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 sho ws high segregational instability during growth in Escherichia coli. Methods By host strain screening and optimization of fermentation, satisfac tory levels of pCMV-CFTR production were achieved. However, the vector was also vulnerable to structural instability manifested by the appearance duri ng fermentation of a more stable mutant form in which the bacterial inserti on sequence ISI had transposed into exon 7 of plasmid-borne CFTR. The insta bility of pCMV-CFTR is attributable to transcription from an upstream crypt ic 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 n atural human 6a-6b intron into pCMV-CFTR. Results The new vector pCMV-CFTR-int6ab is more stable in E. coli than eith er pCMV-CFTR or the ISI mutant, grows to high cell density giving higher DN A yields and expresses CFTR appropriately in transfected cells. Thus, the i ntron 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 betwe en DNA and mature mRNA. The T936C mutation present only in vector DNA has a lso 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 minim ized by rational modification. The introduction of an intron for this purpo se offers the additional advantage of providing a discriminatory RT-PCR ass ay. Copyright (C) 1999 John Wiley & Sons, Ltd.