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Stem Cell Res Ther. 2018 Apr 6;9(1):92. doi: 10.1186/s13287-018-0839-8.

Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system.

Author information

1
State Key Laboratory of Experimental Hematology, Key Laboratory of Gene Therapy of Blood Diseases, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin, 300020, China.
2
Department of Hematology, The First Central Hospital of Tianjin, Tianjin, 300192, China.
3
Department of Transfusion Medicine, Shanghai Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai, 200433, China.
4
Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92350, USA.
5
State Key Laboratory of Experimental Hematology, Key Laboratory of Gene Therapy of Blood Diseases, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin, 300020, China. zlpumc@hotmail.com.

Abstract

BACKGROUND:

Replacement 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.

METHODS:

A 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.

RESULTS:

The 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.

CONCLUSIONS:

PBMNCs 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.

KEYWORDS:

CRISPR-Cas systems; Cellular therapy; Genetic therapy; Hemophilia B; Hepatocyte differentiation; Induced pluripotent stem cells

PMID:
29625575
PMCID:
PMC5889534
DOI:
10.1186/s13287-018-0839-8
[Indexed for MEDLINE]
Free PMC Article

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