Evaluation of Plasmid DNA Removal from Lentiviral Vectors by Benzonase Treatment

Abstract

To improve the purity of lentiviral vector supernatants for clinical studies we have evaluated plasmid DNA removal from lentiviral vectors and also the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol. Optimal conditions of plasmid DNA removal by benzonase treatment were established by varying the temperature, time, and benzonase concentrations in the reaction mix and were determined to be 50 units of benzonase per milliliter of vector supernatant at 37 degrees C, for 15 min. No plasmid DNA was detected, suggesting efficient plasmid degradation was achieved under these experimental conditions. The infectious titer of benzonase-treated lentiviral vector (RRL-CMV-GFP) was nearly identical to the titer of untreated vector (2.3 +/- 0.3 x 10(6) transduction units per milliliter (TU/ml) and 2.7 +/- 0.3 x 10(6) TU/ml, respectively). Analysis of plasmid DNA in concentrated lentiviral vectors shows that concentration substantially decreases the amount of DNA per TU. Analysis of the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol suggests that a minimal amount of plasmid is transferred to transduced cells if the vector supernatant was not previously treated with benzonase. In conclusion, benzonase treatment is effective in eliminating plasmid DNA from vector supernatants and treatment does not affect infectious titers. However, because there is minimal transfer of plasmid DNA to transduced cells under ex vivo transduction conditions, DNA removal from lentiviral vectors may not be essential for all ex vivo clinical applications.

Tubulovesicular Structures within Vesicular Stomatitis Virus G Protein-Pseudotyped Lentiviral Vector Preparations Carry DNA and Stimulate Antiviral Responses via Toll-Like Receptor 9

“…However, most of this activity is attributable not to the viral particles themselves but to contaminating tubulovesicular structures, which largely outnumber virions and carry nucleic acids, including the plasmids used in transfection. The observation that TVS carry DNA may account for previous reports of plasmid DNA presence within LV preparations (54) and implies that TVS can activate pathways of innate defense against DNA viruses. Consistent with this notion, we show that TVS induce strong innate responses via a TLR9-dependent pathway and act as adjuvants in vivo for coadministered antigens.…”

Transcription of Preintegrated HIV-1 cDNA Modulates Cell Surface Expression of Major Histocompatibility Complex Class I via Nef

“…), clarified by centrifugation for 5 min at 470 ϫ g, and passed through a 0.45-m filter. Virus was treated with 50 U/ml benzonase (Sigma) at 37°C for 20 min to digest contaminating plasmid DNA (40) and then stored at Ϫ80°C until use.…”

A Simple and Effective Method to Generate Lentiviral Vectors forEx VivoGene Delivery to Mature Human Peripheral Blood Lymphocytes

“…We have shown previously that a concentrated and purified LVV product is not required for efficient ex vivo transduction of PBLs at clinical scale (Yang et al, 2008b). In addition, we and others (Sastry et al, 2004) have shown that Benzonase treatment or the presence of residual Benzonase in the vector supernatant does not inhibit the ability of LVV to efficiently transduce lymphoid cells (Fig. 3B-D).…”

“…Residual Benzonase was detected by ELISA according to the manufacturer's protocol (EMD Chemicals). Residual lentiviral packaging plasmid DNA was detected by real-time PCR as described previously (Sastry et al, 2004). A standard curve was generated with plasmid pMDLg/pRRE, ranging from 1.0 to 0.0000001 ng.…”

“…Briefly, real-time PCR was used to detect residual lentiviral packaging plasmid DNA (VSV-G) as described previously (Sastry et al, 2004). Detection of adenovirus E1a and simian virus 40 (SV40) DNAs was conducted at the IUVPF by quantitative PCR (qPCR) on PBL genomic DNA 14 days posttransduction with LVV.…”