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Identification and removal of colanic acid from plasmid DNA preparations: implications for gene therapy

Gene Therapy volume 17, pages 1484–1499 (2010)Cite this article

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Abstract

Polysaccharide contaminants in plasmid DNA, including current good manufacturing practices (cGMP) clinical preparations, must be removed to provide the greatest safety and efficacy for use in gene therapy and other clinical applications. We developed assays and methods for the detection and removal of these polysaccharides, our Super Clean DNA (SC-DNA) process, and have shown that these contaminants in plasmid DNA preparations are responsible for toxicity observed post-injection in animals. Furthermore, these contaminants limit the efficacy of low and high doses of plasmid DNA administered by numerous delivery routes. In particular, colanic acid (CA) that is mainly long-chained, branched and has high molecular weight (MW) is most refractory when complexed to cationic delivery vehicles and injected intravenously (IV). Because CA is often extremely large and tightly intertwined with DNA, it must be degraded, in order, to be effectively removed. We have produced a recombinant, truncated colanic acid degrading enzyme (CAE) that successfully accomplishes this task. Initially, we isolated a newly identified CAE from a bacteriophage that required truncation for proper folding while retaining its full enzymatic activity during production. Any plasmid DNA preparation can be digested with CAE and further purified, providing a critical advance to non-viral gene therapy.

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Author notes

  1. W Zhang

    Present address: 4Current address: Division of Medical Genetics, Department of Human Genetics, Emory University School of Medicine, Decatur, GA 30033, USA.,

Authors and Affiliations

  1. Department of Surgery, Baylor College of Medicine, Houston, Texas, USA

    P Firozi, L Chen & N S Templeton

  2. Department of Biochemistry & Molecular Biology, Baylor College of Medicine, Houston, Texas, USA

    W Zhang & F A Quiocho

  3. Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA

    K C Worley

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  1. P Firozi
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Correspondence to N S Templeton.

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Firozi, P., Zhang, W., Chen, L. et al. Identification and removal of colanic acid from plasmid DNA preparations: implications for gene therapy. Gene Ther 17, 1484–1499 (2010). https://doi.org/10.1038/gt.2010.97

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