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Lab-Scale Production of Recombinant Adeno-Associated Viruses (AAV) for Expression of Optogenetic Elements

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Photoswitching Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2173))

Abstract

Optogenetics, that is, the use of photoswitchable/−activatable moieties to precisely control or monitor the activity of cells and genes at unprecedented spatiotemporal resolution, holds tremendous promise for a wide array of applications in fundamental and clinical research. To fully realize and harness this potential, the availability of gene transfer vehicles (“vectors”) that are easily produced and that allow to deliver the essential components to desired target cells in an efficient manner is key. For in vivo applications, it is, moreover, important that these vectors exhibit a high degree of cell specificity in order to reduce the risk of adverse side effects in off-targets and to minimize manufacturing costs. Here, we describe a set of basic protocols for the cloning, production, purification, and quality control of a particular vector that can fulfill all these requirements, that is, recombinant adeno-associated viruses (AAV). The latter are very attractive owing to their apathogenicity, their compatibility with the lowest biosafety level 1 conditions, their occurrence in multiple natural variants with distinct properties, and their exceptional amenability to engineering of the viral capsid and genome. The specific procedures reported here complement alternative protocols for AAV production described by others and us before, and, together, should enable any laboratory to generate these vectors on a small-to-medium scale for ex vivo or in vivo expression of optogenetic elements.

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References

  1. Deisseroth K (2015) Optogenetics: 10 years of microbial opsins in neuroscience. Nat Neurosci 18:1213–1225. https://doi.org/10.1038/nn.4091

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Grosenick L, Marshel JH, Deisseroth K (2015) Closed-loop and activity-guided optogenetic control. Neuron 86:106–139. https://doi.org/10.1016/j.neuron.2015.03.034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bubeck F, Hoffmann MD, Harteveld Z et al (2018) Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. Nat Methods 15:924–927. https://doi.org/10.1038/s41592-018-0178-9

    Article  CAS  PubMed  Google Scholar 

  4. Grimm D, Zolotukhin S (2015) E pluribus Unum: 50 years of research, millions of viruses, and one goal--tailored acceleration of AAV, evolution. Mol Ther 23:1819–1831. https://doi.org/10.1038/mt.2015.173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kotterman MA, Schaffer DV (2014) Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet 15:445–451. https://doi.org/10.1038/nrg3742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Buchholz CJ, Friedel T, Buning H (2015) Surface-engineered viral vectors for selective and cell type-specific gene delivery. Trends Biotechnol 33:777–790. https://doi.org/10.1016/j.tibtech.2015.09.008

    Article  CAS  PubMed  Google Scholar 

  7. Grimm D, Buning H (2017) Small but increasingly mighty: latest advances in AAV vector research, design, and evolution. Hum Gene Ther 28:1075–1086. https://doi.org/10.1089/hum.2017.172

    Article  CAS  PubMed  Google Scholar 

  8. Lee EJ, Guenther CM, Suh J (2018) Adeno-associated virus (AAV) vectors: rational design strategies for capsid engineering. Curr Opin Biomed Eng 7:58–63. https://doi.org/10.1016/j.cobme.2018.09.004

    Article  PubMed  PubMed Central  Google Scholar 

  9. Russell S, Bennett J, Wellman JA et al (2017) Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390:849–860. https://doi.org/10.1016/S0140-6736(17)31868-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ganjawala TH, Lu Q, Fenner MD, Abrams GW, Pan ZH (2019) Improved CoChR variants restore visual acuity and contrast sensitivity in a mouse model of blindness under ambient light conditions. Mol Ther 27:1195–1205. https://doi.org/10.1016/j.ymthe.2019.04.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Khabou H, Garita-Hernandez M, Chaffiol A et al (2018) Noninvasive gene delivery to foveal cones for vision restoration. JCI Insight 3:96029. https://doi.org/10.1172/jci.insight.96029

    Article  PubMed  Google Scholar 

  12. Chaffiol A, Caplette R, Jaillard C et al (2017) A new promoter allows Optogenetic vision restoration with enhanced sensitivity in macaque retina. Mol Ther 25:2546–2560. https://doi.org/10.1016/j.ymthe.2017.07.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sengupta A, Chaffiol A, Mace E et al (2016) Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina. EMBO Mol Med 8:1248–1264. https://doi.org/10.15252/emmm.201505699

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pan ZH, Lu Q, Bi A, Dizhoor AM, Abrams GW (2015) Optogenetic approaches to restoring vision. Annu Rev Vis Sci 1:185–210. https://doi.org/10.1146/annurev-vision-082114-035532

    Article  PubMed  Google Scholar 

  15. Mace E, Caplette R, Marre O et al (2015) Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV restores ON and OFF visual responses in blind mice. Mol Ther 23:7–16. https://doi.org/10.1038/mt.2014.154

