48. 48.

    et al. New mouse lines for the analysis of neuronal morphology using CreER(T)/loxP-directed sparse labeling. PLoS One 4, e7859 (2009).

  49. 49.

    & Genetically encoded indicators of neuronal activity. Nat. Neurosci. 19, 1142–1153 (2016).

  50. 50.

    , , & A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. eLife 5, e14472 (2016).

  51. 51.

    , , , & GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat. Neurosci. 7, 1233–1241 (2004).

  52. 52.

    et al. Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Neuron 71, 142–154 (2011).

  53. 53.

    et al. Olfactory cortical neurons read out a relative time code in the olfactory bulb. Nat. Neurosci. 16, 949–957 (2013).

  54. 54.

    et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012).

  55. 55.

    et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

  56. 56.

    et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).

  57. 57.

    , , & Expression of transgenes in midbrain dopamine neurons using the tyrosine hydroxylase promoter. Gene Ther. 16, 437–440 (2009).

  58. 58.

    , & Myelin basic protein gene contains separate enhancers for oligodendrocyte and Schwann cell expression. J. Cell Biol. 119, 605–616 (1992).

  59. 59.

    , & Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337–347 (2003).

  60. 60.

    , , & GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56, 481–493 (2008).

  61. 61.

    et al. Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration. Curr. Protoc. Neurosci. 4, 4.17, (2011).

  62. 62.

    et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973–985 (1999).

  63. 63.

    et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).

  64. 64.

    et al. ScaleS: an optical clearing palette for biological imaging. Nat. Neurosci. 18, 1518–1529 (2015).

Download references

Acknowledgements

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

Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.

    • Ken Y Chan
    • , Min J Jang
    • , Bryan B Yoo
    • , Alon Greenbaum
    • , Namita Ravi
    • , Wei-Li Wu
    • , Luis Sánchez-Guardado
    • , Carlos Lois
    • , Sarkis K Mazmanian
    • , Benjamin E Deverman
    •  & Viviana Gradinaru

Authors

  1. Search for Ken Y Chan in:

  2. Search for Min J Jang in:

  3. Search for Bryan B Yoo in:

  4. Search for Alon Greenbaum in:

  5. Search for Namita Ravi in:

  6. Search for Wei-Li Wu in:

  7. Search for Luis Sánchez-Guardado in:

  8. Search for Carlos Lois in:

  9. Search for Sarkis K Mazmanian in:

  10. Search for Benjamin E Deverman in:

  11. Search for Viviana Gradinaru in:

Contributions

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.

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.

Corresponding authors

Correspondence to Benjamin E Deverman or Viviana Gradinaru.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1–3

  2. 2.

    Supplementary Methods Checklist

Zip files

  1. 1.

    Supplementary Software

    Supplementary Software

Videos

  1. 1.

    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.