Monogenic autosomal dominant disorders occur through the inheritance of a single copy of a defective gene. Autosomal dominant diseases include many of the serious and more common genetic disorders of adults like, such as Huntington’s Chorea, polycystic kidney disease and Tuberous Sclerosis. However, penetrance may not be complete in some individuals and this results in a milder form of the condition.
To correct double-stranded DNA mutations (dsDNA), different gene editing tools relying on the activity of nucleases have been developed. However, a major concern is that these tools can cause off-target effects in the host genome after treatment. Polypurine reverse Hoogsteen hairpins (PPRHs) are single-stranded, non-modified oligodeoxynucleotides and they are made up of two antiparallel polypurine mirror repeat domains linked by a five-thymidine loop. They have been used for silencing genes involved in resistance to immunotherapy approaches and chemotherapeutic drugs. Studies have shown that PPRHs do not cause off-target effects when a negative DNA hairpin is used and they are neither nephrotoxic nor hepatotoxic in vitro. Repair-PPRHs possess an extension sequence at the 5′ end of the molecule, similar to the DNA sequence to be corrected, containing the wild-type nucleotide instead of the mutated one. In a recent study, repair-PPRHs corrected a single-point mutation in a plasmid containing a mutated version of the dihydrofolate reductase gene.
University of Barcelona scientists: Alex J. Félix, Professors Dr. Carlos J. Ciudad and Dr. Véronique Noé demonstrated the general actions of the repair-PPRH technology. Their findings showed that repair-PPRHs can permanently correct point mutations in the dsDNA in mammalian cells at the endogenous level, with no off-target effects. The study is now published in the Journal Molecular Therapy: Nucleic Acids.
The research team developed three different repair-PPRH, each containing a hairpin core that bound to a particular polypyrimidine sequence close to the mutation. They observed that the repair-PPRHs were able to carry out targeted correction of three different point mutations in the endogenous locus of the aprt gene. In addition, the APRT mRNA levels in the repaired cells were increased compared to those of the mutant cell lines. No major differences were observed in the APRT mRNA levels of clone cells repaired by repair-PPRH with a target near the mutation sequence or farther away (long-distance repair-PPRH). APRT enzymatic activity was also rescued in the repaired clones.
The gene correction frequency was found to be increased by up to 2.5 folds in cells transfected in the S phase compared to those transfected in the asynchronous phase. An analysis of the whole-genome sequencing revealed the absence of off-target effects. The authors also performed gel-shift assays, which showed that repair-PPRHs bind to their target sequence. The binding of the repair-PPRHs to their target sequence resulted in the formation of a displacement-loop structure with the ability to induce the homology directed repair pathway believed to be responsible for the mutation correction observed in this study.
In summary, the study has advanced our knowledge to further the use of the repair-PPRH technology. Its clinical application as an alternative tool for gene editing will prove useful in correcting single point mutations primarily responsible for monogenic disorders.
Véronique Noé and Carlos J. Ciudad, Full Professors, lead the research group on Cancer Therapy, Gene silencing and editing at the School of Pharmacy of the University of Barcelona. Both professors obtained postdoctoral training at Columbia University in New York City.
Most of their research deals with Genomics and drug design. During the last decade they have been developed a new type of molecules termed Polypurine Reverse Hoogsteen hairpins (abbreviated as PPRHs) which have applications in the field of gene silencing, gene correction and gene editing.
PPRHs are non-modified DNA oligonucleotides, thus very easy and economical to synthesize. These hairpins are formed by two stretches of polypurines of about 25 nucleotides of length linked by 5 thymidines. Each arm is reverse to each other with a mirror-repeat structure. The two antiparallel polypurine stretches bind intramolecularly by Hoogsteen bonds resulting in a very stable hairpin structure. Polypurine hairpins then bind in a sequence specific manner to stretches of polypyrimidines in the double stranded DNA and/or to the RNA. This binding occurs by Watson and Crick bonds and forms a triplex. As a result, the fourth strand of the genomic dsDNA is displaced thus stimulating recombination. In this direction, when used for gene correction, the repair-PPRH consists of a clamp bearing at its 5’-end an extension sequence homologous to the sequence to be repaired but incorporating the wild type nucleotide. In addition, the binding of PPRHs to dsDNA can inhibit gene transcription leading to gene silencing.
Félix AJ, Ciudad CJ, Noé V. Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells. Mol Ther Nucleic Acids. 2020 ;19:683-695.Go To Mol Ther Nucleic Acids