    Article  CAS  PubMed  Google Scholar 

  16. Cronin T, Vandenberghe LH, Hantz P et al (2014) Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter. EMBO Mol Med 6:1175–1190. https://doi.org/10.15252/emmm.201404077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Byrne LC, Khalid F, Lee T et al (2013) AAV-mediated, optogenetic ablation of Muller glia leads to structural and functional changes in the mouse retina. PLoS One 8:e76075. https://doi.org/10.1371/journal.pone.0076075

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Fomicheva A, Zhou C, Sun QQ, Gomelsky M (2019) Engineering adenylate cyclase activated by near-infrared window light for mammalian Optogenetic applications. ACS Synth Biol 8:1314–1324. https://doi.org/10.1021/acssynbio.8b00528

    Article  CAS  PubMed  Google Scholar 

  19. Fortuna MG, Kugler S, Hulsmann S (2019) Probing the function of glycinergic neurons in the mouse respiratory network using optogenetics. Respir Physiol Neurobiol 265:141–152. https://doi.org/10.1016/j.resp.2018.10.008

    Article  CAS  PubMed  Google Scholar 

  20. Keaveney MK, Tseng HA, Ta TL, Gritton HJ, Man HY, Han X (2018) A MicroRNA-based gene-targeting tool for virally Labeling interneurons in the rodent cortex. Cell Rep 24:294–303. https://doi.org/10.1016/j.celrep.2018.06.049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Mondello SE, Sunshine MD, Fischedick AE et al (2018) Optogenetic surface stimulation of the rat cervical spinal cord. J Neurophysiol 120:795–811. https://doi.org/10.1152/jn.00461.2017

    Article  CAS  PubMed  Google Scholar 

  22. Redchuk TA, Karasev MM, Omelina ES, Verkhusha VV (2018) Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells. Chembiochem 19:1334–1340. https://doi.org/10.1002/cbic.201700642

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Watanabe M, Narita M, Hamada Y et al (2018) Activation of ventral tegmental area dopaminergic neurons reverses pathological allodynia resulting from nerve injury or bone cancer. Mol Pain 14:1744806918756406. https://doi.org/10.1177/1744806918756406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yazdan-Shahmorad A, Tian N, Kharazia V et al (2018) Widespread optogenetic expression in macaque cortex obtained with MR-guided, convection enhanced delivery (CED) of AAV vector to the thalamus. J Neurosci Methods 293:347–358. https://doi.org/10.1016/j.jneumeth.2017.10.009

    Article  PubMed  Google Scholar 

  25. El-Shamayleh Y, Kojima Y, Soetedjo R, Horwitz GD (2017) Selective optogenetic control of Purkinje cells in monkey cerebellum. Neuron 95:51–62 e54. https://doi.org/10.1016/j.neuron.2017.06.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Mendoza SD, El-Shamayleh Y, Horwitz GD (2017) AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses. J Neurophysiol 117:2004–2013. https://doi.org/10.1152/jn.00780.2016

    Article  PubMed  PubMed Central  Google Scholar 

  27. Jin L, Lange W, Kempmann A et al (2016) High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses. J Biotechnol 233:171–180. https://doi.org/10.1016/j.jbiotec.2016.07.001

    Article  CAS  PubMed  Google Scholar 

  28. Gompf HS, Budygin EA, Fuller PM, Bass CE (2015) Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals. Front Behav Neurosci 9:152. https://doi.org/10.3389/fnbeh.2015.00152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Osawa S, Iwasaki M, Hosaka R et al (2013) Optogenetically induced seizure and the longitudinal hippocampal network dynamics. PLoS One 8:e60928. https://doi.org/10.1371/journal.pone.0060928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ambrosi CM, Sadananda G, Han JL, Entcheva E (2019) Adeno-associated virus mediated gene delivery: implications for scalable in vitro and in vivo cardiac optogenetic models. Front Physiol 10:168. https://doi.org/10.3389/fphys.2019.00168

    Article  PubMed  PubMed Central  Google Scholar 

  31. Keppeler D, Merino RM, de la Morena DL et al (2018) Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos. EMBO J 37:e99649. https://doi.org/10.15252/embj.201899649

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Duarte MJ, Kanumuri VV, Landegger LD et al (2018) Mol Ther 26:1931–1939. https://doi.org/10.1016/j.ymthe.2018.05.023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Vajanthri KY, Yadav P, Poddar S, Mahto SK (2018) Development of optically sensitive liver cells. Tissue Cell 52:129–134. https://doi.org/10.1016/j.tice.2018.05.004

