Helper Dependent Adenovirus Products

CsCl banding $3,852 per construct for non-profit institutions.

Adenoviral vectors are a versatile research tool with a number of beneficial features. They infect dividing and non-dividing cells, have transient, high-level protein expression, and are also easily amplified and purified to high concentrations (1x10¹⁰IGU/mL-1x10¹¹IGU/mL). The original, first generation adenoviral vectors (FGAd) used deletions in the E1 and E3 regions to create space in the adenoviral genome for foreign genes as well as make them replication incompetent. These vectors are extremely useful but have many drawbacks and, over the years, new generations of adenoviral vectors were created with more of the viral genome removed.

Photo by Russell Kightley

Recently the final generation of “gutless” adenoviral vectors has been created. This helper dependent adenovirus (HDAd) has had all viral genes removed. The only viral sequences remaining include the adenoviral ITRs, the adenoviral packaging signal, and a small, un-transcribed portion of the E4 region. This creates a system that can accommodate a very large transgene capacity (up to 30kb), expresses no viral proteins, but still retains the advantages of adenoviral vectors such as the broad tissue tropism and high transgene expression. In order to construct HDAd particles, the HDAd viral genome is transfected into 293 cells that stably express Cre recombinase, and co-infected with a helper virus (an FGAd that has a floxed packaging signal). The HDAd is amplified by sequential passage onto fresh packaging 293 cells with additional helper virus added with each passage. The helper virus provides all of the viral proteins in trans that are required for proper particle construction and packaging of the HDAd. The HDAds are purified using density gradient centrifugation and ion exchange techniques commonly employed for early generation adenovirus. Because the helper virus’ packaging signal is floxed, the majority of helper virus genomes have their packaging signals excised out and thus are not successfully packaged into viral particles while the helper dependent genomes are packaged into active particles. All HDAd preps are subjected to quality control including; sterility testing, quantify the particle to pfu ratio, titer by ddPCR, assay for replication-competent adenovirus, and assessment of fluorophore or enzymatic reporter activity as appropriate.

The VVC HDAd system is based on the system provided by Dr. Brendan Lee at Baylor College of Medicine. The plasmid was modified by Samuel M. Young at the University of Iowa. This system uses various sized pseudo-genomes filled with un-transcribed stuffer sequence into which the transgene cassette is inserted. This creates a final genome that is between 32kb and 37kb long, the most efficient and stable size range for packaging of adenoviral particles. The system is extremely flexible and able to express multiple gene cassettes driven by multiple promoters in a single virus.

Characteristics

Episomal gene expression; therefore little risk of insertional mutagenesis.
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 10¹⁰IGU/ml to 1 x 10¹¹IGU/ml.
Lower host immune response due to no viral proteins being expressed by the helper dependent virions.

Disadvantages and Adverse Effects

The viral particles themselves may cause an immune response, but it is typically 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.
Low level helper virus contamination is present in every preparation. The helper virus is a first generation adenovirus that does not express any transgenes, but does express viral proteins that may be toxic.

Learn More About Helper Dependent Adenovirus

In-stock Helper-Dependent Adenovirus vectors are offered in 25ul aliquots.

If there is a control vector that you would like to purchase and is not on the list below, please inquire to vectors@uiowa.edu as we are attempting to further increase our catalog of HDAd Control Vectors.

Interested in Ordering?

In-Stock Orders

You can browse/order our Helper-Dependent Adenovirus In-Stock Products here.

* Listed prices are for non-profit customers only, additional fees may apply.

** Please note, if you have used our iLabs system in the past, you will need to create a new account to order in-stock products from our new system.

Custom Orders

*** Custom vector orders still need to be submitted in iLabs.

Description

The VVC HDAd system is based on the system created at Baylor College of Medicine. The plasmid was modified by Samuel M. Young. 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 Hepes/Sucrose or A195 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 10¹⁰IGU/ml to 1 x 10¹¹IGU/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 for a nominal fee and shipping cost after a Material Transfer Agreement is signed for academic customers only.

Sample Required

100μg of HDAd plasmid expressing the gene of interest at a concentration greater than 0.25μg/μl. We require FULL sequencing of the plasmid. SnapGene files are preferred that show the sequence with an annotated map and sequence alignment. We require specific gene of interest information in accordance with our biosafety protocols. Please confirm that there are only two PmeI sites flanking the bacterial elements for viral production. PmeI sites in your GOI cassette will need to be modified prior to submission.

Timeline

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

Service Details

Cloning of GOI cassette into the appropriate HDAd5 backbone plasmid can be arranged through Genscript.
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

The yield from a 30 plate prep with a minimum of 500ul of virus in 25ul aliquots at a concentration of at least 1x10¹⁰ IGU/ml.

Vehicle

Helper Dependent Adenovirus vectors are re-suspended in Hepes/Sucrose buffer. A195 buffer may also be used upon request.

Cost

CsCl banding: $3,150 per construct (plus shipping and handling)

Additional 500ul aliquots can be purchased at $2,700 per 500ul. These may take 8-10 weeks for a new prep to be made.

References

Montesinos M.S., Satterfield R., Young S.M. (2016) Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications. In: Schwartzbach S., Skalli O., Schikorski T. (eds) High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol 1474. Humana Press, New York, NY

https://link.springer.com/protocol/10.1007%2F978-1-4939-6352-2_5

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.

Shuttle plasmids are provided for a nominal fee and shipping charges when intended for virus production at the University of Iowa Viral Vector Core.