Gene Knock-outs in Human CD34+ Hematopoietic Stem and Progenitor Cells and in the Human Immune System of Mice

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2023 Jun 28

PMID 37379309

Authors

Daniel A Kuppers

Jonathan Linton

Sergio Ortiz Espinosa

Kelly M McKenna

Anthony Rongvaux

Patrick J Paddison

Affiliations

Soon will be listed here.

Abstract

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate "humanized mice". To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and transplant these modified cells in immunodeficient mice with high engraftment levels and multilineage hematopoietic differentiation. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.

References

Kim M, Yu K, Kenderian S, Ruella M, Chen S, Shin T . Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia. Cell. 2018; 173(6):1439-1453.e19. PMC: 6003425. DOI: 10.1016/j.cell.2018.05.013. View

Allen F, Crepaldi L, Alsinet C, Strong A, Kleshchevnikov V, De Angeli P . Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat Biotechnol. 2018; . PMC: 6949135. DOI: 10.1038/nbt.4317. View

Sidney L, Branch M, Dunphy S, Dua H, Hopkinson A . Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells. 2014; 32(6):1380-9. PMC: 4260088. DOI: 10.1002/stem.1661. View

Amirache F, Levy C, Costa C, Mangeot P, Torbett B, Wang C . Mystery solved: VSV-G-LVs do not allow efficient gene transfer into unstimulated T cells, B cells, and HSCs because they lack the LDL receptor. Blood. 2014; 123(9):1422-4. DOI: 10.1182/blood-2013-11-540641. View

Voillet V, Berger T, McKenna K, Paulson K, Hong Tan W, Smythe K . An In Vivo Model of Human Macrophages in Metastatic Melanoma. J Immunol. 2022; 209(3):606-620. PMC: 9377377. DOI: 10.4049/jimmunol.2101109. View

Freeman S, Kelm S, Barber E, Crocker P . Characterization of CD33 as a new member of the sialoadhesin family of cellular interaction molecules. Blood. 1995; 85(8):2005-12. View

Kuppers D, Arora S, Lim Y, Lim A, Carter L, Corrin P . N-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis. Nat Commun. 2019; 10(1):4596. PMC: 6787028. DOI: 10.1038/s41467-019-12518-6. View

Wu Y, Zeng J, Roscoe B, Liu P, Yao Q, Lazzarotto C . Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat Med. 2019; 25(5):776-783. PMC: 6512986. DOI: 10.1038/s41591-019-0401-y. View

Appelbaum F . Hematopoietic-cell transplantation at 50. N Engl J Med. 2007; 357(15):1472-5. DOI: 10.1056/NEJMp078166. View

10.

Hong S, Hwang D, Yoon S, Isacson O, Ramezani A, Hawley R . Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells. Mol Ther. 2007; 15(9):1630-9. PMC: 2614215. DOI: 10.1038/sj.mt.6300251. View

11.

Ramezani A, Hawley T, Hawley R . Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol Ther. 2000; 2(5):458-69. DOI: 10.1006/mthe.2000.0190. View

12.

Petersdorf S, Kopecky K, Slovak M, Willman C, Nevill T, Brandwein J . A phase 3 study of gemtuzumab ozogamicin during induction and postconsolidation therapy in younger patients with acute myeloid leukemia. Blood. 2013; 121(24):4854-60. PMC: 3682338. DOI: 10.1182/blood-2013-01-466706. View

13.

Rongvaux A, Willinger T, Martinek J, Strowig T, Gearty S, Teichmann L . Development and function of human innate immune cells in a humanized mouse model. Nat Biotechnol. 2014; 32(4):364-72. PMC: 4017589. DOI: 10.1038/nbt.2858. View

14.

Wei J, He C . Chromatin and transcriptional regulation by reversible RNA methylation. Curr Opin Cell Biol. 2021; 70:109-115. PMC: 8119380. DOI: 10.1016/j.ceb.2020.11.005. View

15.

Dull T, Zufferey R, Kelly M, Mandel R, Nguyen M, Trono D . A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998; 72(11):8463-71. PMC: 110254. DOI: 10.1128/JVI.72.11.8463-8471.1998. View

16.

Shapiro J, Iancu O, Jacobi A, McNeill M, Turk R, Rettig G . Increasing CRISPR Efficiency and Measuring Its Specificity in HSPCs Using a Clinically Relevant System. Mol Ther Methods Clin Dev. 2020; 17:1097-1107. PMC: 7251314. DOI: 10.1016/j.omtm.2020.04.027. View

17.

Uchida N, Demirci S, Haro-Mora J, Fujita A, Raines L, Hsieh M . Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production. Mol Ther Methods Clin Dev. 2018; 9:247-256. PMC: 5948232. DOI: 10.1016/j.omtm.2018.03.007. View

18.

Frangoul H, Altshuler D, Cappellini M, Chen Y, Domm J, Eustace B . CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. N Engl J Med. 2020; 384(3):252-260. DOI: 10.1056/NEJMoa2031054. View

19.

Conant D, Hsiau T, Rossi N, Oki J, Maures T, Waite K . Inference of CRISPR Edits from Sanger Trace Data. CRISPR J. 2022; 5(1):123-130. DOI: 10.1089/crispr.2021.0113. View

20.

Shi X, Shou J, Mehryar M, Li J, Wang L, Zhang M . Cas9 has no exonuclease activity resulting in staggered cleavage with overhangs and predictable di- and tri-nucleotide CRISPR insertions without template donor. Cell Discov. 2019; 5:53. PMC: 6796948. DOI: 10.1038/s41421-019-0120-z. View