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Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

Figure 4

Production of tat/rev shRNA-containing lentiviral vectors at high titer using a modified helper plasmid.

(A) Three synonymous mutations indicated by an underline were introduced into rev in the lentiviral helper plasmid pCMVRdelta8.74 which expresses Gag-Pol and Rev to create pCMVRdelta8.74-sIM, which contains three mismatches at the tat/rev site I shRNA target sequence. (B) Lentiviral vectors were prepared by transient transfection using either the lentiviral helper plasmid pCMVRdelta8.74, which contains the wild-type rev sequence (wt-rev), or the pCMVRdelta8.74-sIM helper, which contains the mutated (sIM-rev) and titered using the EGFP reporter. The RSC-UsI-SC46-IMPGW vector containing the tat/rev shRNA is produced at low titer using the wt rev helper, but is produced at 38-fold higher titer using the sIM-rev helper.

Figure 4