Evaluation of Plasmid DNA Removal from Lentiviral Vectors by Benzonase Treatment

Lakshmi Sastry, Yi Xu, Ryan Cooper, Karen Pollok, Kenneth Cornetta

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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°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 106 transduction units per milliliter (TU/ml) and 2.7 ± 0.3 x 106 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.

Original languageEnglish
Pages (from-to)221-226
Number of pages6
JournalHuman Gene Therapy
Volume15
Issue number2
DOIs
StatePublished - Feb 2004

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Serratia marcescens nuclease
Plasmids
DNA

ASJC Scopus subject areas

  • Genetics

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Evaluation of Plasmid DNA Removal from Lentiviral Vectors by Benzonase Treatment. / Sastry, Lakshmi; Xu, Yi; Cooper, Ryan; Pollok, Karen; Cornetta, Kenneth.

In: Human Gene Therapy, Vol. 15, No. 2, 02.2004, p. 221-226.

Research output: Contribution to journalArticle

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