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

• Published in: Gene therapy Vol. 20; no. 6; pp. 658 - 669
• Main Authors: McNeer, N A, Schleifman, E B, Cuthbert, A, Brehm, M, Jackson, A, Cheng, C, Anandalingam, K, Kumar, P, Shultz, L D, Greiner, D L, Mark Saltzman, W, Glazer, P M
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2013; Nature Publishing Group
• Subjects: 631/1647/2300; 631/1647/350/354; 692/700/565/201; Animals; Biodegradability; Biomedical and Life Sciences; Biomedicine; Bone marrow; CCR2 protein; CCR5 protein; Cell Biology; Cell Line; DNA - administration & dosage; DNA - chemistry; DNA - genetics; Gene Expression; Gene Targeting; Gene Therapy; Gene Transfer Techniques; Genetic aspects; Genetic Therapy; Genetically modified organisms; Genome editing; Green fluorescent protein; Health aspects; Hematopoietic stem cells; Hematopoietic Stem Cells - cytology; Human Genetics; Humans; Intravenous administration; Mice; Monocyte chemoattractant protein 1; mRNA; Nanoparticles; Nanoparticles - administration & dosage; Nanoparticles - chemistry; Nanotechnology; original-article; Peptide nucleic acids; Peptide Nucleic Acids - administration & dosage; Peptide Nucleic Acids - chemistry; Peptide Nucleic Acids - genetics; Physiological aspects; Receptors, CCR5 - genetics; Rodents; Single-stranded DNA; Spleen; Stem cells; United States; gene delivery; gene modification; nanoparticles; triple-forming oligonucleotide; peptide nucleic acid
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/gt.2012.82

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 IL2rγ tm1Wjl (abbreviated NOD- scid IL2rγ 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 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 .