I am familiar with bicistronic AAV gene therapy vectors that have two cistrons with an IRES between them. Since this is known to result in less efficient expression of the second gene why not simply create vectors with the structure ITR-promoter-gene1-promoter2-gene2-WPRE-polyA-ITR or ITR-promoter-gene1-promoter2-pA-gene2-WPRE-polyA-ITR if this fits the 4800 KB limit? I can find no published examples of this design being used in primate studies and little in rodent studies. Are there known issues with this approach?
One of the reason should be 'transcription interference', I think. There are some ways to avoid it but the capacity of AAV restricts its application.
I think what you imagine will work. An even more efficient solution would be a 2A peptide.
If your using 2 promoter system, i agree with Takefumi that transcriptional interference may be an issue which can be resolved by changing direction of one of the cassettes. Also to prevent rearrangements, you may want to use unique promoters. I prefer to John's suggestion where you can co-express using a 2A peptide.
Hi Mike, I just recently tried the approach you suggested (CMV-gene1-pA-CMV-gene2-pA) and found that gene2 was expressed much less efficiently than gene1 in cell culture, probably due to transcriptional interference, as already pointed out by Takefumi Sone and Tin Mao. As John said, using the 2A system is an elegant and well working approach, provided that you can accept the presence of residual amino acids remaining with your proteins after cleavage. A third alternative would be the use of bidirectional promoters, e.g. BI-CMV1 (clontech), which worked quite well in my hands...
I think with regard to gene therapy applications, the major restrictions are:
- for "your" design: risk of rearrangement due to promoter homology.
- for the 2A design: risk of immunogenicity due to 2A fragments or low amounts of uncut fusion protein
- Badges
- Science topic
- Similar topics
- Correction