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Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo

Abstract

In vivo delivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD.Cg-PrkdcscidIL2rγtm1Wjl (abbreviated NOD-scid IL2rγnull) mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an enhanced green fluorescent protein-β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

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Acknowledgements

We thank Hanspeter Neiderstrasser and Faye Rogers for their technical assistance and helpful discussions. We also thank John Overton, Francesc Lopez and Jennifer Yamtich for assistance with deep sequencing and analysis. This work was supported by the NIGMS Medical Scientist Training Program T32GM07205 (to NAM), the NIH Genetics Training Grant T32 GM007499 (to EBS) and the National Institute of Health grants R01HL082655 (to PMG) and R01EB000487 (to WMS). This work was also supported by F30HL110372 from the National Heart, Lung and Blood institute, and the content is solely the responsibility of the authors and does not necessarily represent the official views of the NHLBI. This work was also supported by grants from National Institutes of Health Research AI46629 (DLG, LDS, MAB), AI083911 (MAB), HL077642 (LDS), CA34196 (LDS), AI073871 (DLG, LDS), DK32520 (DLG, LDS), P30 AI042845 (MAB), and grants from the Juvenile Diabetes Foundation, International (DLG, LDS, MAB) and the Helmsley Foundation (DLG, LDS, MAB). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

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Correspondence to W Mark Saltzman or P M Glazer.

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McNeer, N., Schleifman, E., Cuthbert, A. et al. Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo. Gene Ther 20, 658–669 (2013). https://doi.org/10.1038/gt.2012.82

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