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Abstract
The common IVSI-110 (G>A) β-thalassemia mutation is a paradigm for intronic disease-causing mutations and their functional repair by non-homologous end joining-mediated disruption. Such mutation-specific repair by disruption of aberrant regulatory elements (DARE) is highly efficient, but to date, no systematic analysis has been performed to evaluate disease-causing mutations as therapeutic targets. Here, DARE was performed in highly characterized erythroid IVSI-110(G>A) transgenic cells and the disruption events were compared with published observations in primary CD34+ cells. DARE achieved the functional correction of β-globin expression equally through the removal of causative mutations and through the removal of context sequences, with disruption events and the restriction of indel events close to the cut site closely resembling those seen in primary cells. Correlation of DNA-, RNA-, and protein-level findings then allowed the extrapolation of findings to other mutations by in silico analyses for potential repair based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, Cas12a, and transcription activator-like effector nuclease (TALEN) platforms. The high efficiency of DARE and unexpected freedom of target design render the approach potentially suitable for 14 known thalassemia mutations besides IVSI-110(G>A) and put it forward for several prominent mutations causing other inherited diseases. The application of DARE, therefore, has a wide scope for sustainable personalized advanced therapy medicinal product development for thalassemia and beyond.
Highlights
Thalassemias are caused by the deficient production of globins
In order to allow the clonal analysis of the effect of indel events on HBBIVSI-110(G>A)-derived expression from a single locus and without the interference of endogenous HBB-derived expression, we drew on the humanized transgenic polyclonal Murine Erythroid Leukemia (MEL)-HBBIVS cell line published elsewhere [19]
MEL-HBBIVS cells express ≈5% of functional globin chain levels observed in their normal counterpart MEL-HBB (VCN ≈ 2), similar to the ratio seen in peripheral blood of HBBIVSI-110(G>A) homozygous patients compared to normal controls [19]
Summary
Beta-thalassemia has particular clinical relevance and as a monogenic blood disorder affecting the expression of β-globin (HBB), is an ideal target for therapy development based on gene addition or gene correction It is a frequent testbed for more widely applicable advanced therapy medicinal product (ATMP) technology, including the establishment of efficient mutation-specific therapies, which in most cases employ precise sequence repair by homology-directed repair (HDR) or the nascent base or prime editing technologies [1,2]. Despite the early application of zinc finger nucleases (ZFN) to the HBB locus [3,4] and rapid method development for gene editing since, precise correction of mutations is, usually still too inefficient for clinical use [5] This is true for ZFN-based tools, the redesign of which for new targets is notoriously difficult [6], as for the two more recent and more accessible editing platforms, transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nucleases (Cas). Our study vindicated the DARE approach for application to many other disease targets, by highlighting an unexpected degree of flexibility for the functional correction of splicing
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