Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9-Enhanced Gene Targeting

Chia-Wei Chang; Yi-Shin Lai; Erik Westin; Alireza Khodadadi-Jamayran; Kevin M Pawlik; Lawrence S Lamb Jr; Frederick D Goldman; Tim M Townes

doi:10.1016/j.celrep.2015.08.013

Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9-Enhanced Gene Targeting

Cell Rep. 2015 Sep 8;12(10):1668-77. doi: 10.1016/j.celrep.2015.08.013. Epub 2015 Aug 28.

Authors

Chia-Wei Chang¹, Yi-Shin Lai¹, Erik Westin¹, Alireza Khodadadi-Jamayran¹, Kevin M Pawlik¹, Lawrence S Lamb Jr², Frederick D Goldman³, Tim M Townes⁴

Affiliations

¹ Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA; UAB Stem Cell Institute, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
² Department of Medicine, Division of Hematology/Oncology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Cell Therapy Lab, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA; UAB Stem Cell Institute, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
³ Department of Pediatrics, Division of Hematology/Oncology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA; UAB Stem Cell Institute, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
⁴ Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA; UAB Stem Cell Institute, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: ttownes@uab.edu.

PMID: 26321643
DOI: 10.1016/j.celrep.2015.08.013

Abstract

Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T(-)B(+)NK(-)). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.

MeSH terms

Bacterial Proteins / genetics
Base Sequence
CRISPR-Associated Protein 9
Cells, Cultured
Child, Preschool
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Mutational Analysis
Endonucleases / genetics
Gene Targeting
Genetic Therapy*
Humans
Induced Pluripotent Stem Cells / enzymology
Janus Kinase 3 / genetics*
Male
Mutation, Missense
Proto-Oncogene Proteins c-bcl-2 / metabolism
Severe Combined Immunodeficiency / genetics
Severe Combined Immunodeficiency / therapy*
T-Lymphocytes / physiology

Substances

BCL2 protein, human
Bacterial Proteins
Proto-Oncogene Proteins c-bcl-2
JAK3 protein, human
Janus Kinase 3
CRISPR-Associated Protein 9
Cas9 protein, Francisella novicida
Endonucleases

Associated data

SRA/SRP056149