Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy

Sridhar Selvaraj; Neha R. Dhoke; James Kiley; Alba Judith Mateos-Aierdi; Sudheer Tungtur; Ricardo Mondragon-Gonzalez; Grace Killeen; Vanessa K.P. Oliveira; Adolfo López de Munain; Rita C.R. Perlingeiro

doi:10.1016/j.ymthe.2019.08.011

Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy

Sridhar Selvaraj, Neha R. Dhoke, James Kiley, Alba Judith Mateos-Aierdi, Sudheer Tungtur, Ricardo Mondragon-Gonzalez, Grace Killeen, Vanessa K.P. Oliveira, Adolfo López de Munain, Rita C.R. Perlingeiro

Medicine - Cardiology Division

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

35 Scopus citations

Abstract

Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives in vitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.

Original language	English (US)
Pages (from-to)	2147-2157
Number of pages	11
Journal	Molecular Therapy
Volume	27
Issue number	12
DOIs	https://doi.org/10.1016/j.ymthe.2019.08.011
State	Published - Dec 4 2019

Bibliographical note

Funding Information:
We thank the generous support from ADVault, Inc. and MyDirectives.com (R.C.R.P.). This project was also supported by funds from the NIH , grants R01 AR055299 and AR071439 (R.C.R.P.), the Coalition to Cure Calpain 3 (R.C.R.P.), and Instituto Carlos III, Fondo Investigaciones Sanitarias-ALM, PI17/01841 (A.J.M.-A and A.L.d.M). The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH grant P30 CA077598-09 . The monoclonal antibody to MHC was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We thank Melissa Spencer (UCLA) for the C3KO mice and Michael Kyba for critical reading of the manuscript.

Funding Information:
We thank the generous support from ADVault, Inc. and MyDirectives.com (R.C.R.P.). This project was also supported by funds from the NIH, grants R01 AR055299 and AR071439 (R.C.R.P.), the Coalition to Cure Calpain 3 (R.C.R.P.), and Instituto Carlos III, Fondo Investigaciones Sanitarias-ALM, PI17/01841 (A.J.M.-A and A.L.d.M). The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH grant P30 CA077598-09. The monoclonal antibody to MHC was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We thank Melissa Spencer (UCLA) for the C3KO mice and Michael Kyba for critical reading of the manuscript.

Publisher Copyright:
© 2019 The American Society of Gene and Cell Therapy

Keywords

CAPN3
Calpain 3
autologous cell therapy
gene correction
genome editing
iPSCs
induced pluripotent stem cells
limb girdle muscular dystrophy type 2A
myogenic progenitors

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Selvaraj, S., Dhoke, N. R., Kiley, J., Mateos-Aierdi, A. J., Tungtur, S., Mondragon-Gonzalez, R., Killeen, G., Oliveira, V. K. P., López de Munain, A., & Perlingeiro, R. C. R. (2019). Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy. Molecular Therapy, 27(12), 2147-2157. https://doi.org/10.1016/j.ymthe.2019.08.011

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abstract = "Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives in vitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.",

keywords = "CAPN3, Calpain 3, autologous cell therapy, gene correction, genome editing, iPSCs, induced pluripotent stem cells, limb girdle muscular dystrophy type 2A, myogenic progenitors",

author = "Sridhar Selvaraj and Dhoke, {Neha R.} and James Kiley and Mateos-Aierdi, {Alba Judith} and Sudheer Tungtur and Ricardo Mondragon-Gonzalez and Grace Killeen and Oliveira, {Vanessa K.P.} and {L{\'o}pez de Munain}, Adolfo and Perlingeiro, {Rita C.R.}",

note = "Funding Information: We thank the generous support from ADVault, Inc. and MyDirectives.com (R.C.R.P.). This project was also supported by funds from the NIH , grants R01 AR055299 and AR071439 (R.C.R.P.), the Coalition to Cure Calpain 3 (R.C.R.P.), and Instituto Carlos III, Fondo Investigaciones Sanitarias-ALM, PI17/01841 (A.J.M.-A and A.L.d.M). The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH grant P30 CA077598-09 . The monoclonal antibody to MHC was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We thank Melissa Spencer (UCLA) for the C3KO mice and Michael Kyba for critical reading of the manuscript. Funding Information: We thank the generous support from ADVault, Inc. and MyDirectives.com (R.C.R.P.). This project was also supported by funds from the NIH, grants R01 AR055299 and AR071439 (R.C.R.P.), the Coalition to Cure Calpain 3 (R.C.R.P.), and Instituto Carlos III, Fondo Investigaciones Sanitarias-ALM, PI17/01841 (A.J.M.-A and A.L.d.M). The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH grant P30 CA077598-09. The monoclonal antibody to MHC was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We thank Melissa Spencer (UCLA) for the C3KO mice and Michael Kyba for critical reading of the manuscript. Publisher Copyright: {\textcopyright} 2019 The American Society of Gene and Cell Therapy",

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doi = "10.1016/j.ymthe.2019.08.011",

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AU - Dhoke, Neha R.

AU - Kiley, James

AU - Mateos-Aierdi, Alba Judith

AU - Tungtur, Sudheer

AU - Mondragon-Gonzalez, Ricardo

AU - Killeen, Grace

AU - Oliveira, Vanessa K.P.

AU - López de Munain, Adolfo

AU - Perlingeiro, Rita C.R.

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N2 - Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives in vitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.

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KW - Calpain 3

KW - autologous cell therapy

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KW - genome editing

KW - iPSCs

KW - induced pluripotent stem cells

KW - limb girdle muscular dystrophy type 2A

KW - myogenic progenitors

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Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy

Abstract

Bibliographical note

Keywords

UN SDGs

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