Creating Higher Titer Lentivirus with Caffeine

Abstract

The use of lentiviral vectors extends from the laboratory, where they are used for basic studies in virology and as gene transfer vectors gene delivery, to the clinic, where clinical trials using these vectors for gene therapy are currently underway. Lentiviral vectors are useful for gene transfer because they have a large cloning capacity and a broad tropism. Although procedures for lentiviral vector production have been standardized, simple methods to create higher titer virus during production would have extensive and important applications for both research and clinical use. Here we present a simple and inexpensive method to increase the titer by 3- to 8-fold for both integration-competent lentivirus and integration-deficient lentivirus. This is achieved during standard lentiviral production by the addition of caffeine to a final concentration of 2-4 mM. We find that sodium butyrate, a histone deacetylase inhibitor shown previously to increase viral titer, works only ∼50% as well as caffeine. We also show that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) inhibitor NU7026 can also increase viral titer, but that the combination of caffeine and NU7026 is not more effective than caffeine alone. We show that the time course of caffeine treatment is important in achieving a higher titer virus, and is most effective when caffeine is present from 17 to 41 hr posttransfection. Last, although caffeine increases lentiviral vector titer, it has the opposite effect on the titer of adeno-associated virus type 2 vector. Together, these results provide a novel, simple, and inexpensive way to significantly increase the titer of lentiviral vectors.

Fibrotic extracellular matrix activates a profibrotic positive feedback loop

“…DNA complexes were incubated with the cells for 4 hours (37°C, 5% CO2). Medium was removed and replaced with 5 ml DMEM, 10% FBS, and antibiotic medium containing 2 mM caffeine (62). The transfected cells were incubated for 24 hours (37°C, 5%CO2), and media were removed and replaced with 5 ml DMEM, 10% FBS, and antibiotic medium containing 2 mM caffeine.…”

Overexpression of tumor suppressor gene ZNF750 inhibits oral squamous cell carcinoma metastasis

“…For the generation of LV-PGK and LV-ZNF750 lentivirus, 7 µg PGK or ZNF750 lentiviral vector plasmid together with packaging plasmids (7 µg psPax2, 3 µg pRSV-Rev and 3 µg VSV-G) were co-transfected into 70-80% confluent 293T cells, using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Lipofectamine ® 2000/DNA complexes were added to 293T cells in DMEM with 10% FBS and 1% penicillin/streptomycin to which caffeine (4 mM) and sodium butyrate (1 mM) were added to increase the lentiviral titer (13). At 48 and 72 h post-transfection, the virus particles present in the cell supernatant were harvested and centrifuged at 5,000 x g for 30 min to remove cell debris, then filtered through a Steriflip-HV 0.45 µm polyvinylidene fluoride (PVDF) filter unit (EMD Millipore, Billerica, MA, USA) and concentrated using PEG-it virus precipitation solution (System Biosciences, Inc., Palo Alto, CA, USA) to obtain virus particles.…”

Efficient intratracheal delivery of airway epithelial cells in mice and pigs

“…7A). The relatively lower infection efficiency of pSicoR-CFTR-GFP lentivirus compared with pSicoR-GFP lentivirus is mostly due to the large size of the CFTR-GFP gene (5.2 kb) (29) and may be improved by increasing viral titration as described (13). In addition, sorting for GFP ϩ cells could potentially allow for production of a pure population of cells expressing CFTR-GFP.…”