The effect of a cryptic splice acceptor (cSA) site located at the end of the extended packaging signal in murine leukemia virus (MLV)-based vectors was investigated, Although this cSA is also present in wild type MLV, it was found to result in a smaller transcript in which the packaging signal (Psi) had been removed by splicing only in MLV-derived vectors. Splicing occurs both in packaging cells producing the MLV-vectors as well as in the infected target cells. Transcripts lacking the Psi-sequence (Psi(-)) are packaged relatively efficiently into virus particles. even in the presence of wild type Psi(+)-vector transcripts, The Psi(-)- viral RNA is reverse transcribed in vector transduced cells as is any other retroviral genome. The titer obtained From the T(-)-vector was only 1000-fold reduced in comparison to the same Psi(+)-vector. These results suggest that Psi(-)-transcripts may be packaged more frequently than previously supposed and that splicing patterns should be carefully analysed on an individual basis for retroviral vectors used in gene therapy. (C) 2002 Elsevier Science (USA). All rights reserved.