Custom Helper Dependent Adenovirus Constructs

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Description

The VVC HDAd system is based on the system created at Baylor College of Medicine.  This system uses various sized pseudo-genomes filled with un-transcripted stuffer sequence into which the transgene cassette is inserted.  This creates a final genome that is between 31kb and 37kb long, the most efficient and stable sizes for the packaging of adenoviral particles. The system is extremely flexible and able to express multiple gene cassettes driven by multiple promoters in a single virus.  Once a genome with a transgene cassette has been cloned, it is transfected into 293 cells that express the Cre recombinase protein.  After transfection the helper virus is added and the helper dependent vector is allowed to grow.  The helper virus provides all the needed viral proteins while the transfected genomes are packaged into the viral particles.  The HDAd is then amplified with more helper viruses being added at each amplification step.  The final amplification is then purified by double CsCl gradients, dialyzed to our A-195 storage buffer, and frozen at -80C for long term storage.

Characteristics

  • Episomal gene expression.
  • Infects dividing and non-dividing cells.
  • Transient high-level protein expression.
  • Very large capacity, accommodates inserts of up to 30kb.
  • High viral titer can be produced: 1 x 1010pfu/ml to 1 x 1011pfu/ml.
  • Lower host immune response due to no viral proteins being expressed in the helper dependent virions.

Disadvantages and Adverse Effects

  • The viral particles themselves will cause an immune response, but it is delayed and less intense than a first generation adenovirus.
  • Viral particles can be neutralized by the host immune response.
  • Transient expression of the transgene due to lack of integration into the host genome. .
  • Helper virus contamination is present in every preparation.  Every batch is tested for the contamination level.  The helper virus is a first generation adenovirus that does not express any transgenes, but does express viral proteins.

Producing New Helper Dependent Adenoviral Vector from a Shuttle Plasmid

Cloning of GOI into HDAd5 backbone, Transfection, amplification, purification and titer

Shuttle

A cloning/expression shuttle vector is provided free of charge except for shipping after a Material Transfer Agreement is signed. 

Sample Required

30μg of Adenovirus plasmid expressing the gene of interest at a concentration greater than 0.25μg/μl. We also require a sequence, map, and specific gene of interest information in accordance with our biosafety protocols.

Timeline

10-14 weeks from the time the plasmid is received.

Service Details

  • Cloning of GOI cassette into the appropriate HDAd5 backbone plasmid
  • Transfection and recombination
  • Large-scale amplification
  • Purification
  • With every new lot an infectious titer (infectous genome units igu/ml) is provided.
  • Each lot is checked for wild-type virus contamination (RCA) and helper virus contamination.

Material Provided

1 ml of virus in a 1mL aliquot at a concentration of at least 1x1010 pfu/ml.

Vehicle

Adenovirus vectors are re-suspended in A 195 buffer (see reference below).

Cost

CsCl banding: $2,734 per construct (plus shipping and handling)

Additional 1ml aliquots can be purchased at $1051 per ml. These may take 8-10 weeks for a new prep to be made.

References

A195 Buffer: Evans RK, Nawrocki DK, Isopi LA, Williams DM, Casimiro DR, Chin S, Chen M, Zhu DM, Shiver JW, Volkin DB. Development of stable liquid formulations for adenovirus-based vaccines. J Pharm Sci. 2004 Oct;93(10):2458-75.·         

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.