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

Abstract

In vivodelivery 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-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 EGFP- 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.

Transient loading of CD34⁺ hematopoietic progenitor cells with polystyrene nanoparticles

“…[9][10][11][12] Additionally, the delivery of peptide nucleic acids by poly(lactic-co-glycolic acid) NPs for genome editing in HSCs is currently under investigation. 13,14 With the available knowledge, limited attention has been paid to the loading behavior of HSCs and HPCs with NPs, not to mention the kinetics involved. Since this determines the efficacy and safety of nanotechnology-based applications, it is definitely worthwhile to consider these aspects in more detail.…”

Genome Editing for Neuromuscular Diseases

“…Moreover, these complexes can be used to deliver therapeutic peptides or RNA in addition to conventional gene transfer via DNA. Notably, it has been demonstrated that nanoparticles can mediate in vivo gene editing by simultaneously delivering both the gene editing agent and a suitable donor template [ 155 ]. Thus, non-viral methods may be a promising method to deliver short bursts of gene editing drugs .…”

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery

“…17 Silica nanoparticles have been linked to PNA by disulfide bond to achieve delivery and release of antisense PNAs in HeLa cells; 18 biodegradable PLGA formulations have been successfully employed for the delivery in vivo of PNA−polylysine conjugates in hematopoietic cells. 19 The main drawback of the cited strategies derives from the need of synthesizing special building blocks to be used in the solid phase synthesis or to conjugate the carriers on solid phase or in solution, ending up in very large molecules.…”