title: Nanoparticles deliver triplex-forming PNAs for site-specific genomic recombination in CD34+ human hematopoietic progenitors.
abstract: Triplex-forming peptide nucleic acids (PNAs) are powerful gene therapy agents that can enhance recombination of short donor DNAs with genomic DNA, leading to targeted and specific correction of disease-causing genetic mutations. Therapeutic use of PNAs is severely limited, however, by challenges in intracellular delivery, particularly in clinically relevant targets such as hematopoietic stem and progenitor cells. Here, we demonstrate efficient and nontoxic PNA-mediated recombination in human CD34(+) cells using poly(lactic-co-glycolic acid) (PLGA) nanoparticles for intracellular oligonucleotide delivery. Treatment of progenitor cells with nanoparticles loaded with PNAs and DNAs targeting the β-globin locus led to levels of site-specific modification in the range of 0.5-1% in a single treatment, without detectable loss in cell viability, resulting in a 60-fold increase in modified and viable cells as compared to nucleofection. As well, the differentiation capacity of the progenitor cells treated with nanoparticles did not change relative to untreated progenitor cells, indicating that nanoparticles are safe and minimally disruptive delivery vectors for PNAs and DNAs to mediate gene modification in human primary cells. This is the first demonstration of the use of biodegradable nanoparticles to deliver genome-editing agents to human primary cells, and provides a strong rationale for systemic delivery of complex nucleic acid mixtures designed for gene correction.
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: McNeer
authors.forename: Joanna Y
authors.initials: JY
authors.lastname: Chin
authors.forename: Erica B
authors.initials: EB
authors.lastname: Schleifman
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Fields
authors.forename: Peter M
authors.initials: PM
authors.lastname: Glazer
authors.forename: W Mark
authors.initials: WM
authors.lastname: Saltzman
mesh_terms: D018952:Antigens, CD34 / Q000096:biosynthesis*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D016678:Genome; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D019344:Lactic Acid / Q000494:pharmacology; D053758:Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D009841:Oligonucleotides / Q000494:pharmacology; D010316:Particle Size; D020135:Peptide Nucleic Acids / Q000008:administration & dosage* / Q000235:genetics; D011100:Polyglycolic Acid / Q000494:pharmacology; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D019713:Receptors, CCR5 / Q000235:genetics; D011995:Recombination, Genetic; D052416:Targeted Gene Repair; D055544:beta-Globins / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D019713:Receptors, CCR5; D055544:beta-Globins; C051802:triplex DNA; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D004247:DNA