CRISPR/Cas9 β-globin Gene Targeting in Human Haematopoietic Stem Cells

Overview

Journal Nature

Specialty Science

Date 2016 Nov 8

PMID 27820943

Citations 444

Authors

Daniel P Dever

Rasmus O Bak

Andreas Reinisch

Joab Camarena

Gabriel Washington

Carmencita E Nicolas

Mara Pavel-Dinu

Nivi Saxena

Alec B Wilkens

Sruthi Mantri

Nobuko Uchida

Ayal Hendel

Anupama Narla

Ravindra Majeti

Kenneth I Weinberg

Matthew H Porteus

Affiliations

Soon will be listed here.

Abstract

The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

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References

Naldini L . Gene therapy returns to centre stage. Nature. 2015; 526(7573):351-60. DOI: 10.1038/nature15818. View

Romero Z, Urbinati F, Geiger S, Cooper A, Wherley J, Kaufman M . β-globin gene transfer to human bone marrow for sickle cell disease. J Clin Invest. 2013; . PMC: 4011030. DOI: 10.1172/JCI67930. View

Voit R, Hendel A, Pruett-Miller S, Porteus M . Nuclease-mediated gene editing by homologous recombination of the human globin locus. Nucleic Acids Res. 2013; 42(2):1365-78. PMC: 3902937. DOI: 10.1093/nar/gkt947. View

SMITHIES O, Gregg R, Boggs S, Koralewski M, Kucherlapati R . Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985; 317(6034):230-4. DOI: 10.1038/317230a0. View

Weidner C, Walenda T, Lin Q, Wolfler M, Denecke B, Costa I . Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture. Sci Rep. 2013; 3:3372. PMC: 3842544. DOI: 10.1038/srep03372. View

Rouet P, Smih F, Jasin M . Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci U S A. 1994; 91(13):6064-8. PMC: 44138. DOI: 10.1073/pnas.91.13.6064. View

de Ravin S, Reik A, Liu P, Li L, Wu X, Su L . Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol. 2016; 34(4):424-9. PMC: 4824656. DOI: 10.1038/nbt.3513. View

Doudna J, Charpentier E . Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014; 346(6213):1258096. DOI: 10.1126/science.1258096. View

Bonini C, Grez M, Traversari C, Ciceri F, Marktel S, Ferrari G . Safety of retroviral gene marking with a truncated NGF receptor. Nat Med. 2003; 9(4):367-9. DOI: 10.1038/nm0403-367. View

10.

Wang J, DeClercq J, Hayward S, Li P, Shivak D, Gregory P . Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res. 2015; 44(3):e30. PMC: 4756813. DOI: 10.1093/nar/gkv1121. View

11.

Sather B, Romano Ibarra G, Sommer K, Curinga G, Hale M, Khan I . Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci Transl Med. 2015; 7(307):307ra156. PMC: 4790757. DOI: 10.1126/scitranslmed.aac5530. View

12.

Cradick T, Fine E, Antico C, Bao G . CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 2013; 41(20):9584-92. PMC: 3814385. DOI: 10.1093/nar/gkt714. View

13.

Bonini C, Ferrari G, Verzeletti S, Servida P, Zappone E, Ruggieri L . HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science. 1997; 276(5319):1719-24. DOI: 10.1126/science.276.5319.1719. View

14.

Brinkman E, Chen T, Amendola M, van Steensel B . Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014; 42(22):e168. PMC: 4267669. DOI: 10.1093/nar/gku936. View

15.

Mukherjee S, Thrasher A . Gene therapy for PIDs: progress, pitfalls and prospects. Gene. 2013; 525(2):174-81. PMC: 3725417. DOI: 10.1016/j.gene.2013.03.098. View

16.

Barzel A, Paulk N, Shi Y, Huang Y, Chu K, Zhang F . Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature. 2014; 517(7534):360-4. PMC: 4297598. DOI: 10.1038/nature13864. View

17.

Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I . Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods. 2012; 23(1):18-28. DOI: 10.1089/hgtb.2011.034. View

18.

Hendel A, Kildebeck E, Fine E, Clark J, Punjya N, Sebastiano V . Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. Cell Rep. 2014; 7(1):293-305. PMC: 4015468. DOI: 10.1016/j.celrep.2014.02.040. View

19.

Gu A, Torres-Coronado M, Tran C, Vu H, Epps E, Chung J . Engraftment and lineage potential of adult hematopoietic stem and progenitor cells is compromised following short-term culture in the presence of an aryl hydrocarbon receptor antagonist. Hum Gene Ther Methods. 2014; 25(4):221-31. PMC: 4142829. DOI: 10.1089/hgtb.2014.043. View

20.

Jenq R, van den Brink M . Allogeneic haematopoietic stem cell transplantation: individualized stem cell and immune therapy of cancer. Nat Rev Cancer. 2010; 10(3):213-21. DOI: 10.1038/nrc2804. View