Journal
GENE THERAPY
Volume 20, Issue 6, Pages 658-669Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/gt.2012.82
Keywords
gene modification; peptide nucleic acid; nanoparticles; gene delivery; CCR5; triple-forming oligonucleotide
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Funding
- NIGMS Medical Scientist Training Program [T32GM07205]
- NIH Genetics Training Grant [T32 GM007499]
- National Institute of Health [R01HL082655, R01EB000487]
- National Heart, Lung and Blood institute [F30HL110372]
- National Institutes of Health Research [AI46629, AI083911, HL077642, CA34196, AI073871, DK32520, P30 AI042845]
- Juvenile Diabetes Foundation, International
- Helmsley Foundation
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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-Prkdc(scid)IL2r gamma(tm1Wjl) (abbreviated NOD-scid IL2r gamma(null)) 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 beta-globin gene using nanoparticles carrying beta-globin-targeted PNAs/DNAs, demonstrating this method's versatility. In vivo testing in an enhanced green fluorescent protein-beta-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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