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Differences in vector-genome processing and illegitimate integration of non-integrating lentiviral vectors

Gene Therapy volume 24, pages 12–20 (2017)Cite this article

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Abstract

A variety of mutations in lentiviral vector expression systems have been shown to generate a non-integrating phenotype. We studied a novel 12 base-pair U3-long terminal repeats (LTR) integrase (IN) attachment site deletion (U3-LTR att site) mutant and found similar physical titers to the previously reported IN catalytic core mutant IN/D116N. Both mutations led to a greater than two log reduction in vector integration; with IN/D116N providing lower illegitimate integration frequency, whereas the U3-LTR att site mutant provided a higher level of transgene expression. The improved expression of the U3-LTR att site mutant could not be explained solely based on an observed modest increase in integration frequency. In evaluating processing, we noted significant differences in unintegrated vector forms, with the U3-LTR att site mutant leading to a predominance of 1-LTR circles. The mutations also differed in the manner of illegitimate integration. The U3-LTR att site mutant vector demonstrated IN-mediated integration at the intact U5-LTR att site and non-IN-mediated integration at the mutated U3-LTR att site. Finally, we combined a variety of mutations and modifications and assessed transgene expression and integration frequency to show that combining modifications can improve the potential clinical utility of non-integrating lentiviral vectors.

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Acknowledgements

We thank Teresa Johnson for her assistance with vector production, David Gilley who provided the MO59K and MO59J cell lines and Troy Hawkins and Hongyu Gao for assistance with bioinformatics analysis. The study was funded in part by a grant from the NHLBI (P40HL11621). GJ was supported by Edward T Harper Scholar—funded by NIH NIGMS on 1R25 GM079657-01, Indiana University Initiative for Maximizing Graduate Student Diversity. AS was supported by the Joe and Shirley Christian Graduate Student Education Fund.

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Author notes

  1. A M Shaw and G L Joseph: These authors contributed equally to this work

Authors and Affiliations

  1. Departments of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA

    A M Shaw, A C Jasti, L Sastry-Dent & K Cornetta

  2. Departments of Microbiology, Indiana University School of Medicine, Indianapolis, IN, USA

    G L Joseph & K Cornetta

  3. Department of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital, Cincinnati, OH, USA

    S Witting

  4. Departments of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

    K Cornetta

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Correspondence to K Cornetta.

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Dr Cornetta is a consultant for Cook Regentec but there are no collaborative or financial relationships with the work contained in this manuscript. Dr Shaw, Dr Joseph, Mrs Jasti, Dr Sastry and Dr Witting declare no potential conflict of interest.

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Shaw, A., Joseph, G., Jasti, A. et al. Differences in vector-genome processing and illegitimate integration of non-integrating lentiviral vectors. Gene Ther 24, 12–20 (2017). https://doi.org/10.1038/gt.2016.69

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