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

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 BanPaul S. Weiss, Donald B. Kohn, Kai Chen, Shih Hwa Chiou, Hsian Rong Tseng^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

23 Scopus citations

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.

Original language	English
Article number	abb7107
Journal	Science Advances
Volume	6
Issue number	43
DOIs	https://doi.org/10.1126/sciadv.abb7107
State	Published - 23 Oct 2020

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Yang, P., Chou, S. J., Li, J., Hui, W., Liu, W., Sun, N., Zhang, R. Y., Zhu, Y., Tsai, M. L., Lai, H. I., Smalley, M., Zhang, X., Chen, J., Romero, Z., Liu, D., Ke, Z., Zou, C., Lee, C. F., Jonas, S. J., ... Tseng, H. R. (2020). Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies. Science Advances, 6(43), Article abb7107. https://doi.org/10.1126/sciadv.abb7107

@article{bba44c66879c4b0a8c2e71ab0c760508,

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

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.",

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

note = "Publisher Copyright: Copyright {\textcopyright} 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).",

year = "2020",

month = oct,

day = "23",

doi = "10.1126/sciadv.abb7107",

language = "English",

volume = "6",

journal = "Science Advances",

issn = "2375-2548",

publisher = "American Association for the Advancement of Science",

number = "43",

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Yang, P, Chou, SJ, Li, J, Hui, W, Liu, W, Sun, N, Zhang, RY, Zhu, Y, Tsai, ML, Lai, HI, Smalley, M, Zhang, X, Chen, J, Romero, Z, Liu, D, Ke, Z, Zou, C, Lee, CF, Jonas, SJ, Ban, Q, Weiss, PS, Kohn, DB, Chen, K, Chiou, SH & Tseng, HR 2020, 'Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies', Science Advances, vol. 6, no. 43, abb7107. https://doi.org/10.1126/sciadv.abb7107

TY - JOUR

T1 - Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies

AU - Yang, Peng

AU - Chou, Shih Jie

AU - Li, Jindian

AU - Hui, Wenqiao

AU - Liu, Wenfei

AU - Sun, Na

AU - Zhang, Ryan Y.

AU - Zhu, Yazhen

AU - Tsai, Ming Long

AU - Lai, Henkie I.

AU - Smalley, Matthew

AU - Zhang, Xinyue

AU - Chen, Jiayuan

AU - Romero, Zulema

AU - Liu, Dahai

AU - Ke, Zunfu

AU - Zou, Chang

AU - Lee, Chin Fa

AU - Jonas, Steven J.

AU - Ban, Qian

AU - Weiss, Paul S.

AU - Kohn, Donald B.

AU - Chen, Kai

AU - Chiou, Shih Hwa

AU - Tseng, Hsian Rong

N1 - Publisher Copyright: 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).

PY - 2020/10/23

Y1 - 2020/10/23

N2 - 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.

AB - 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.

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U2 - 10.1126/sciadv.abb7107

DO - 10.1126/sciadv.abb7107

M3 - Article

C2 - 33097539

AN - SCOPUS:85094685256

SN - 2375-2548

VL - 6

JO - Science Advances

JF - Science Advances

IS - 43

M1 - abb7107

ER -

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

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