Adenovirus Production Services

RAPAd® Technology: High titer virus that's fast and RCA-free

Our RAPAd® expression system enables our team to produce your custom adenovirus particles in as little as 3 weeks. The majority of inserts can be subcloned in a single step in an easy to use shuttle plasmid that contains strong viral promoters for maximal gene expression. If tissue-specific promoters are of interest, we can provide you with a promoterless shuttle to clone in your promoter of choice!

Subcloning options - your clone or ours?

Our team can subclone with cDNA supplied by your lab or we can procure the cDNA for you. Our subcloning services include sequencing the 5’ and 3’ ends or the entire insert if PCR was used as part of the cloning strategy. If you prefer to carry out the subcloning, we can send our shuttle plasmids to you and provide technical assistance with the cloning strategy.

If you need a synthetic product, shRNA, miRNA, mutations, or modifications to your construct, we can do that too. Turnaround time on synthesis is typically a week or so.

Dual expressers - utilizing the E3 region

We can make your novel adenovirus with a reporter gene, or the gene of your choice, in the E3 region of the backbone. This eliminates the need for dual infection and guarantees that each cell receives both expression cassettes. Dual expressers can also be useful when delivering secreted gene products, heterodimers, or multivalent vaccines.

Recombination and amplification

Once confirmed by sequencing or functional analysis, our team will recombine the shuttle plasmid with the appropriate backbone to generate virus particles. We then do the initial transfection and serial amplifications to obtain large scale lysates for purification on CsCl gradients. If you have an adenovirus that you constructed in-house or received from a collaborator, we can utilize your seed stock to amplify and purify the particles.

Purification strategies

Our purification protocol includes 2 rounds of CsCl gradients, dialysis against the buffer of your choice, and then the final formulation. Our typical concentration is 1e12 pts/mL in 1 mL aliquots. The purified particles are suitable for direct injection for in-vivo experiments or cell culture. We can formulate the virus at higher concentrations and in smaller aliquots at the time of manufacture. This is generally used for in vivo experiments that require high virus load in low volumes, and eliminates any freeze-thaw cycles on the finished product. If you have specific buffer requirements, we can formulate your virus prep in the storage buffer of your choice.

Quality control

We QC all virus preparations made in our facility. Prior to shipping, all viruses are tested for E1a sequences using DNA-based PCR to ensure that there are no revertants in the virus preparation. We also confirm PCR data with a cell based assay and determine PFU titer, guaranteeing ≥ 1e10 PFU/mL and no detectable RCA. A 50 uL tester is included with every new lot number, this enables you to check for gene expression while we complete the cell based assays. Final characterization forms are emailed when all assays are complete.

Pre-GMP Production

We can supply high-quality and referenced product for your GMP facility. These services include:

• Plaque purification
• Sterility (FTM and TSB)
• PFU titer
• Particle:PFU ratio

We track all reagents and consumables in the intial production of the virus destined for the clinic. Please inquire.