Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies

Peng Yang; Shih-Jie Chou; Jindian Li; Wenqiao Hui; Wenfei Liu; Na Sun; Ryan Y Zhang; Yazhen Zhu; Ming-Long Tsai; Henkie I Lai; Matthew Smalley; Xinyue Zhang; Jiayuan Chen; Zulema Romero; Dahai Liu; Zunfu Ke; Chang Zou; Chin-Fa Lee; Steven J Jonas; Qian Ban; Paul S Weiss; Donald B Kohn; Kai Chen; Shih-Hwa Chiou; Hsian-Rong Tseng

doi:10.1126/sciadv.abb7107

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies

Sci Adv. 2020 Oct 23;6(43):eabb7107. doi: 10.1126/sciadv.abb7107. Print 2020 Oct.

Authors

Peng Yang^{1

2}, Shih-Jie Chou^{3

4}, Jindian Li⁵, Wenqiao Hui⁶, Wenfei Liu⁷, Na Sun², Ryan Y Zhang², Yazhen Zhu², Ming-Long Tsai^{3

4}, Henkie I Lai^{3

4}, Matthew Smalley², Xinyue Zhang², Jiayuan Chen², Zulema Romero⁸, Dahai Liu⁹, Zunfu Ke¹⁰, Chang Zou¹¹, Chin-Fa Lee¹², Steven J Jonas¹³, Qian Ban¹⁴, Paul S Weiss¹⁵, Donald B Kohn¹⁶, Kai Chen¹⁷, Shih-Hwa Chiou^{18

4

19}, Hsian-Rong Tseng²⁰

Affiliations

¹ Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei 230601, China.
² Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging (CIMI), California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Division of Basic Research, Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.
⁴ Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
⁵ Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9061, USA.
⁶ Institute of Animal Husbandry and Veterinary Medicine, Anhui Academy of Agriculture Sciences, Hefei 230031, China.
⁷ Department of Chemistry and Biochemistry, Department of Bioengineering, Department of Materials Science and Engineering, and California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Department of Microbiology, Immunology and Molecular Genetics, DGSOM, Division of Pediatric Hematology-Oncology, Department of Pediatrics, David Geffen School of Medicine, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ School of Stomatology and Medicine, Foshan University, Foshan 528000, China.
¹⁰ Department of Pathology, Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
¹¹ Clinical Medical Research Center, The First Affiliated Hospital of Southern University, Shenzhen People's Hospital, Shenzhen 518020, China.
¹² Department of Chemistry, i-Center for Advanced Science and Technology, Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University (NCHU), 145 Xingda Road, South Dist., Taichung 402, Taiwan.
¹³ Department of Pediatrics, David Geffen School of Medicine, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Children's Discovery and Innovation Institute, California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁴ Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei 230601, China. hrtseng@mednet.ucla.edu shchiou@vghtpe.gov.tw chenkai@med.usc.edu dkohn1@mednet.ucla.edu psw@cnsi.ucla.edu bqashi@ahu.edu.cn.
¹⁵ Department of Chemistry and Biochemistry, Department of Bioengineering, Department of Materials Science and Engineering, and California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA 90095, USA. hrtseng@mednet.ucla.edu shchiou@vghtpe.gov.tw chenkai@med.usc.edu dkohn1@mednet.ucla.edu psw@cnsi.ucla.edu bqashi@ahu.edu.cn.
¹⁶ Department of Microbiology, Immunology and Molecular Genetics, DGSOM, Division of Pediatric Hematology-Oncology, Department of Pediatrics, David Geffen School of Medicine, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. hrtseng@mednet.ucla.edu shchiou@vghtpe.gov.tw chenkai@med.usc.edu dkohn1@mednet.ucla.edu psw@cnsi.ucla.edu bqashi@ahu.edu.cn.
¹⁷ Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9061, USA. hrtseng@mednet.ucla.edu shchiou@vghtpe.gov.tw chenkai@med.usc.edu dkohn1@mednet.ucla.edu psw@cnsi.ucla.edu bqashi@ahu.edu.cn.
¹⁸ Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan. hrtseng@mednet.ucla.edu shchiou@vghtpe.gov.tw chenkai@med.usc.edu dkohn1@mednet.ucla.edu psw@cnsi.ucla.edu bqashi@ahu.edu.cn.
¹⁹ Genomic Research Center, Academia Sinica, Taipei, Taiwan.
²⁰ Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging (CIMI), California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA 90095, USA. hrtseng@mednet.ucla.edu shchiou@vghtpe.gov.tw chenkai@med.usc.edu dkohn1@mednet.ucla.edu psw@cnsi.ucla.edu bqashi@ahu.edu.cn.

Abstract

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems*
Gene Editing
Genetic Vectors / genetics
Hemoglobinopathies* / genetics
Hemoglobinopathies* / therapy
Hemoglobins / genetics
Mice

Substances

Hemoglobins

Abstract

Publication types

MeSH terms

Substances

Grants and funding