HiPerform expression vectors were compared to vectors not containing the cppt and WPRE but otherwise identical. Vectors were transfected into 293FT cells and packaged into the virus in 10 mL adherent cultures. Culture supernatants were collected at 48 hours, filtered, diluted, and applied to cultures of HT1080 cells to determine the concentration of functional viral particles.
Titer performance of the pLenti6.3/V5-TOPO vector was compared to a similar vector lacking the enhancing elements. Constructs containing two different genes (erythropoietin or EmGFP) were evaluated. Both HiPerform constructs revealed a substantial increase in blasticidin-resistant transductants, effectively yielding up to twofold more functional virus particles per mL of viral supernatant over vectors lacking these elements (Figure 1).
Titer was estimated using erythropoietin constructs with either blasticidin selection in pLent6.3 or the EmGFP marker in pLenti7.3. Similar titers were observed using both reporter systems, as each marker signals successful transduction and expression (Figure 1).
In many systems, the WPRE enhancer is placed downstream of the selection marker, distal to the cloning site, where it provides only minor benefit to the cloned gene. HiPerform vectors were created with the WPRE element placed immediately downstream of the cloning site where it provides maximum expression of the gene of interest. Expression of EmGFP and erythropoietin from pLenti6.3 was greater than 10-fold higher than from the same vector lacking the WPRE element (Figure 2 A, C, and D). The expression benefit of HiPerform’s enhancer configuration was reiterated with EmGFP.
Cells transduced with the HiPerform vector generated at least a 10-fold greater EmGFP fluorescence signal than the cells transduced with a vector lacking the HiPerform elements (Figure 2 C and D). On a per particle basis, HiPerform offers higher expression levels stemming from robust support of the expression cassette. Using equal volumes of viral preparations reveals that HiPerform yields more functional virus per mL of viral supernatant (Figure 2 C, red bars).
To demonstrate the stability of the integrated pseudovirus, cells transduced with and expressing the erythropoietin construct were propagated without blasticidin selection for 30 days. Erythropoietin expression was found to be essentially unchanged after 30 days, reflecting the stability of the integrant and its resistance to silencing (Figure 2B).
In RNAi evaluations using a luciferase reporter, HiPerform viral preps offered two- to threefold more robust gene knockdown over previous generation vectors stemming from an increase in viral titer (Figure 3).
The benefit from the HiPerform vector modifications is an increase in yield of functional virus, higher transduction efficiency, higher gene expression, and stronger knockdown performance. Optimizations to the HiPerform system continue with an eye toward increasing the productivity and utility of lentiviral technologies for both research and therapeutic discovery.