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: 6.23). 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.

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    • "Recombinant AAVs of serotype 1 and 2 were generated as described [27] and purified by AVB Sepharose affinity chromatography [43]. For each virus the genomic titer was determined by real-time PCR using primers and a probe against WPRE: WPRE forward primer: 5′-TGCCCGCTGCTGGAC-3′; WPRE reverse primer: 5′-CCGACAACACCACGGAATTG-3′; and WPRE probe: 5′-CAGTGCCCAACAGCC-3′ (1.0–6.0 × 1012 viral genomes (vg)/ml, TaqMan Assay, Applied Biosystems). "
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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
    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.
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      [Show abstract] [Hide abstract]
      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.
      Full-text · Article · Jul 2009 · Molecular Therapy