Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Systemic AAV vectors for widespread and targeted gene delivery in rodents

A Publisher Correction to this article was published on 16 July 2019

This article has been updated

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of the protocol.
Fig. 2: AAV-PHP.eB and gene regulatory elements enable cell type–specific gene expression in the brain.
Fig. 3: AAV-PHP.S transduces neurons throughout the PNS.
Fig. 4: AAV-PHP.S for mapping the anatomy and physiology of the heart.
Fig. 5: AAV-PHP.B and AAV-PHP.eB can be used in several mouse and rat strains.
Fig. 6: A modular AAV toolbox for cell type–specific gene expression.
Fig. 7: Time line and AAV harvest procedure.
Fig. 8: AAV purification procedure.

Change history

  • 16 July 2019

    During the production process, the authors of this paper supplied revised versions of Figs. 2–5, Supplementary Tables 1–4, and Supplementary Videos 1–3, but because of publisher error, these revised items were not included in the final published version of the protocol. The figures have been updated in the PDF and HTML versions of the paper, and the revised Supplementary Information files are now available online. We note that the figures have been revised to improve their resolution only; the content of the figures and the data reflected remain unchanged. Also, print requirements impose some limits on figure resolution, but the authors have made very high-resolution versions of Figs. 2–5 available at as Source data.

References

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

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Bedbrook, C. N., Deverman, B. E. & Gradinaru, V. Viral strategies for targeting the central and peripheral nervous systems. Annu. Rev. Neurosci. 41, 323–348 (2018).

    Article  CAS  Google Scholar 

  5. Reed, S. E., Staley, E. M., Mayginnes, J. P., Pintel, D. J. & Tullis, G. E. Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J. Virol. Methods 138, 85–98 (2006).

    Article  CAS  Google Scholar 

  6. Wright, J. F. Transient transfection methods for clinical adeno-associated viral vector production. Hum. Gene Ther. 20, 698–706 (2009).

    Article  CAS  Google Scholar 

  7. Xiao, X., Li, J. & Samulski, R. J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72, 2224–2232 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Ayuso, E. et al. High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther. 17, 503–510 (2010).

    Article  CAS  Google Scholar 

  9. Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Gray, S. J. et al. Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration. Curr. Protoc. Neurosci. 57, Chapter 4, Unit 4.17 (2011).

  12. Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M. & Hoogstraten-Miller, S. Retro-orbital injections in mice. Lab Anim. 40, 155–160 (2011).

    Article  Google Scholar 

  13. Lerner, T. N. et al. Intact-brain analyses reveal distinct information carried by SNc dopamine subcircuits. Cell 162, 635–647 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W. & Sanes, J. R. Improved tools for the Brainbow toolbox. Nat. Methods 10, 540–547 (2013).

    Article  CAS  Google Scholar 

  16. Morabito, G. et al. AAV-PHP.B-mediated global-scale expression in the mouse nervous system enables GBA1 gene therapy for wide protection from synucleinopathy. Mol. Ther. 25, 2727–2742 (2017).

    Article  CAS  Google Scholar 

  17. Zelikowsky, M. et al. The neuropeptide Tac2 controls a distributed brain state induced by chronic social isolation stress. Cell 173, 1265–1279.e19 (2018).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Hillier, D. et al. Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex. Nat. Neurosci. 20, 960–968 (2017).

    Article  CAS  Google Scholar 

  20. Chang, R. B., Strochlic, D. E., Williams, E. K., Umans, B. D. & Liberles, S. D. Vagal sensory neuron subtypes that differentially control breathing. Cell 161, 622–633 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Bruegmann, T. et al. Optogenetic control of heart muscle in vitro and in vivo. Nat. Methods 7, 897–900 (2010).

    Article  CAS  Google Scholar 

  23. Guettier, J. M. et al. A chemical-genetic approach to study G protein regulation of beta cell function in vivo. Proc. Natl. Acad. Sci. USA 106, 19197–19202 (2009).

    Article  CAS  Google Scholar 

  24. Jain, S. et al. Chronic activation of a designer G(q)-coupled receptor improves beta cell function. J. Clin. Invest. 123, 1750–1762 (2013)..

    Article  CAS  Google Scholar 

  25. Li, J. H. et al. A novel experimental strategy to assess the metabolic effects of selective activation of a G(q)-coupled receptor in hepatocytes in vivo. Endocrinology 154, 3539–3551 (2013).

