Gene Correction of Point Mutations Using PolyPurine Reverse Hoogsteen Hairpins Technology

Abstract

Monogenic disorders are often the result of single point mutations in specific genes,leading to the production of non-functional proteins. Different blood disorders suchas ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, andHemophilia A and B are usually caused by point mutations. Gene editing toolsincluding TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correctmutations responsible for different diseases. However, alternative molecular tools suchas triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), notrelying on nuclease activity, have also demonstrated theirability to correct mutationsin the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins(PPRHs) technology, which can represent an alternative gene editing tool within thisfield. Repair-PPRHs are non-modified single-stranded DNA molecules formed by twopolypurine mirror repeat sequences linked by a five-thymidine bridge, followed by anextended sequence at one end of the molecule which is homologous to the DNAsequence to be repaired but containing the corrected nucleotide. The two polypurinearms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between thepurines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tractslocated relatively near the target mutation in the dsDNA in asequence-specific manner byWatson-Crick bonds, thus producing a triplex structure which stimulates recombination.This technology has been successfully employed to repair a collection of mutants ofthedhfrandaprtgenes within their endogenouslociin mammalian cells and could besuitable for the correction of mutations responsible for blood disorders.