Abstract
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-EF1α-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.
Highlights
Replacement therapy for hemophilia remains a lifelong treatment
Generation of induced pluripotent stem cell (iPSC) of the hemophilia B (HB) patient The patient with HB was clinically and genetically confirmed in the Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences, Tianjin, China. iPSC lines were generated from peripheral blood mononuclear cells (PBMNCs), using four episomal vectors, as described previously [22]
The efficiency of CRISPRCas9 in pluripotent stem cells (PSCs) is relatively low, we successfully knocked the human full-length F9 cDNA into the AAVS1 locus of the iPSCs without off-target effects. It is of far-reaching significance to construct single guide RNA (sgRNA) with high efficiency, especially when they are used in PSCs
Summary
Replacement therapy for hemophilia remains a lifelong treatment. 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. Hemophilia, caused by mutations of coagulation factors VIII (F8) and IX (F9), is one of the best-known hereditary hemorrhagic disorders. The current standard treatment is replacement therapy, which is effective but runs the risk of viral infection, and remains a lifelong treatment. Gene therapy can cure hemophilia at a fundamental level [1]. Adeno-associated viral (AAV) vectors are widely used in the gene therapy for HB. Immune responses to the AAV capsid limit the wider application of this approach
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