    Article  CAS  Google Scholar 

  26. Gradinaru, V. Overriding sleep. Science 358, 457 (2017).

    Article  Google Scholar 

  27. Robinson, J. E. & Gradinaru, V. Dopaminergic dysfunction in neurodevelopmental disorders: recent advances and synergistic technologies to aid basic research. Curr. Opin. Neurobiol. 48, 17–29 (2018).

    Article  CAS  Google Scholar 

  28. Jackson, K. L., Dayton, R. D., Deverman, B. E. & Klein, R. L. Better targeting, better efficiency for wide-scale neuronal transduction with the synapsin promoter and AAV-PHP.B. Front. Mol. Neurosci. 9, 116 (2016).

  29. Giannelli, S. G. et al. Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery. Hum. Mol. Genet. 27, 761–779 (2018).

    Article  CAS  Google Scholar 

  30. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  Google Scholar 

  31. Yang, Y. 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).

    Article  CAS  Google Scholar 

  32. Senis, E. et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol. J. 9, 1402–1412 (2014).

    Article  CAS  Google Scholar 

  33. Yang, Q. et al. AAV-based shRNA silencing of NF-kappaB ameliorates muscle pathologies in mdx mice. Gene Ther. 19, 1196–1204 (2012).

    Article  CAS  Google Scholar 

  34. Kotterman, M. A. & Schaffer, D. V. Engineering adeno-associated viruses for clinical gene therapy. Nat. Rev. Genet. 15, 445–451 (2014).

    Article  CAS  Google Scholar 

  35. Choi, J. H. et al. Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons. Mol. Brain 7, 17 (2014).

    Article  Google Scholar 

  36. Paterna, J. C., Moccetti, T., Mura, A., Feldon, J. & Bueler, H. Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain. Gene Ther. 7, 1304–1311 (2000).

    Article  CAS  Google Scholar 

  37. Xu, R. et al. Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes. Gene Ther. 8, 1323–1332 (2001).

    Article  CAS  Google Scholar 

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

  39. Gray, S. J. et al. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum. Gene Ther. 22, 1143–1153 (2011).

    Article  CAS  Google Scholar 

  40. Chamberlain, K., Riyad, J. M. & Weber, T. Expressing transgenes that exceed the packaging capacity of adeno-associated virus capsids. Hum. Gene Ther. Methods 27, 1–12 (2016).

    Article  CAS  Google Scholar 

  41. Broderick, J. A. & Zamore, P. D. MicroRNA therapeutics. Gene Ther. 18, 1104–1110 (2011).

    Article  CAS  Google Scholar 

  42. Xie, J. et al. MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol. Ther. 19, 526–535 (2011).

    Article  CAS  Google Scholar 

  43. Nayak, S. & Herzog, R. W. Progress and prospects: immune responses to viral vectors. Gene Ther. 17, 295–304 (2010).

    Article  CAS  Google Scholar 

  44. Gao, K. et al. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Mol. Ther. Methods Clin. Dev. 1, 9 (2014).

    Article  Google Scholar 

  45. Mingozzi, F. & High, K. A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23–36 (2013).

    Article  CAS  Google Scholar 

  46. Strobel, B., Miller, F. D., Rist, W. & Lamla, T. Comparative analysis of cesium chloride- and iodixanol-based purification of recombinant adeno-associated viral vectors for preclinical applications. Hum. Gene Ther. Methods 26, 147–157 (2015).

    Article  CAS  Google Scholar 

  47. Rincon, M. Y. et al. Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector. Gene Ther. 25, 83–92 (2018).

    Article  CAS  Google Scholar 

  48. Hordeaux, J. et al. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol. Ther. 26, 664-668 (2018).

    Article  CAS  Google Scholar 

  49. Dayton, R. D., Grames, M. S. & Klein, R. L. More expansive gene transfer to the rat CNS: AAV PHP.EB vector dose-response and comparison to AAV PHP.B. Gene Ther. 25, 392-400 (2018).

  50. Gray, S. J. et al. Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol. Ther. 19, 1058–1069 (2011).

    Article  CAS  Google Scholar 

  51. Chakrabarty, P. et al. Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain. PLoS ONE 8, e67680 (2013).

    Article  CAS  Google Scholar 

  52. Maguire, C. A. et al. Mouse gender influences brain transduction by intravascularly administered AAV9. Mol. Ther. 21, 1469–1470 (2013).

