Abstract
The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% β-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.
Highlights
The blood disorder, b-thalassaemia, is considered an attractive target for gene correction
To quantitatively assay for gene editing, we used a mouse model with a b-globin/ green fluorescent protein (GFP) fusion transgene consisting of human b-globin intron 2 carrying a thalassaemia-associated IVS2-654 (C-T) mutation embedded within the GFP coding sequence, resulting in incorrect splicing of b-globin/GFP mRNA and lack of GFP expression[19]
We made the substitutions in the Watson–Crick domains because in prior work gPNAs have been shown to enhance strand invasion and DNA binding in the Watson–Crick binding mode due to helical pre-organization enforced by the modification[16]
Summary
The blood disorder, b-thalassaemia, is considered an attractive target for gene correction. We report that triplex-forming peptide nucleic acids (PNAs) substituted at the g position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human b-thalassaemia. GPNA treatment yielded gene editing in human CD34 þ HSCs ex vivo at a frequency of 5% with a single treatment, with very low off-target effects These results indicate that chemical advances in PNA design and in vivo delivery via polymer NPs can mediate clinically relevant levels of gene editing. They identify SCF treatment as a potential pharmacologic strategy to increase gene editing that may be applicable to triplex-forming PNAs and to approaches such as CRISPR/Cas[9]
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