Lentiviral packaging constructs

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, cell culture, virology, and the like which are in the skill of one in the art.
These techniques are fully disclosed in current literature and reference in made specifically to Sambrook, Fritsch and Maniatis eds.
, "Molecular Cloning, A Laboratory Manual", 2nd Ed.
, Cold Spring Harbor Laboratory Press (1989); Celis J.
E.
"Cell Biology, A Laboratory Handbook" Academic Press, Inc.
(1994) and Bahnson et al.
, J.
of Virol.
Methods, 54:131 143 (1995).
All publications and patent applications cited in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are hereby incorporated by reference in their entirety.
The present invention is concerned with novel lentivirus-based packaging constructs that are useful for the establishment of stable packaging cell line and producer cell lines.
Surprisingly it is found that mutations in the active site of the respective lentiviral protease gene enable the construction of lentiviral packaging vectors which are useful to establish stable packaging cell lines for the production of lentiviral vectors.
The catalytic center of HIV protease includes a three amino acid motif, Asp-Thr-Gly (Konvalinka, J.
et al.
,.
J.
Virol.
69:7180 7186, 1995) These three amino acids are conserved among HIV and SIV isolates documented so far (Korber B, Theiler J, Wolinsky S Science 1998 Jun.
19 280: 5371 1868 71).
Konvalinka, J.
et al.
mutated the Thr residue (corresponding to amino acid number 26 from the start of Protease in HIV isolate HXB2) to a Ser.
They found that the mutated HIV protease has a significantly reduced toxicity while preserving the protease activity.
It has been surprisingly found that this information makes it possible for one to generate a stable cell line to express high levels of lentiviral Gag/Pol proteins.
Expression of these proteins is absolutely necessary in order to establish a stable packaging cell line for lentiviral vectors, in particular for HIV- or BIV-based lentiviral vectors