Insertion of natural intron 6a-6b into a human cDNA-derived gene therapy vector for cystic fibrosis improves plasmid stability and permits facile RNA/DNA discrimination.

Background: The gene therapy vector pCMV-CFTR containing human CFTR cDNA shows high segregational instability during growth in Escherichia coli.

Methods: By host strain screening and optimization of fermentation, satisfactory levels of pCMV-CFTR production were achieved. However, the vector was also vulnerable to structural instability manifested by the appearance during fermentation of a more stable mutant form in which the bacterial insertion sequence IS1 had transposed into exon 7 of plasmidborne CFTR. The instability of pCMV-CFTR is attributable to transcription from an upstream cryptic promoter leading to the production of CFTR peptide fragments known to be toxic when expressed in E. coli. To address this, we inserted the 1.1 kb natural human 6a-6b intron into pCMV-CFTR.

Results: The new vector pCMV-CFTR-int6ab is more stable in E. coli than either pCMV-CFTR or the IS1 mutant, grows to high cell density giving higher DNA yields and expresses CFTR appropriately in transfected cells. Thus, the intron has a stabilizing effect comparable to the IS1 insertion yet retains full functionality for gene therapy. We describe a PCR assay using primers directed to sequences flanking the intron that allows differentiation between DNA and mature mRNA. The T936C mutation present only in vector DNA has also been exploited to allow transgene CFTR to be distinguished and its dose-dependent expression to be detected in human cellular backgrounds.

Conclusions: Instability of a plasmid vector for gene therapy has been minimized by rational modification. The introduction of an intron for this purpose offers the additional advantage of providing a discriminatory RT-PCR assay.