FACTOR VIII MUTATION REPAIR AND TOLERANCE INDUCTION AND RELATED cDNAs, COMPOSITIONS, METHODS AND SYSTEMS

摘要

The present disclosure relates to methods, systems, and compositions to repair one or more mutations in a Factor VIII gene sequence of a subject by introducing into a cell of the subject one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) and one or more repair vehicles (RVs) containing at least a cDNA-repair sequence (RS) such that insertion of the cDNA-RS through homologous recombination with the F8 gene of the subject (sF8) provides a repaired F8 gene (rF8), the repaired F8 gene (rF8) upon expression forming a functional FVIII conferring improved coagulation functionality to the FVIII protein encoded by the sF8. The present disclosure also relates to cells derived using the methods, systems and compositions described.

主权项

1. A method for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject, the method comprising
introducing into a cell of the subject one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) and one or more repair vehicles (RVs) containing at least a cDNA-repair sequence (RS) flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA donor within each of the one or more repair vehicles (RVs), wherein
the DNA-SE is selected to be capable of targeting a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by the cDNA-RS,the cDNA-RS comprises a repaired version of the F8 gene sequence of the subject comprising the one or more mutations within a cDNA sequence encoding for a truncated Factor VIII, andthe upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene, and wherein introducing into a cell of the subject one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) and one or more repair vehicles (RVs) is performed to allow insertion of the cDNA-RS through homologous recombination of the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS) with the F8 gene of the subject (sF8) to provide a repaired F8 gene (rF8), the repaired F8 gene (rF8) upon expression forming a functional FVIII conferring improved coagulation functionality to the FVIII protein encoded by the sF8.