Correction of a Factor VIII genomic inversion with designer-recombinases

Abstract

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 a 140 kb genomic inversion of the F8 gene, which is frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we developed a fused 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, resulted in prominent correction of the inversion and restored Factor VIII mRNA expression. Our data suggests that designer-recombinases may represent efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

Pairing of single mutations yields obligate Cre-type site-specific recombinases

“…Previously described plasmids containing the target sites of loxF8, loxF8L and loxF8R were used for evolution (pEVO-loxF8, pEVO-loxF8L and pEVO-loxF8R, respectively) ( Supplementary Figure S1A ) (Preprint) ( 9 ). To verify the presence of the vector within the bacteria, the vector contains a chloramphenicol resistance gene driven by the CAT promoter.…”

“…Significant progress has been made to engineer novel tyrosine SSRs capable of recombination on a range of DNA substrates ( 13–15 ), including non-symmetric sites ( 8 , 9 , 16 ). Approaches focusing on the modular nature of the recombinases have been effectively employed to alter specificity in both serine and tyrosine recombinases.…”

“…The distinct variants were then expressed together forming a functional heterotetramer capable of specifically excising a DNA fragment flanked by the desired asymmetric human target sites ( 8 ). More recently, this approach was used to correct a chromosomal inversion causing a genetic human disorder (Preprint) ( 9 ). By combining two designer-recombinases targeting the asymmetric loxF8 sequence located on the human X-chromosome, Lansing et al.…”

“…By combining two designer-recombinases targeting the asymmetric loxF8 sequence located on the human X-chromosome, Lansing et al. showed that the heterotetramer (D7) could efficiently correct the genomic int1h inversion to reestablish factor VIII expression in patient-derived cells (Preprint) ( 9 ). The D7 SSR is composed of two unique Cre-type subunits, D7L and D7R, each evolved to bind to their corresponding half site, loxF8L and loxF8R, respectively (Figure 1A ).…”

“…By using several Cre-derived recombinases with different specificities, there is an increased possibility of subunit assembly into undesired functional complexes, including homotetramers, and heterotetramers of unequal monomer combinations, that could cause recombination events at non-target sites. To mitigate these potential off-target effects, approaches to assure that only SSR heterotetramers with the desired monomer combination have catalytic activity are critical for increasing their safety in therapeutic applications (Preprint) ( 9 ). To improve the applied properties of the heterospecific D7/loxF8 recombinase system, we set out to identify mutations that render the monomers functionally inactive in isolation, while they can form active heterotetramers when co-expressed in cells.…”