The inclusion of retrovirus-derived introns within retrovirus-based express
ion vectors leads to a fraction of the resulting transcripts being spliced,
Such splicing has been shown to markedly improve expression (W.J. Krall et
al,, Gene Ther, 3:37-48, 1996), One way to improve upon this. still furthe
r might involve the use of more efficient introns instead of those from the
provirus. Currently, however, incorporation of such introns remains self-d
efeating since they are removed in the nucleus of the producer cell. In the
past, elaborate ways to overcome this problem have included the use of alp
haviruses to make the vector transcripts within the cytoplasm, thus avoidin
g the nuclear splicing machinery during vector production (K. J. Li and H.
Garoff, Proc. Natl, Acad, Sci. USA 95:3650-3654, 1998), We now present a no
vel design for the inclusion of introns within a retroviral vector. In esse
nce, this is achieved by exploiting the retroviral replication process to c
opy not only the U3 promoter but also a synthetic splice donor to the 5'-lo
ng-terminal-repeat position during reverse transcription. Once copied, synt
hesized transcripts then:contain a splice donor at their 5' end capable of
interacting with a consensus splice acceptor engineered downstream of the p
ackaging signal. Upon transduction, we demonstrate these vectors to produce
enhanced expression from near fully spliced land thus packaging signal min
us) transcripts. The unique design of these high titer and high-expression
retroviral vectors may be of use in a number of gene therapy applications.