A new chemical complex can rapidly concentrate lentivirus and significantly enhance gene transduction

Abstract

In this study, we developed a new purification method using chondroitin sulfate C (CSC) and protamine sulfate (PS) to concentrate lentivirus. To evaluate the efficiency of this new method, we compared it with several previously described purification protocols, including virus concentrated by ultracentrifugation (Ultra), precipitated by polyethylene glycol (PEG), and sedimented by CSC combined with polybrene (PB). After using the different methods to purify and concentrate equivalent amounts of lentivirus supernatant, the virus pellets precipitated by the different methods were resuspended using the equivalent volumes of DMEM. Subsequently, 10 μl of each lentivirus stock carrying EGFP gene was used to transduce two types of cells, human embryonic kidney 293T (HEK293T) cells and mouse mesenchymal stem cells (mMSC). It was obvious that HEK293T and mMSC appeared much intensiver green fluorescence through virus transduction from PS method than from other methods. To quantitate the transduction efficiency of the viruses, we examined virus titer in the cells after transduction using a real-time PCR-based analysis. Accordingly, we verified that PS precipitation could generate virus with a higher titer (4.39 × 10 IU/ml) than PB (2.43 × 10 IU/ml), Ultra (1.16 × 10 IU/ml), and PEG (0.56 × 10 IU/ml) in HEK293T cells. As for HEK293T cells in mMSC, the PS method also generated virus with a higher titer (4.66 × 10 IU/ml) than the Ultra method (2.36 × 10 IU/ml), and a much higher titer than those of the other chemical-based precipitation methods using PB (4.82 × 10 IU/ml) and PEG (8.98 × 10 IU/ml). Furthermore, the HEK293T cells and mMSC transduced by PS(1X)-virus appeared to have higher cell growth ratios, respectively, than the HEK293T cells and mMSC transduced by lentivirus using the other methods. We conclude that our new method for purifying lentivirus is cost-effective, time-saving, and highly efficient, and that lentivirus purification by this means could possibly be used to transduce a variety of cells, including stem cells.

Materials promoting viral gene delivery

“…243 TE could be enhanced even further by combining protamine sulfate with chondroitin sulfate. 358 Retronectin is a 574 amino acid long recombinant polypeptide based on the fibronectin fragment CH296. It is one of the first commercially available enhancers for VV delivery and has already been tested in clinical trials.…”

The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes

“…This kit calculates the transduction units (TU) per mL based on an equation that convert genome copies per mL (GC/mL) values to TU/mL (see Materials and Methods for details). The value obtained using this formula is generally very close to the amount of effective particles per mL, 57 although this can vary depending on the vector backbone. The relative amounts of reverse transcription products of the different vectors were quantified by qPCR using the U3Fw/PBSRev primers pair (see Figure S2 ).…”

Lentiviral Vector Bioprocessing

“…LVs can be precipitated by the addition of excipients into the vector-containing media which can be utilised for concentration purposes. Precipitating agents such as polyethylene glycol (PEG) [65,204], poly-L-lysine [205], a mixture of chondroitin sulphate C and protamine sulphate [206] and calcium phosphate [207] have been employed. In most cases, the vector is incubated with the material before pelleting by centrifugation prior to resuspension and dissociating the precipitates followed by further processing such as TFF or dialysis.…”

“…Mode of action is a decrease in solubility due to reduced solvent availability leading to vector precipitation or aggregation due to charges. However, the use of precipitating agents may require additional processing for their removal, as well as extended time to fully precipitate the vector particles and to pellet the precipitate with up to 16 h incubation common [65,204,206]. Furthermore, it may be problematic to resuspend and dissociate precipitates, with EDTA treatment required when using calcium phosphate [207].…”