Article

A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells

Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
Molecular Therapy (Impact Factor: 6.43). 07/2009; 17(11):1888-96. DOI: 10.1038/mt.2009.128
Source: PubMed

ABSTRACT Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 x 10(4) purified rAAV particles per cell (range: 3.7 x 10(4) to 9.6 x 10(4)) from suspension cultures of recombinant baculovirus-infected insect cells.

0 Followers
 · 
225 Views
  • Source
    • "24. Hauck B, Murphy SL, Smith PH, et al. 2009. Undetectable transcription of cap in a clinical AAV vector: implications for preformed capsid in immune responses. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cell yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based, scalable production system for high per particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.
    Human gene therapy 07/2015; DOI:10.1089/hum.2015.050 · 3.62 Impact Factor
  • Source
    • "With the exception of AAV.ZFN EB and AAV.ZFN BA , all AAV vectors were produced and purified as previously described (Gellhaus et al., 2010). Vectors AAV.ZFN EB and AAV.ZFN BA were generated using the baculovirus system, as described in detail elsewhere (Smith et al., 2009). AAV vector titers were determined by quantitative real-time PCR (LightCycler, Roche) using SYBR Green (DyNAmo TM Capillary SYBR Green, Finnzymes) and appropriate primers, as previously described (Gellhaus et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Zinc-finger nucleases (ZFNs) have become a valuable tool for targeted genome engineering. Based on the enzyme's ability to create a site-specific DNA double-strand break, ZFNs promote genome editing by activating the cellular DNA damage response, including homology-directed repair (HDR) and nonhomologous end-joining. The goal of this study was (i) to demonstrate the versatility of combining the ZFN technology with a vector platform based on adeno-associated virus (AAV), and (ii) to assess the toxicity evoked by this platform. To this end, human cell lines that harbor enhanced green fluorescence protein (EGFP) reporters were generated to easily quantify the frequencies of gene deletion, gene disruption, and gene correction. We demonstrated that ZFN-encoding AAV expression vectors can be employed to induce large chromosomal deletions or to disrupt genes in up to 32% of transduced cells. In combination with AAV vectors that served as HDR donors, the AAV-ZFN platform was utilized to correct a mutation in EGFP in up to 6% of cells. Genome editing on the DNA level was confirmed by genotyping. Although cell cycle profiling revealed a modest G2/M arrest at high AAV-ZFN vector doses, platform-induced apoptosis could not be detected. In conclusion, the combined AAV-ZFN vector technology is a useful tool to edit the human genome with high efficiency. Because AAV vectors can transduce many cell types relevant for gene therapy, the ex vivo and in vivo delivery of ZFNs via AAV vectors will be of great interest for the treatment of inherited disorders.
    Human gene therapy 12/2011; 23(3):321-9. DOI:10.1089/hum.2011.140 · 3.62 Impact Factor
  • Source
    • "Baculovirus-derived influenza virus vaccines, either as protein subunits or as VLPs, may follow in the near future (Cox and Hollister, 2009; van Oers, 2006; Wen et al., 2009). Baculovirus expression technology is also being used for the production of adeno-associated virus (AAV) vectors carrying transgenes for gene therapy against for instance muscle diseases (Smith et al., 2009; Urabe et al., 2002) or familial lipoprotein lipase deficiency, for which product registration has been initiated by the European Medical Agency (EMA) in January 2010 (AMT's marketing authorization application for Glybera 1 , http://www.amtbv.com/news-and-events/ news-2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel baculovirus-based protein expression strategy was developed to produce recombinant proteins in insect cells without contaminating baculovirus virions. This novel strategy greatly simplifies the downstream processing of biopharmaceuticals produced in insect cells. The formation of these virions is prevented by deletion of a baculovirus gene essential for virion formation. The deletion is trans-complemented in a transgenic insect cell line in which the baculovirus seed stock is produced. The Autographa californica multicapsid nucleopolyhedrovirus vp80 gene was selected for this purpose, as absence of VP80 prevented the formation of budded virus as well as occlusion-derived virus, while foreign gene expression was not affected. Sf9 insect cells were engineered to functionally complement the vp80 deletion in the expression vector virus during seed stock production. The trans-complemented vp80-deletion baculovirus seed produced an amount of recombinant protein similar to that produced with conventional baculovirus vectors but without contaminating virions. This novel expression method obviates the need to purify the virions away from the biopharmaceuticals.
    Biotechnology and Bioengineering 05/2011; 108(5):1056-67. DOI:10.1002/bit.23028 · 4.16 Impact Factor
Show more

Questions & Answers about this publication

  • Mario Mietzsch added an answer in Adeno-associated Virus:
    Simple and effective method for purifying rAAV crude lysate for in vivo infection?
    I have produced recombinant AAV virus using Agilent's AAV Helper-free kit (transfected 293 cells with pHelper, pRep/Cap, and plasmid with LTR-flanked gene of interest, freeze/thaw cells 72hr post-transfection, collect cell lysate).

    Now, I would like to do an intra-vitreal injection in mice to infect the retinal ganglion cells. Is purification/concentration always required before in vivo use? If so, I'd like to know what the easiest/cheapest/most simple method is currently. I have read that you can do CsCl or iodixanol gradients (Zolotukhin et al, 1999). Is one easier or simpler than the other? Do both need to be followed by some type of purification and/or concentration step? It seems like 1999 was a long time ago and there must have been improvements in the method since then. Could anybody provide advice and/or a detailed protocol?
    Mario Mietzsch · Charité Universitätsmedizin Berlin
    In my opinion, AVB sepharose affinity chromatography is the easiest way to purify AAV from crude lysates.
    http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/de/GELifeSciences/28411201

    We are using it on a HPLC machine but it can also be without it.