I would like to develop a lentiviral delivery plasmid that contains two transgenes with independent promoters. I have two main questions regarding the design:
The specific promoters are CMV and SV40.
First, in what direction should the two transgenes be placed, one after the other (so promoter 1, transgene1, promoter 2, transgene 2), in opposite directions (promoter 1, transgene 1, transgene 2 (antisense) promoter 2 (antisense), or in opposite directions with the promoters in the middle (transgene 1 (antisense), promoter 1 (antisense), promoter 2, transgene 2)? All these options will be between 5`LTR+lentiviral components and the 3`LTR)
Second, depending on the answer above, should I add an SV40 early Poly A signal to the transgenes not ending in the `3 LTR? SV40 early poly A between the two LTRs reduces titer count when using the CMV promoter (according to: Article An Internal Polyadenylation Signal Substantially Increases E...
)? What would a good strategy to design a plasmid so that both genes can be delivered at a single locus?While it would be preferable to keep both transgenes expressed by seperate promoters, if this is not a possibility then I suppose there is always the option of adding a 2A cleaving linker, but due to this having the effect of some proteins still fused together, I would like to avoid it.
Any thoughts? Thanks in advance!
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