Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR Cas9 system
BackgroundReplacement therapy for hemophilia remains a lifelong treatment. Only gene therapy can cure hemophilia at a fundamental level. The clustered regularly interspaced short palindromic repeats CRISPR associated nuclease 9 (CRISPR Cas9) system is a versatile and convenient genome editing tool which can be applied to gene therapy for hemophilia.MethodsA patient's induced pluripotent stem cells (iPSCs) were generated from their peripheral blood mononuclear cells (PBMNCs) using episomal vectors. The AAVS1 Cas9 sgRNA plasmid which targets the AAVS1 locus and the AAVS1 EF1a F9 cDNA puromycin donor plasmid were constructed, and they were electroporated into the iPSCs. When insertion of F9 cDNA into the AAVS1 locus was confirmed, whole genome sequencing (WGS) was carried out to detect the off target issue. The iPSCs were then differentiated into hepatocytes, and human factor IX (hFIX) antigen and activity were measured in the culture supernatant. Finally, the hepatocytes were transplanted into non obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice through splenic injection.ResultsThe patient's iPSCs were generated from PBMNCs. Human full length F9 cDNA was inserted into the AAVS1 locus of iPSCs of a hemophilia B patient using the CRISPR Cas9 system. No off target mutations were detected by WGS. The hepatocytes differentiated from the inserted iPSCs could secrete hFIX stably and had the ability to be transplanted into the NOD/SCID mice in the short term.ConclusionsPBMNCs are good somatic cell choices for generating iPSCs from hemophilia patients. The iPSC technique is a good tool for genetic therapy for human hereditary diseases. CRISPR Cas9 is versatile, convenient, and safe to be used in iPSCs with low off target effects. Our research offers new approaches for clinical gene therapy for hemophilia.
Stem Cell Research Therapy