Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans

Abstract

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

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Fig. 1: In vivo cell-type targeting in mouse retina.
Fig. 2: AND/OR logic for cell-type targeting by AAVs.
Fig. 3: Recording and modulating activity of AAV-targeted cells.
Fig. 4: In vivo cell-type targeting in NHP retina.
Fig. 5: In vitro cell-type targeting in the human retina.
Fig. 6: Quantitative metrics of the similarity of AAV expression in retinal cell groups in mice, NHPs and humans.

Data availability

The AAV expression patterns of synthetic promoters described here have been made available in a public database (https://data.fmi.ch/promoterDB/). Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, B. Roska (botond.roska@iob.ch) on signing a material transfer agreement.

Code availability

The computer codes and algorithms used in this study are available upon reasonable request.

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Acknowledgements

We thank the following people: A.E. Kacso for the multielectrode array recording analyses; Z. Raics and D. Hillier for developing the recording software; N. Ledergerber for assistance in mouse breeding and maintenance; A. Drinnenberg for providing the AAV-ProA1-GCaMP6s confocal images; N. Gerber-Hollbach for help with the human eye donations; A. Police Reddy for assistance with cloning; X.W. Cheng for the eye injections; L. Vandenberghe for advice on small-scale virus preparation; D. Gaidatzis for support in ProB synthetic promoters design; T. Siegmann and R. Schmidt for creating the AAV database; C. Cepko, V. Gradinaru, E. Bamberg and K. Deisseroth for providing the plasmids; and W. Baehr for providing the anti-CAR antibody. We thank P. King, S. Oakeley and E. Macé for commenting on the manuscript. This work was supported by the Swiss National Science Foundation (grant no. CRS115_173728), the National Centre of Competence in Research (NCCR) ‘Molecular Systems Engineering’ (grant no. 51NF40-182895), a European Research Council Advanced Grant (funding under the European Union’s Horizon 2020 research and innovation program RETMUS grant no. 669157) and a Gebert-Rüf grant (grant no. GRS-039/12) to B.R.; the NCCR ‘Molecular Systems Engineering’ (grant no. 51NF40-182895), the Wellcome Trust (grant no. 210572/Z/18/Z) and the Foundation Fighting Blindness Clinical Research Institute (grant no. NNCC-CL-0816-0097-UBAS-NC) to H.P.N.S.; the National Natural Science Foundation of China (grant no. 81522014), National Key Research and Development Program of China (grant no. 2017YFA0105300) and Zhejiang Provincial Natural Science Foundation of China (grant no. LQ17H120005) to Z.-B.J. We also thank Lynn and Diana Lady Dougan for a personal donation to the Institute of Molecular and Clinical Ophthalmology.

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Authors

Contributions

J.J., J.K. and B.R. designed and supervised the study. J.J., A.S., A.K., A.L., J.N., Z.Z.N., D.G. and H.P.N.S. optimized, performed and coordinated experiments on human retina culture. J.J., B.G.-S., C.P.P.-A., Ö.K. and R.I.H. performed experiments. R.K.M., S.B.R., P.H. and F.E. performed two-photon imaging or multielectrode array experiments. J.J., T.S., C.S.C., T.A., K.-C.W., R.-H.W. L.X., X.-L.F., Z.-B.J. and P.W.H. coordinated and performed experiments on NHPs. A.B. performed statistical analyses. D.H., A.R.K. and D.S. contributed to the synthetic promoter design. J.J., A.B., J.K. and B.R. wrote the paper.

Corresponding authors

Correspondence to Jacek Krol or Botond Roska.

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The authors declare no competing interests.

Additional information

Peer review information: Nature Neuroscience thanks Liqun Luo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Integrated supplementary information

Supplementary Figure 1 AAV-mediated sparse cell-type targeting in mouse retina.

(a) Confocal images of AAV-infected retinas. Left, CatCh-GFP (green); middle-left, immunostaining with marker (magenta) indicated above; middle-right, CatCh-GFP and marker; right, CatCh-GFP and marker and nuclear stain (Höchst, white). (b) Left, confocal images of AAV-infected retinas (top view), CatCh-GFP (black). Middle, quantification of CatCh-GFP+ cell density as a percentage of target cell-type or cell-class density, values are means ± SEM from n = 12 confocal images. Right, quantification of AAV-targeting specificity shown as a percentage of the major (black) and minor (grey) cell types among cells expressing the transgene.

Supplementary Figure 2 AAV-mediated GCaMP6s or CatCh-GFP expression in wild-type or rd1 retinas.

Confocal images of AAV-infected retinas. Left, GCaMP6s or CatCh-GFP (green); middle-left, immunostaining with marker (magenta) indicated above; middle-right, GCaMP6s or CatCh-GFP and marker; right, GCaMP6s or CatCh-GFP and marker and nuclear stain (Höchst, white). Images show representative reproducible results from n = 3 independent experiments.

Supplementary Figure 3 AAV-mediated cell-type targeting in NHP retina.

(a) Confocal images of AAV-infected retinas (top view). Left, GFP or CatCh-GFP (green); middle, immunostaining with marker (magenta) indicated above or nuclear stain (Höchst, white); right, GFP or CatCh-GFP and marker or nuclear stain. Images show representative reproducible results from n = 2 independent experiments. (b) Quantification of the dendritic field diameter of cells targeted by AAV-ProB15 and AAV-ProA5 with means (red line) indicated. (c) Left, quantification of CatCh-GFP+ cell density as a percentage of target cell-type or cell-class density, values are means ± SEM from n = 10 confocal images. Right, quantification of AAV-targeting specificity shown as a percentage of the major (black) and minor (grey) cell types among cells expressing the transgene. Viral titer values are shown as genome copies per ml.

Supplementary Figure 4 AAV-mediated cell-type targeting in human retina.

Confocal images of AAV-infected retinas (top view). Left, GFP or CatCh-GFP (green); middle, immunostaining with marker (magenta) indicated above; right, GFP or CatCh-GFP and marker. Images show representative reproducible results from n = 2 independent experiments.

Supplementary information

Supplementary Figs. 1–4 and Supplementary Table Notes.

Reporting Summary

Supplementary Table 1

AAVs targeting mouse retinal cells.

Supplementary Table 2

AAVs targeting non-human primate retinal cells.

Supplementary Table 3

AAVs targeting human retinal cells.

Supplementary Table 4

Metric of AAV activity and specificity across species.

Supplementary Table 5

AAVs with retained selectivity in targeting at least one retinal cell class across species.

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Jüttner, J., Szabo, A., Gross-Scherf, B. et al. Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans. Nat Neurosci 22, 1345–1356 (2019). https://doi.org/10.1038/s41593-019-0431-2

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