    Article  CAS  PubMed  Google Scholar 

  34. Yu L, Zhou L, Cao G et al (2017) Optogenetic modulation of cardiac sympathetic nerve activity to prevent ventricular arrhythmias. J Am Coll Cardiol 70:2778–2790. https://doi.org/10.1016/j.jacc.2017.09.1107

    Article  PubMed  Google Scholar 

  35. Richter C, Bruegmann T (2019) No light without the dark: perspectives and hindrances for translation of cardiac optogenetics. Prog Biophys Mol Biol. https://doi.org/10.1016/j.pbiomolbio.2019.08.013

  36. Vogt CC, Bruegmann T, Malan D, Ottersbach A, Roell W, Fleischmann BK, Sasse P (2015) Systemic gene transfer enables optogenetic pacing of mouse hearts. Cardiovasc Res 106:338–343. https://doi.org/10.1093/cvr/cvv004

    Article  CAS  PubMed  Google Scholar 

  37. Fakhiri J, Nickl M, Grimm D (2019) Rapid and simple screening of CRISPR guide RNAs (gRNAs) in cultured cells using Adeno-associated viral (AAV) vectors. Methods Mol Biol 1961:111–126. https://doi.org/10.1007/978-1-4939-9170-9_8

    Article  CAS  PubMed  Google Scholar 

  38. Sanmiguel J, Gao G, Vandenberghe LH (2019) Quantitative and digital droplet-based AAV genome titration. Methods Mol Biol 1950:51–83. https://doi.org/10.1007/978-1-4939-9139-6_4

    Article  CAS  PubMed  Google Scholar 

  39. Grimm D, Kern A, Pawlita M, Ferrari F, Samulski R, Kleinschmidt J (1999) Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther 6:1322–1330. https://doi.org/10.1038/sj.gt.3300946

    Article  CAS  PubMed  Google Scholar 

  40. Kuck D, Kern A, Kleinschmidt JA (2007) Development of AAV serotype-specific ELISAs using novel monoclonal antibodies. J Virol Methods 140:17–24. https://doi.org/10.1016/j.jviromet.2006.10.005

    Article  CAS  PubMed  Google Scholar 

  41. McCarty DM, Monahan PE, Samulski RJ (2001) Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther 8:1248–1254. https://doi.org/10.1038/sj.gt.3301514

    Article  CAS  PubMed  Google Scholar 

  42. Carvalho LS, Turunen HT, Wassmer SJ, Luna-Velez MV, Xiao R, Bennett J, Vandenberghe LH (2017) Evaluating efficiencies of dual AAV approaches for retinal targeting. Front Neurosci 11:503. https://doi.org/10.3389/fnins.2017.00503

    Article  PubMed  PubMed Central  Google Scholar 

  43. Deverman BE, Pravdo PL, Simpson BP et al (2016) Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34:204–209. https://doi.org/10.1038/nbt.3440

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We kindly acknowledge support of this work by the German Research Foundation (DFG, EXC81 [Cluster of Excellence CellNetworks]; SFB1129 [Collaborative Research Center 1129] to D.G. [TP2/16, Projektnummer 240245660]; and TRR179 [Transregional Collaborative Research Center 179] to D.G. [TP18, Projektnummer 272983813]). D.G. is moreover grateful for funding and other support from the MYOCURE project and the ERA-NET Neuron project KARTLE. MYOCURE has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 667751. D.G. is also thankful for support by the German Center for Infection Research (DZIF, BMBF [TTU-HIV 04.803 and TTU-HIV 04.815]). Finally, we apologize to all authors whose work on optogenetic elements and/or AAV vector technologies we unfortunately had to omit due to space constraint.

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Authors and Affiliations

  1. Department of Infectious Diseases/Virology, Heidelberg University Hospital, Heidelberg, Germany

    Janina Haar, Chiara Krämer & Dirk Grimm

  2. BioQuant Center, University of Heidelberg, Heidelberg, Germany

    Janina Haar & Chiara Krämer

  3. German Center for Infection Research (DZIF) and German Center for Cardiovascular Research (DZHK), Heidelberg, Germany

    Dirk Grimm

Authors
  1. Janina Haar
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  2. Chiara Krämer
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  3. Dirk Grimm
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Correspondence to Dirk Grimm .

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  1. IPMB and BioQuant, Heidelberg University, Heidelberg, Baden-Württemberg, Germany

    Dominik Niopek

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Haar, J., Krämer, C., Grimm, D. (2020). Lab-Scale Production of Recombinant Adeno-Associated Viruses (AAV) for Expression of Optogenetic Elements. In: Niopek, D. (eds) Photoswitching Proteins . Methods in Molecular Biology, vol 2173. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0755-8_5

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  • DOI: https://doi.org/10.1007/978-1-0716-0755-8_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0754-1

  • Online ISBN: 978-1-0716-0755-8

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