RAPAd® Technology - Fast Adenovirus Production
High titer virus that's fast and RCA-free
RAPAd® adenovirus production
RAPAd® technology was pioneered by our scientists while at the University of Iowa. In contrast to traditional homologous recombination virus production using Ad5 mutant dl309 which can take up to 18 weeks, the RAPAd® system eliminates the need for multiple plaque purifications and for bacterial in vitro recombination. This streamlined system can prepare adenovirus in as little as 3 weeks!
The RAPAd® system removes these issues by using an adenoviral backbone incapable of packaging and replication on its own. Read our scientists' seminal paper published in Gene Therapy on the development of the RAPAd® technique here. (US Patent No. 6,830,920 B2)
RCA (replication competent adenovirus E1+) "wild-type" contamination or revertants can occur when amplifying adenovirus in HEK-derived cells. Since this is a rare but possible event, we screen every prep for RCA using PCR and cell-based assays.
RAPAd® vs. AdEasy adenovirus production timeline
RAPAd® technology can deliver your virus construct in 3-6 weeks!
RAPAd® innovations to eliminate wild-type contamination
The RAPAd® system uses a two-plasmid transfection: a viral shuttle plasmid and a novel adenovirus backbone. The viral shuttle plasmid contains the gene of interest and a portion of the viral genome sequences. Read more about our shuttle plasmids here. The viral backbone contains the remaining viral sequences including a region identical to the viral shuttle plasmid.
The true innovation in the RAPAd® system is in the novel backbone. This plasmid is engineered with left-hand ITR, E1a, and partial E1b sequence deletions, which greatly reduces wild-type occurrence in the final vector preparations. This eliminates the need for time-consuming plaque purification steps. For more information on the RAPAd® backbone, please see our publication in Gene Therapy on the development of the RAPAd® technique here.
RAPAd®-produced viruses show no wild-type contamination
During the development of the RAPAd® system, we tested for wild type contamination. First, we constructed an eGFP-expressing virus. The shuttle plasmid VQAd CMVeGFP and VQAd 9.2-100 were digested with PacI and the mixture subsequently transfected into HEK293 cells using standard calcium phospate methods. As a control, HEK293 cells were transfected with VQAd 9.2-100 alone.
Generation of recombinant adenovirus
At left: VQAd CMVeGFP and VQAd 9.2–100 (15 and 4 mg, respectively) were digested independently with PacI and precipitated in transfection buffer. The precipitate was added to 60 mm plates containing 50% confluent HEK293 cells and fresh DMEM containing 2% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin Homologous recombination in HEK293 cells lead to generation of VQAd CMVeGFP.
Viral foci were observed in the transfected HEK293 cells via fluorsecence microscopy beginning on day 6. The cells were harvested 8 days after transfection, and the virus was isolated, amplified and purified. In the control plate, foci were not evident and virus could not be amplified, confirming an absence of any recombinant or wild-type virus genome. Read the full article here.
eGFP Expression in HEK293 cells
At left: EGFP expression detected after cotransfection of VQAd CMVeGFP and VQAd 9.2–100 into HEK293 cells. Plates were examined from 1 to 8 days for the presence of viral foci by fluorescence microscopy using an inverted fluorescence microscope. As shown in the representative photomicrographs, VQAd CMVeGFP foci produce a comet-like appearance at approximately 6 days (magnification ×10).
RAPAd® E3 expressing backbones offer even more options!
After linearization and transfection into HEK293 cells (which provide the needed E1a protein in-trans), the plasmids undergo homologous recombination to produce a full length adenoviral genome with the gene of interest inserted into the E1 region. We offer several backbones with additional E3 deletions and/or reporter gene cassettes.
Right: an E3 deletion in the backbone (top) allows for ntlacZ and eGFP inserts.
Read more about our Dual Expresser systems here.
The RAPAd® Process - high titer virus that's fast and RCA-free!
Anderson RD, Haskell RE, Xia H, Roessler BJ, Davidson BL.
Gene Ther. 2000 Jun;7(12):1034-8. doi: 10.1038/sj.gt.3301197. PMID: 10871752.
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