Helper Dependent Adenovirus (Register and log in to see discounts)

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 (1x10¹⁰pfu/mL-1x10¹¹pfu/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.  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 QPCR, 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.  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 10¹⁰pfu/ml to 1 x 10¹¹pfu/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.