Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.
|Number of pages
|Published - 20 Jan 2022
DFG Classification of Subject Areas according to Review Boards
Sustainable Development Goals
ASJC Scopus subject areas
- Amino Acid Sequence, Base Sequence, Cell Differentiation, Chromosome Inversion/genetics, Clone Cells, Directed Molecular Evolution, Endothelial Cells/cytology, Exons/genetics, Factor VIII/genetics, HEK293 Cells, HeLa Cells, Humans, Induced Pluripotent Stem Cells/metabolism, Inverted Repeat Sequences/genetics, Recombinases/metabolism, Recombination, Genetic/genetics, Substrate Specificity, Whole Genome Sequencing