    Article  Google Scholar 

  53. Chen, Y. H., Chang, M. & Davidson, B. L. Molecular signatures of disease brain endothelia provide new sites for CNS-directed enzyme therapy. Nat. Med. 15, 1215–1218 (2009).

    Article  CAS  Google Scholar 

  54. Powell, S. K., Rivera-Soto, R. & Gray, S. J. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy. Discov. Med. 19, 49–57 (2015).

    PubMed  PubMed Central  Google Scholar 

  55. Franks, K. M. et al. Recurrent circuitry dynamically shapes the activation of piriform cortex. Neuron 72, 49–56 (2011).

    Article  CAS  Google Scholar 

  56. Resendez, S. L. et al. Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat. Protoc. 11, 566–597 (2016).

    Article  CAS  Google Scholar 

  57. Treweek, J. B. et al. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat. Protoc. 10, 1860–1896 (2015).

    Article  CAS  Google Scholar 

  58. Mahmood, T. & Yang, P. C. Western blot: technique, theory, and trouble shooting. N. Am. J. Med. Sci. 4, 429–434 (2012).

    Article  Google Scholar 

  59. Greenbaum, A., Jang, M. J., Challis, C. & Gradinaru, V. Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology? BMC Biol. 15, 87 (2017).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  61. Richardson, D. S. & Lichtman, J. W. Clarifying tissue clearing. Cell 162, 246–257 (2015).

    Article  CAS  Google Scholar 

  62. Gradinaru, V., Treweek, J., Overton, K. & Deisseroth, K. Hydrogel-tissue chemistry: principles and applications. Annu. Rev. Biophys. 47, 355–376 (2018).

    Article  CAS  Google Scholar 

  63. Treweek, J. B. & Gradinaru, V. Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors. Curr. Opin. Biotechnol. 40, 193–207 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  65. Day, R. N. & Davidson, M. W. The fluorescent protein palette: tools for cellular imaging. Chemi. Soc. Rev. 38, 2887–2921 (2009).

    Article  CAS  Google Scholar 

  66. Gray, D. A. & Woulfe, J. Lipofuscin and aging: a matter of toxic waste. Sci. Aging Knowledge Environ. 2005, re1 (2005).

    PubMed  Google Scholar 

  67. Kupferschmidt, D. A., Cody, P. A., Lovinger, D. M. & Davis, M. I. Brain BLAQ: post-hoc thick-section histochemistry for localizing optogenetic constructs in neurons and their distal terminals. Front. Neuroanat. 9, 6 (2015).

    Article  Google Scholar 

  68. Petri, K. et al. Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats. Biotechniques 56, 269–273 (2014).

    Article  CAS  Google Scholar 

  69. Masters, J. R. & Stacey, G. N. Changing medium and passaging cell lines. Nat. Protoc. 2, 2276–2284 (2007).

    Article  CAS  Google Scholar 

  70. JoVE Science Education Database. Anesthesia induction and maintenance. Lab Animal Research. https://www.jove.com/science-education/10263/anesthesia-induction-and-maintenance (2018).

  71. Huang, X. et al. AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications. J. Virol. Methods 193, 270–277 (2013).

    Article  CAS  Google Scholar 

  72. Lock, M. et al. Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum. Gene Ther. 21, 1259–1271 (2010).

    Article  CAS  Google Scholar 

  73. Gage, G. J., Kipke, D. R. & Shain, W. Whole animal perfusion fixation for rodents. J. Vis. Exp. 2012, https://doi.org/10.3791/3564 (2012).

  74. Park, J. J. & Cunningham, M. G. Thin sectioning of slice preparations for immunohistochemistry. J. Vis. Exp., 2007, https://doi.org/10.3791/194 (2007).

  75. Iulianella, A. Cutting thick sections using a vibratome. Cold Spring Harb. Protoc. 2017, https://doi.org/10.1101/pdb.prot094011 (2017).

    Article  Google Scholar 

  76. Hancock, J. F., Cadwallader, K., Paterson, H. & Marshall, C. J. A CAAX or a CAAL motif and a 2nd signal are sufficient for plasma-membrane targeting of ras proteins. EMBO J. 10, 4033–4039 (1991).

