Here we describe a novel "super-split" lentiviral packaging system in which the overlapping Gag and Pol polyprotein precursors are completely separated and supplied independently to produce high titer virus. This approach also brings further evidence that Vpr can be used as a vehicle to incorporate the Pol components, PR and RT/IN, effectively into viral particles, as we and others have successfully used Vpr fusions to supply proteins in trans to viral particles [24-28]. Vpr has also been used to supply RT/IN as part of a safer lentiviral packaging system in which Gag-PR and RT/IN functions were delivered by separate plasmids . In this safer system, Wu, et al. showed that the lentiviral packaging functions could be supplied from separate plasmids, although they did not truly physically split the Gag-Pol gene. The Gag-PR plasmid they used had a stop codon at the start of RT and IN to prevent the expression of RT and IN, but the RT and IN sequences remained as part of their Gag-PR expression plasmid. This configuration was exposing to residual risk of RCL formation by sequence read-through, reversion or recombination. In contrast, the split packaging systems presented here establish the functionality of creating a true physical split of the Gag-Pol gene, where neither Gag-PR nor Gag expression plasmids contains RT or IN sequences. Tat and Rev are also provided from completely separated expression plasmids.
In our first attempt at constructing this split-packaging system, the Gag-Pol polyproteins were expressed using two expression plasmids: one for Gag-PR and the second expressing RT/IN. As was shown in the Results, this first generation system (the 6 plasmid system) produces infectious viral particles at titers 9 fold lower than those generated by the conventional lentiviral packaging system in which Gag-Pol is supplied intact from a single expression plasmid. After examining the profile of viral proteins from virions produced by the 6 plasmid system, we determined that RT/IN was not efficiently processed although it was incorporated into the viral particles. The same phenomenon was observed for the Gag p55 precursor. Because PR is central to processing the precursor polyproteins, the reduced processing of Gag and RT/IN suggested that the low titers might be explained by a defect in the activation and release of PR. Another contributing factor that could explain the low titers is the accumulation of uncleaved Vpr-RT/IN fusion proteins. We have previously shown that incorporation of Vpr fused heterologous amino-acid sequence affected the infectivity of HIV-1 viral particles .
To improve upon this first generation split-packaging system, we then developed a new "super-split" system (the 7 plasmid system) in which Gag is not only separated from Pol, but PR is separated from Gag and supplied independently in trans. It was our hope that, by supplying PR in trans, we could increase the amount of active PR and improve processing of the precursor proteins. This approach raised two theoretical concerns: the potentially enhanced cytotoxic effect of PR [9,14,17] and the possible premature processing of the precursor polyproteins [5,14,17]. To address the issue of cytotoxicity, we used a mutant PR with slightly reduced protease activity and none of the cytotoxic effects seen with the wt PR . We found that supplying PR in trans as part of the 7 plasmid system resulted in titer improvement comparatively to those obtained with the 6 plasmid system. Furthermore, the mutant PR supplied in trans yielded viral titers higher than those obtained with the use of wt PR. A concurrent improvement upon processing of both the Pr55Gag and RT/IN polyproteins was also observed. When the 7 plasmid system was compared to the conventional lentiviral packaging system, the viral titers were only 3 fold lower with a mean titer of 1.0 × 106 TU/ml for unconcentrated virus. In addition to the data presented here and to demonstrate that the viral particles generated by the 7 plasmid system can be concentrated and used to transduce dividing and non-dividing cells, the efficacy of this "super-split" packaging system with the PR supplied in trans was demonstrated by us in a published study where the 7 plasmid system was used to transduce human cord blood hematopoietic stem cells with a complex beta-globin expressing lentiviral vector assessed in NOD-SCID mouse transplant studies .
In order to generate the safest possible lentiviral packaging system, we incorporated several safety features into the 7-plasmid system. These features include: (i) splitting the Gag, PR, and RT/IN functions into separate plasmids, (ii) eliminating the frameshift, so that even in the event of a recombination event, Pol could not be produced, and (iii) minimizing the overlapping sequences that existed between the lentiviral vector (packaging signal) and the Gag expression plasmid (from 542 to 55 bases), and between the Gag and Pol (from 208 to 54 bases). In addition to the packaging systems, we also compared viral titers produced by the wt-LTR and SIN lentiviral vectors and found that the SIN vector produced equivalent or higher viral titers for all three packaging systems, possibly due to the benefit conferred by the "ideal" poly(A) sequence that we substituted for U5 within the 5' LTR. Iwakuma, et al., also reported an increase in viral titers when they replaced the U5 region of their SIN vector with a bGH poly(A) sequence . Most importantly, this increase resulted in viral titers for the 7 plasmid system in conjunction with the SIN vector that were only 2 fold lower than those obtained by the conventional system using a wt-LTR vector, 1.0 × 106 TU/ml and 2.2 × 106 TU/ml, respectively.