Abstract
Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques in any well-equipped molecular neurobiology laboratory. With this protocol we typically obtain research batches of 100–200 μL that range in titer from 5 × 1012 to 2 × 1013 genomic copies/mL.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Murlidharan G, Samulski RJ, Asokan A (2014) Biology of adeno-associated viral vectors in the central nervous system. Front Mol Neurosci 7:76
Xiao X, Li J, Samulski RJ (1998) Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72:2224–2232
Grimm D, Kay MA, Kleinschmidt JA (2003) Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6. Mol Ther 7:839–850
Zolotukhin S, Potter M, Zolotukhin I et al (2002) Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28:158–167
Grimm D, Kern A, Rittner K et al (1998) Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 9:2745–2760
Gao GP, Alvira MR, Wang L et al (2002) Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A 99:11854–11859
Mietzsch M, Grasse S, Zurawski C et al (2014) OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1–12 vectors for gene therapy. Hum Gene Ther 25:212–222
Smith RH, Levy JR, Kotin RM (2009) A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells. Mol Ther 17:1888–1896
Grieger JC, Soltys SM, Samulski RJ (2016) Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector. Mol Ther 24:287–297
Hermens WT et al (1999) Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum Gene Ther 10:1885–1891
Zolotukhin S, Byrne BJ, Mason E et al (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6:973–985
Blits B, Derks S, Twisk J et al (2010) Adeno-associated viral vector (AAV)-mediated gene transfer in the red nucleus of the adult rat brain: comparative analysis of the transduction properties of seven AAV serotypes and lentiviral vectors. J Neurosci Methods 185:257–263
Drummond ES, Muhling J, Martins RN et al (2013) Pathology associated with AAV mediated expression of beta amyloid or C100 in adult mouse hippocampus and cerebellum. PLoS One 8:e59166
Ruitenberg MJ, Blits B, Dijkhuizen PA et al (2004) Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury. Neurobiol Dis 15:394–406
Ruitenberg MJ, Eggers R, Boer GJ et al (2002) Adeno-associated viral vectors as agents for gene delivery: application in disorders and trauma of the central nervous system. Methods 28:182–194
Harvey AR, Kamphuis W, Eggers R et al (2002) Intravitreal injection of adeno-associated viral vectors results in the transduction of different types of retinal neurons in neonatal and adult rats: a comparison with lentiviral vectors. Mol Cell Neurosci 21:141–157
Hellstrom M, Ruitenberg MJ, Pollett MA et al (2009) Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection. Gene Ther 16:521–532
You SW, Hellström M, Pollett MA et al (2016) Large-scale reconstitution of a retina-to-brain pathway in adult rats using gene therapy and bridging grafts: an anatomical and behavioral analysis. Exp Neurol 279:197–211
Muhlfriedel R, Michalakis S, Garcia Garrido M et al (2013) Optimized technique for subretinal injections in mice. Methods Mol Biol 935:343–349
Qi Y, Dai X, Zhang H et al (2015) Trans-corneal subretinal injection in mice and its effect on the function and morphology of the retina. PLoS One 10:e0136523
Acknowledgment
We acknowledge the support of the International Spinal Research Trust (STR 111), a ZonMW TOP grant, and the ZonMW dementia research and innovation program Memorabel (grant no. 733050106) for financial support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Verhaagen, J. et al. (2018). Small Scale Production of Recombinant Adeno-Associated Viral Vectors for Gene Delivery to the Nervous System. In: Boon, C., Wijnholds, J. (eds) Retinal Gene Therapy. Methods in Molecular Biology, vol 1715. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7522-8_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7522-8_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7521-1
Online ISBN: 978-1-4939-7522-8
eBook Packages: Springer Protocols