Genome editing for human gene therapy.
The rapid advancement of genome-editing techniques holds much promise for the field of human gene therapy. From bacteria to model organisms and human cells, genome editing tools such as zinc-finger nucleases (ZNFs), TALENs, and CRISPR/Cas9 have been successfully used to manipulate the respective genomes with unprecedented precision. With regard to human gene therapy, it is of great interest to test the feasibility of genome editing in primary human hematopoietic cells that could potentially be used to treat a variety of human genetic disorders such as hemoglobinopathies, primary immunodeficiencies, and cancer. In this chapter, we explore the use of the CRISPR/Cas9 system for the efficient ablation of genes in two clinically relevant primary human cell types, CD4+ T cells and CD34+ hematopoietic stem and progenitor cells. By using two guide RNAs directed at a single locus, we achieve highly efficient and predictable deletions that ablate gene function. The use of a Cas9-2A-GFP fusion protein allows FACS-based enrichment of the transfected cells. The ease of designing, constructing, and testing guide RNAs makes this dual guide strategy an attractive approach for the efficient deletion of clinically relevant genes in primary human hematopoietic stem and effector cells and enables the use of CRISPR/Cas9 for gene therapy.
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
Torsten B
TB
Meissner
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.
Pankaj K
PK
Mandal
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
Leonardo M R
LM
Ferreira
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA; Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA; Harvard Stem Cell Institute, Sherman Fairchild Biochemistry, Cambridge, Massachusetts, USA.
Derrick J
DJ
Rossi
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA; Harvard Stem Cell Institute, Sherman Fairchild Biochemistry, Cambridge, Massachusetts, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA. Electronic address: chad_cowan@harvard.edu.
Chad A
CA
Cowan
D018952:Antigens, CD34 / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D015894:Genome, Human; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans
D018952:Antigens, CD34
CD34+ HSPCs; CD4+ T Cells; CRISPR/Cas9; Dual guide strategy; Gene therapy; Genome editing