    Article  CAS  Google Scholar 

  77. Jovicic, A. et al. Comprehensive expression analyses of neural cell-type-specific miRNAs identify new determinants of the specification and maintenance of neuronal phenotypes. J. Neurosci. 33, 5127–5137 (2013).

    Article  CAS  Google Scholar 

  78. Lagos-Quintana, M. et al. Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739 (2002).

    Article  CAS  Google Scholar 

  79. Fosque, B. F. et al. Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators. Science 347, 755–760 (2015).

    Article  CAS  Google Scholar 

  80. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).

  81. Kim, J. et al. mGRASP enables mapping mammalian synaptic connectivity with light microscopy. Nat. Methods 9, 96–102 (2012).

    Article  CAS  Google Scholar 

  82. Kim, E. J. & Sheng, M. PDZ domain proteins of synapses. Nat. Rev. Neurosci. 5, 771–781 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Fabiszak (W. Freiwald lab, Rockefeller University) and N.C. Flytzanis (V. Gradinaru lab) for the images in Fig. 5d and e, respectively. We also thank M.S. Ladinsky at the Biological and Cryogenic Transmission Electron Microscopy Center (California Institute of Technology (Caltech)) for preparing transmission electron microscopy samples and for acquiring the image shown in Fig. 7b. We are grateful to Y. Lei for help with cloning and K. Lencioni for performing tail-vein injections in rats. The images in Fig. 5a,b were acquired in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. AAV-PHP capsids are dedicated to the memory of Paul H. Patterson (P.H.P.), who passed away during the preparation of the manuscript describing AAV-PHP.B[3]. This work was primarily supported by the National Institutes of Health (NIH) through grants to V.G.: Director’s New Innovator grant DP2NS087949 and PECASE; National Institute on Aging grant R01AG047664; BRAIN grant U01NS090577; SPARC grant OT2OD023848-01 (to V.G. and K.S.); and the Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO). Additional funding included the NSF NeuroNex Technology Hub grant 1707316, and funds from the Curci Foundation, the Beckman Institute, and the Rosen Center at Caltech. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute. R.C.C. was supported by an American Heart Association Postdoctoral Fellowship (17POST33410404). C.C. was funded by the National Institute on Aging (F32AG054101), and P.S.R. was funded by the National Heart, Lung, and Blood Institute (F31HL127974).

Author information

Authors and Affiliations

Authors

Contributions

R.C.C. and V.G. wrote the manuscript with input from all coauthors. S.R.K., K.Y.C., K.B., and B.E.D. optimized the viral production and purification protocols. R.C.C., S.R.K., K.Y.C., C.C., H.K., P.S.R., J.D.T., K.S., B.E.D., and V.G. designed and performed the experiments, analyzed the data, and prepared the figures. M.J.J. analyzed the data in Fig. 2c. V.G. supervised all aspects of the project. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Viviana Gradinaru.

Ethics declarations

Competing interests

The California Institute of Technology has filed patent applications related to (but not on) this work: Recombinant AAV Capsid Protein (US patent no. 9,585,971); Selective Recovery (US patent application no. 15/422,259); Targeting Peptides for Directing Adeno-Associated Viruses (AAVs) (US patent application no. 15/374,596). The authors declare no other competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Deverman, B. E. et al. Nat. Biotechnol. 34, 204–209 (2016): https://doi.org/10.1038/nbt.3440

Chan, K. Y. et al. Nat. Neurosci. 20, 1172–1179 (2017): https://doi.org/10.1038/nn.4593

Supplementary information

Supplementary Tables 1 and 3

Supplementary Table 2

Transfection calculator

Supplementary Table 4

Titration calculator

Supplementary Video 1

Steps 16A and 18: Pouring the density gradients and loading the virus. In Step 16A, use a 2-ml serological pipette to pour the gradients. Next, load the virus (also shown in Step 16B (Supplementary Video 2))

Supplementary Video 2

Steps 16B and 18: Pouring the density gradients and loading the virus. In Step 16B, use a 5-ml serological pipette to pour the gradients. Next, load the virus (also shown in Step16A (Supplementary Video 1))

Supplementary Video 3

Steps 26–27: Collecting the virus

Source Data, Figures 2-5

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Challis, R.C., Ravindra Kumar, S., Chan, K.Y. et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat Protoc 14, 379–414 (2019). https://doi.org/10.1038/s41596-018-0097-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-018-0097-3

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research