| First |
3 plasmids system |
Expression cassette LTR sequence from HIV-1 genome, encapsulation sequence, RRE sequence, and a promoter to drive transgene expression. |
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Envelope plasmid Replaced HIV-1 envelope with VSV-G (vesicular stomatitis virus glycoprotein) envelope to transduce a wide range of cell types. |
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| Packaging plasmid Sequences of regulatory proteins (tat, rev), structural proteins (gag, pol), and accessory proteins (vif, vpr, vpu, nef) |
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| Second |
3 plasmids system |
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Packaging plasmid Removed all accessory proteins (vif, vpr, vpu, nef) that are non-essential proteins involving in viral replication |
| Third |
4 plasmids system |
Addition of HIV-1 cPPt and CTS pol gene -Important for the central DNA flap production -Enable viral DNA nuclear import and infection of non-dividing cells. -Rev protein is provided by a different plasmid. -increased vector transduction efficiency |
Packaging plasmid -removed all accessory proteins (vif, vpr, vpu, nef) and 1 regulatory gene (tat). -reduce insertional mutagenesis -reduce RCL formation |
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Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) -Enhances mRNA transcript stability. -Increases transgene expression |
Expression cassette -inactivation of LTR -substitution of U3 region by CMV/RSV promoters from HIV-1 5′ LTR -reduce insertional mutagenesis -eliminates tat dependence |
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Replacement of Rev/RRE by autonomous RNA export signals (aka constitutive RNA transport elements (CTE) |
TATA box, Sp1 and NF-kB transcription factor -from HIV-1 3′ LTR -Known as self-inactivating vectors (SIN) |
| Fourth |
5 plasmid system |
gag-pro and pol are further separated into two plasmids -The pol gene is fused to vpr to ensure transport of the reverse transcriptase/integrase protein into the recombinant viral particle. -High expression of essential viral components are driven by 2 separate plasmids: the Tet-Off and Tat transactivators. |
-reduce the recombination events that leads to RCL formations -reduce autonomous replication of virus |
