Lentiviral vectors are quickly becoming avector of choice for many in vivo gene deliveryand RNA interference applications. However,lentiviral vector production remains a timeconsuming and relatively low yield process. Inthis study we compared the efficiency oflentiviral production using a standard calciumphosphate protocol versus a protocol using ahigh efficiency, low toxicity, lipopolyplexformulation (TransIT®-LT1 Reagent). The goalof this work was to determine the overall titerand time course of lentivirus formation using athree plasmid co-transfection strategy.

As expected, the results of theseexperiments demonstrated that bothmethodologies could be used to generateinfectious lentiviral vectors. However, thelipopolyplex mediated transfection protocol(TransIT-LT1 Reagent) facilitated higherlentiviral titers in a significantly shorter timecourse. In addition to the more rapidtimecourse of lentiviral production, higheroverall titers were obtained at 24 and 48 hourspost transfection and there was an addedbenefit of not needing to change the mediumduring the viral grow-up phase when using thelow toxicity lipopolyplex formulation.

Figure 2. Transfection at 70% Confluency Is Suitable for Both Transfection Methods

High Efficiency Production of Lentiviral Vectors in HEK 293FT Cells Using Plasmid Co-transfection: Calcium Phosphate vs. Lipopolyplex Transfection

Steven S. Hahn 1 and James E. Hagstrom 2

1 Waisman Center, University of Wisconsin-Madison, Madison, WI 53705 and 2 Mirus Bio Corporation, 505 S. Rosa Rd., Madison, WI 53719www.mirusbio.com

© 2006 Mirus Bio Corporation. Please visit www.mirusbio.com for the most updated patent and trademark information.

Abstract Results and Discussion

General Methods

Conclusions

Lentiviruses expressing EGFP were producedby transfecting three lentiviral plasmids (EGFP-expressing lentiviral vector, gag-pol expressionplasmid, and a VSV-G envelope plasmid) into HEK293FT cells in 6-well tissue culture plates usingeither calcium phosphate co-precipitation orlipopolyplex-mediated transfection with theTransIT-LT1 Transfection Reagent (Mirus Bio).Figure 1 illustrates the two transfection protocols.

Standard plasmid cocktails used for calciumphosphate transfection contained the followingamounts of each of the three plasmids:

EGFP-lentiviral plasmid - 5 µggag-pol plasmid - 3.75 µgVSV G envelope plasmid - 1.5 µg

10.25 µg totalTransfections using TransIT-LT1 Reagent usedhalf the amount of each plasmid listed to be morein line with the manufacturer’s recommendations.

Lentivirus titers were assayed by harvestingculture supernatants at various timepoints post-transfection, serially diluting the supernatants, andinfecting fresh HEK 293FT cells with the variousdilutions. Four days post-infection, the number ofEGFP expressing cells was determined, and usedto back-calculate the number of infectious lentiviralparticles per ml of original culture supernatant.

HEK 293FT cells were transfected usingeither TransIT-LT1 Reagent (15 µl / 5 µglentiviral plasmid DNA), or calciumphosphate co-precipitation (10 µg or 5 µg oflentiviral plasmid DNA). As shown, theTransIT-LT1 Reagent transfections yieldedsignificantly higher lentiviral titers at allharvest times post-transfection comparedto calcium phosphate transfectionsperformed with equal or 2X more lentiviralplasmid DNA.

TransIT-LT1 ReagentCalcium Phosphate

Our previously optimized calcium phosphate protocolrecommends transfecting cells at 90% confluency; theTransIT-LT1 Reagent protocol recommends transfectingcells at 50%-80% confluency. To test the effect of cellconfluency on lentivirus yield, we transfected cells at both70% and 90% confluency, using both methods. Twenty-four hours post-transfection culture supernatants wereharvested and assayed for infectious lentivirus. Virus yieldwas about 20% greater when transfecting cells at 70%confluency using the TransIT-LT1 Reagent. The differencein confluency had a minimal effect on lentivirus yield whenusing the calcium phosphate method.

Figure 1. Transfection Protocols

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TransIT-LT1 Reagent(5 µg total DNA)

Calcium Phosphate(10 µg total DNA)

Calcium phosphate and TransIT-LT1 Reagenttransfections produce high lentiviral titers,but use of the TransIT-LT1 Reagent offersseveral benefits over calcium phosphate co-precipitation.

1. 30-300% increase in lentiviral titers(depending on harvest time) when using theTransIT-LT1 Reagent.

2. TransIT-LT1 Reagent transfections require50% less viral plasmid DNA to produceincreased lentivirus yields.

3. The TransIT-LT1 Reagent protocol has fewersteps than the calcium phosphate protocolincluding no wash step, no media additionafter complex addition, and no media change24 hours post-transfection.

Figure 3. A 3:1 Ratio of TransIT-LT1 Reagent: Viral DNA is Optimal

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To determine the optimal ratio of TransIT-LT1Reagent to lentiviral plasmid DNA, two differentratios were tested. The first transfectioncontained 10 µl of TransIT-LT1 Reagent per 5µg of total lentiviral plasmid DNA (2:1 ratio). Thesecond transfection used 15 µl of TransIT-LT1Reagent per 5 µg of total lentiviral plasmid DNA(3:1 ratio). Both transfection complexes wereformed in parallel and transfected into the HEK293FT cells. At 24, 48, and 72 hours post-transfection, the culture supernatants wereharvested and assayed for lentivirus. As shown,the 3:1 ratio of TransIT-LT1 Reagent to lentiviralplasmid DNA produced significantly higherlentiviral titers than the cells transfected usingthe 2:1 ratio of TransIT-LT1 Reagent to lentiviralplasmids.

Figure 4. Increased Lentivirus Production When Using TransIT-LT1 Reagent to Transfect the Lentiviral

Plasmids

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TransIT-LT1 Reagent (5 µg viral DNA)Calcium Phosphate (10 µg viral DNA)Calcium Phosphate (5 µg viral DNA)

3:1 Ratio (3 µl/µg viral DNA)2:1 Ratio (2 µl/µg viral DNA)

Optimal Conditions

Optimal lentiviral plasmid transfection conditions using TransIT-LT1 Reagent:- 70% confluent HEK 293FT cells- 5 µg of total viral plasmid DNA transfected/well- 15 µl of TransIT-LT1 Reagent/5 µg plasmid DNA- Harvest culture supernatants 72 hours post-

transfection for maximal viral yield.