A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy

Neha Dhoke; Hyunkee Kim; Sridhar Selvaraj; Karim Azzag; Haowen Zhou; Nelio A.J. Oliveira; Sudheer K Tungtur; Carolina Ortiz Cordero; James Kiley; Qi Long Lu; Anne G. Bang; Rita C.R. Perlingeiro

doi:10.1016/j.celrep.2021.109360

A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy

Neha Dhoke, Hyunkee Kim, Sridhar Selvaraj, Karim Azzag, Haowen Zhou, Nelio A.J. Oliveira, Sudheer K Tungtur, Carolina Ortiz Cordero, James Kiley, Qi Long Lu, Anne G. Bang, Rita C.R. Perlingeiro

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

11 Scopus citations

Abstract

Mutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRP^P448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs.

Original language	English (US)
Article number	109360
Journal	Cell reports
Volume	36
Issue number	2
DOIs	https://doi.org/10.1016/j.celrep.2021.109360
State	Published - Jul 13 2021

Bibliographical note

Funding Information:
This project was supported by NIH grants R01 AR071439 and AR055299 (to R.C.R.P.) and the LGMD2I Research Funds (to R.C.R.P. and A.G.B.). We are grateful to Jiri Vajsar for providing the WWS patient sample. We thank Lila Habib for her contribution in the generation of WWS iPS cells. The monoclonal antibody to MHC and the IIH6 antibody were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa. The authors are grateful to Cynthia Faraday for graphic design.

Funding Information:
This project was supported by NIH grants R01 AR071439 and AR055299 (to R.C.R.P.) and the LGMD2I Research Funds (to R.C.R.P. and A.G.B.). We are grateful to Jiri Vajsar for providing the WWS patient sample. We thank Lila Habib for her contribution in the generation of WWS iPS cells. The monoclonal antibody to MHC and the IIH6 antibody were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa. The authors are grateful to Cynthia Faraday for graphic design. N.R.D. designed and performed the experiments, analyzed the data, and wrote the manuscript. H.K. S.S. and K.A. designed and performed the experiments and analyzed the data. H.Z. N.A.J.O. S.T. and J.K. performed the experiments and analyzed the data. C.O.-C. contributed to design and data analysis. Q.L.L. and A.G.B. provided the reagents and contributed to the interpretation of the data and the writing of the manuscript. R.C.R.P. contributed to the experimental design, interpretation of the data, and writing of the manuscript. The authors declare no competing interests.

Publisher Copyright:
© 2021 The Author(s)

Keywords

CRISPR-Cas9
FKRP mutations
WWS
gene editing
muscle engraftment
patient-specific iPS cells
phenotype rescue
satellite cell engraftment
universal correction
α-dystroglycan

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10.1016/j.celrep.2021.109360

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Dhoke, N., Kim, H., Selvaraj, S., Azzag, K., Zhou, H., Oliveira, N. A. J., Tungtur, S. K., Ortiz Cordero, C., Kiley, J., Lu, Q. L., Bang, A. G., & Perlingeiro, R. C. R. (2021). A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy. Cell reports, 36(2), Article 109360. https://doi.org/10.1016/j.celrep.2021.109360

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title = "A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy",

abstract = "Mutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRPP448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs.",

keywords = "CRISPR-Cas9, FKRP mutations, WWS, gene editing, muscle engraftment, patient-specific iPS cells, phenotype rescue, satellite cell engraftment, universal correction, α-dystroglycan",

author = "Neha Dhoke and Hyunkee Kim and Sridhar Selvaraj and Karim Azzag and Haowen Zhou and Oliveira, {Nelio A.J.} and Tungtur, {Sudheer K} and {Ortiz Cordero}, Carolina and James Kiley and Lu, {Qi Long} and Bang, {Anne G.} and Perlingeiro, {Rita C.R.}",

note = "Funding Information: This project was supported by NIH grants R01 AR071439 and AR055299 (to R.C.R.P.) and the LGMD2I Research Funds (to R.C.R.P. and A.G.B.). We are grateful to Jiri Vajsar for providing the WWS patient sample. We thank Lila Habib for her contribution in the generation of WWS iPS cells. The monoclonal antibody to MHC and the IIH6 antibody were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa. The authors are grateful to Cynthia Faraday for graphic design. Funding Information: This project was supported by NIH grants R01 AR071439 and AR055299 (to R.C.R.P.) and the LGMD2I Research Funds (to R.C.R.P. and A.G.B.). We are grateful to Jiri Vajsar for providing the WWS patient sample. We thank Lila Habib for her contribution in the generation of WWS iPS cells. The monoclonal antibody to MHC and the IIH6 antibody were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa. The authors are grateful to Cynthia Faraday for graphic design. N.R.D. designed and performed the experiments, analyzed the data, and wrote the manuscript. H.K. S.S. and K.A. designed and performed the experiments and analyzed the data. H.Z. N.A.J.O. S.T. and J.K. performed the experiments and analyzed the data. C.O.-C. contributed to design and data analysis. Q.L.L. and A.G.B. provided the reagents and contributed to the interpretation of the data and the writing of the manuscript. R.C.R.P. contributed to the experimental design, interpretation of the data, and writing of the manuscript. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2021 The Author(s)",

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doi = "10.1016/j.celrep.2021.109360",

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T1 - A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy

AU - Dhoke, Neha

AU - Kim, Hyunkee

AU - Selvaraj, Sridhar

AU - Azzag, Karim

AU - Zhou, Haowen

AU - Oliveira, Nelio A.J.

AU - Tungtur, Sudheer K

AU - Ortiz Cordero, Carolina

AU - Kiley, James

AU - Lu, Qi Long

AU - Bang, Anne G.

AU - Perlingeiro, Rita C.R.

N1 - Funding Information: This project was supported by NIH grants R01 AR071439 and AR055299 (to R.C.R.P.) and the LGMD2I Research Funds (to R.C.R.P. and A.G.B.). We are grateful to Jiri Vajsar for providing the WWS patient sample. We thank Lila Habib for her contribution in the generation of WWS iPS cells. The monoclonal antibody to MHC and the IIH6 antibody were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa. The authors are grateful to Cynthia Faraday for graphic design. Funding Information: This project was supported by NIH grants R01 AR071439 and AR055299 (to R.C.R.P.) and the LGMD2I Research Funds (to R.C.R.P. and A.G.B.). We are grateful to Jiri Vajsar for providing the WWS patient sample. We thank Lila Habib for her contribution in the generation of WWS iPS cells. The monoclonal antibody to MHC and the IIH6 antibody were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa. The authors are grateful to Cynthia Faraday for graphic design. N.R.D. designed and performed the experiments, analyzed the data, and wrote the manuscript. H.K. S.S. and K.A. designed and performed the experiments and analyzed the data. H.Z. N.A.J.O. S.T. and J.K. performed the experiments and analyzed the data. C.O.-C. contributed to design and data analysis. Q.L.L. and A.G.B. provided the reagents and contributed to the interpretation of the data and the writing of the manuscript. R.C.R.P. contributed to the experimental design, interpretation of the data, and writing of the manuscript. The authors declare no competing interests. Publisher Copyright: © 2021 The Author(s)

PY - 2021/7/13

Y1 - 2021/7/13

N2 - Mutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRPP448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs.

AB - Mutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRPP448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs.

KW - CRISPR-Cas9

KW - FKRP mutations

KW - WWS

KW - gene editing

KW - muscle engraftment

KW - patient-specific iPS cells

KW - phenotype rescue

KW - satellite cell engraftment

KW - universal correction

KW - α-dystroglycan

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A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy

Abstract

Bibliographical note

Keywords

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