In Situ Genetic Correction of F8 Intron 22 Inversion in Hemophilia A Patient-Specific iPSCs

Sci Rep. 2016 Jan 8;6:18865. doi: 10.1038/srep18865.

Abstract

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5' part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23-26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Differentiation
  • Codon, Nonsense
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism
  • Exons
  • Factor VIII / biosynthesis
  • Factor VIII / genetics*
  • Factor VIII / metabolism
  • Gene Expression
  • Genetic Engineering
  • Genetic Therapy / methods*
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Hemophilia A / blood
  • Hemophilia A / genetics*
  • Hemophilia A / pathology
  • Hemophilia A / therapy
  • Homologous Recombination
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism*
  • Integrases / genetics*
  • Integrases / metabolism
  • Introns
  • Male
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / metabolism
  • Middle Aged
  • Primary Cell Culture
  • Promoter Regions, Genetic
  • Sequence Inversion*
  • Transcription Activator-Like Effector Nucleases / genetics*
  • Transcription Activator-Like Effector Nucleases / metabolism

Substances

  • Codon, Nonsense
  • Factor VIII
  • Cre recombinase
  • Integrases
  • Transcription Activator-Like Effector Nucleases