The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements

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.

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia

“…Moreover, based on the novel multiplex RT-qPCR protocol, this study revealed that aberrant RNA constitutes 60.9 ± 7.4% of total HBB-derived mRNA in primary erythroid cell cultures of HBB IVSI−110 patients, with residual expression of (differentiation-normalized) normal HBB mRNA reaching 37.2% × 2.1 ±1 of levels observed in corresponding healthy cultures. This first accurate measurement of missplicing and of normal HBB mRNA amounts in HBB IVSI−110 -homozygous erythroid cells underpins our previous observations that absolute HBB mRNA levels are not limiting for HBB production and that removal of aberrant transcripts alone may be sufficient to achieve a therapeutic effect in HBB IVSI−110 patients [13][14][15], even in the absence of a functional HBB trans-or endogene [13].…”

“…To quantify the apparent effect of the HBB IVSI−110 locus on the one hand and to allow correlation of HBB mRNA and protein levels in the efficiency evaluation of novel therapies on the other, it is necessary to monitor accurately both, steady-state total HBB-derived mRNA levels and the ratio of normal to aberrantly spliced RNA. For instance, optimization of measurement would improve the evaluation of the potential of β-thalassemia HBB IVSI−110 -specific gene therapy approaches [13][14][15]. In this study, we have, therefore, developed a duplex reverse-transcription quantitative PCR (RT-qPCR) assay for robust measurement of both mRNA species in patient-derived cells, and have employed it for mRNA measurements and correlation with HBB protein levels for proof of principle.…”

DNA Damage: From Threat to Treatment

“…In the attempt to restore normal splicing, both CRISPR-Cas and TALENs have been used to introduce indel mutations to disrupt the aberrant splice site. The high efficiency of this approach and the large number of mutations that could be treated using such strategies are promising to prompt future investigation [ 119 ]. Recently, genome editing for the treatment of beta-hemoglobinopathies has entered clinics with two ongoing trials.…”

Efficient CRISPR-Cas9-based genome editing of β-globin gene on erythroid cells from homozygous β039-thalassemia patients

“…4,5,12,13 To the best of our knowledge, no data are available in the literature on the CRISPR-Cas9-based gene correction of the b 0 39-thalassemia mutation. Most of the gene-editing intervention for b-thalassemia is focusing on other mutations (e.g., IVS2-654 or IVS I-110) 4,5,[18][19][20][21][22] or on the reactivation of fetal globin gene expression by disrupting the g-globin gene repressor BCL11A. 4,5,[23][24][25][26][27][28][29][30][31][32][33] An example is the paper by Antoniani et al 24 They designed a CRISPR-Cas9 strategy to disrupt a 13.6-kb genomic region encompassing the dand b-globin genes and a putative g-d intergenic HbF.…”