Nanoparticle‐mediated genome editing in single‐cell embryos via peptide nucleic acids

Abstract

Through preimplantation genetic diagnosis, genetic diseases can be detected during the early stages of embryogenesis, but effective treatments for many of these disorders are lacking. Gene editing could allow for correction of the underlying mutation during embryogenesis to prevent disease pathogenesis or even provide a cure. Here, we demonstrate that administration of peptide nucleic acids and single‐stranded donor DNA oligonucleotides encapsulated in poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles to single‐cell embryos allows for editing of an eGFP‐beta globin fusion transgene. Blastocysts from treated embryos exhibit high levels of editing (~94%), normal physiological development, normal morphology, and no detected off‐target genomic effects. Treated embryos reimplanted to surrogate moms show normal growth without gross developmental abnormalities and with no identified off‐target effects. Mice from reimplanted embryos consistently show editing, characterized by mosaicism across multiple organs with some organ biopsies showing up to 100% editing. This proof‐of‐concept work demonstrates for the first time the use of peptide nucleic acid (PNA)/DNA nanoparticles as a means to achieve embryonic gene editing.

Recent Advancements in Development and Therapeutic Applications of Genome-Targeting Triplex-Forming Oligonucleotides and Peptide Nucleic Acids

“…The mice from the re-implanted embryos consistently exhibited editing. This research is the first example of embryonic-gene editing using PNA [126]. In these above experiments, gene editing was very site-specific, with considerably low levels of off-target sequence modification.…”

Gene therapy for Cystic Fibrosis: recent advances and future prospects

“…Интерес представляет также использование биодеградируемых триплексформирующих пептидно-нуклеиновых кислот (PNA, Peptide nucleic acid), которые связываются с геномной ДНК и формируют триплексы PNA/ ДНК/PNA, стимулирующие восстановление эндогенной ДНК. Доставка таких комплексов вместе с корректирующим геном приводит к сайтспецифическому исправлению гена [70]. В этом случае введение донорской ДНК in vivo в носовые пазухи и легкие гомозиготных мышей ΔF508del приводило к существенной коррекции мутации в эпителии дыхательных путей и облегчало течение заболевания [71].…”

Facile prepared microfluidic chip for multiplexed digital RT‐qPCR test

“…[1][2][3] Quantitative nucleic acid amplification has been widely accepted as a reliable route to inspect the expression level of these biomarkers. [4][5][6] Although the conventional real-time polymerase chain reaction (real-time PCR) does not rely on additional downstream processes and is highly versatile, trace nucleic acid detection limitations restrict their further applications. [7] Digital polymerase chain reaction (dPCR) is a precise and efficient technology to quantify the targets with high sensitivity and specificity through dividing bulk mix into isolated microcompartments of nL-pL volumes and performing massively parallel PCR assays in each microunit at the single-molecule level.…”

“…[ 1–3 ] Quantitative nucleic acid amplification has been widely accepted as a reliable route to inspect the expression level of these biomarkers. [ 4–6 ] Although the conventional real‐time polymerase chain reaction (real‐time PCR) does not rely on additional downstream processes and is highly versatile, trace nucleic acid detection limitations restrict their further applications. [ 7 ]…”