Custom Adeno-Associated Virus Constructs

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Please view our ordering information for custom constructs to learn how to register for an account, place orders for in-stock vectors and make payment through our online system.

Pricing

New Custom Vectors: $2,640 - $3,000 for 500ul purified virus in 100ul aliquots, depending on production method.

*Re-order Custom Vectors: $2,376 - $3,000 for 500ul purified virus in 100ul aliquots, depending on production method.

Description

We offer two systems for AAV production.

 

 

Triple Transfection System- $3,000

The vectors are prepared by a triple plasmid transfection in HEK-293 cells. The cis-acting AAV plasmid carries the AAV expression cassette containing the gene of interest flanked by the AAV2 inverted terminal repeats (ITRs). A second plasmid provides the genes for the structural proteins, rep and cap. The third pHelper plasmid provides the adenovirus helper genes. The triple transfection system works with any AAV plasmid from any source. 

Material provided: 500 µl in 100 µl aliquots (Titer: 1x1012 to 1x1013vg/ml, viral genomes/ml). Additional aliquots may be ordered with a minimum order of 500ul.

Serotypes: AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/6.2, AAV2/8, AAV2/9, AAV2/DJ, AAV2/rh10. Modified capsids are considered on a case by case basis. 

Shuttle: A cloning/expression AAV shuttle vector is provided after a Material Transfer Agreement is signed. Other outside pAAV shuttle vectors are also compatible with this system.

Sample required: 1500 µg of AAV plasmid expressing the gene of interest at a concentration greater than 0.25 µg/µl.

Timeline: 3-4 weeks from the time the plasmid is received depending on the current queue and serotype.

Services include:

  • Transfection.
  • Large-scale amplification of AAV vectors.
  • Purification performed using a variety of methods depending on the serotype. Most often this is an iodixanol gradient, followed by either an ion-exchange filter membrane or affinity chromatography. CsCl purification is also commonly used.
  • Quality control assays include a physical titer (vg/ml) by QPCR, transduced titer by FACs when appropriate, and silver stain to further assess virus titer and purity.
  • Vehicle: The vectors are provided in F68/PBS

Baculovirus System- $2,640

The vectors are prepared by a dual baculovirus infection in SF-9 insect cells. The cis-acting AAV baculovirus carries the AAV expression cassette containing the gene of interest flanked by the AAV inverted terminal repeats (ITR). The genes for the structural proteins rep and cap and the adenovirus helper genes are encoding the proteins in trans, and are not likely to be packaged inappropriately in the virus particles. The baculovirus system is compatible with our shuttle plasmids based on the pFastBac backbone engineered with AAV ITRs by Dr. Robert Kotin.



Baculovirus production is robust, easily repeatable, and can be scaled up. We recommend this system for higher titer, large volume, and certain serotype requests.  The baculovirus system is recommended for ITR to ITR cassettes pushing the limit of the ~4.7kb AAV packaging capacity as the large bacmid backbone is unlikely to be packaged inappropriately. The baculovirus system is also suggested for genes that may be toxic or effect cell growth as most promoters are not active in the insect cells. The disadvantages of the baculovirus system are that it takes a little longer and is not compatible with all available AAV vectors.

Material provided: 500μl in 100μl aliquots (Titer: 1x1012 to 1x1013vg/ml). Additional aliquots may be ordered with a minimum order of 500ul.

Serotypes: AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, AAV2/9. (AAV2/6.2, AAV2/DJ, AAV2/rh10 only offered in the Triple Transfection system). Modified capsids are considered on a case by case basis. Please contact us for Rep/Cap cloning plasmids for the baculovirus system. 

Shuttle: A cloning/expression AAV shuttle vector compatible with this system based on the pFastBac vector with transposable elements is provided after a Material Transfer Agreement is signed. Outside pAAV shuttle plasmids may not be compatible with this system.

Sample required: 10μg of AAV plasmid expressing the gene of interest at a concentration greater than 0.25ug/ul.

Timeline: 5-6 weeks from the time the plasmid is received depending on the current queue and serotype.

Services include:

  • Transposition.
  • Transfection for construction of baculovirus stock.
  • Titer of P1 baculovirus stock. Amplification to P2 baculovirus stock if needed.
  • Dual baculovirus infection for large-scale amplification of AAV vectors.
  • Purification performed using a variety of methods depending on the serotype. Most often this is an iodixanol gradient, followed by either an ion-exchange filter membrane or affinity chromatography. CsCl purification is also commonly used.
  • Quality control assays include a physical titer (vg/ml) by QPCR, transduced titer by FACs when appropriate, and silver stain to further assess virus titer and purity.
  • Vehicle: The vectors are provided in F68/PBS. 

References:

Urabe M, Ding C, Kotin RM “Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors” Human Gene Therapy 13:1935-1943 (November 1, 2002).


Plasmid Quality Control Upon Submission

Every effort is made to deliver a high titer, high quality viral vector. Viral vector preps are repeated a second time if they do not pass our quality control at no charge, provided that the plasmid submitted meets several criteria. We require gene information, a sequence, map, and ITR digest upon submission of a new project. Preps will not be repeated and charges will apply to the first prep regardless of outcome in the following situations:

  • Constructs that are over packaging capacity. This is ~4.7 kb from ITR to ITR for single stranded AAV and ~2.2 kb from ITR to ITR for self-complementary AAV.
  • Where the shuttle backbone size is equal to or smaller than the ITR to ITR transgene. 
  • Involve a novel or modified capsid.
  • Constructs where the sequence does not match the ITR digest. We highly suggest that you perform your own ITR digest before submission of a project. This is especially important on plasmids you may obtain from another lab. This diagnostic digest can be performed by our lab for a fee.

Investigators will be notified when constructs are flagged as problematic and we will discuss the pros, cons, and finances of proceeding with the project. Sometimes the gamble to proceed with plasmids that fall under these criteria are warranted and we will do our best to guide you in the decision process.

Despite best efforts, some genes of interest may confer cellular toxicity that results in lower vector titers. We will notify investigators of progress and problems and discuss the next step if problems are noted.