Abstract
Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employ a combination of the viral vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.
| Original language | English |
|---|---|
| Article number | 3714 |
| Journal | Nature communications |
| Volume | 14 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Dec 2023 |
| Externally published | Yes |
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In: Nature communications, Vol. 14, No. 1, 3714, 12.2023.
Research output: Contribution to journal › Article › Academic › peer-review
TY - JOUR
T1 - Striated muscle-specific base editing enables correction of mutations causing dilated cardiomyopathy
AU - Grosch, Markus
AU - Schraft, Laura
AU - Chan, Adrian
AU - Küchenhoff, Leonie
AU - Rapti, Kleopatra
AU - Ferreira, Anne Maud
AU - Kornienko, Julia
AU - Li, Shengdi
AU - Radke, Michael H.
AU - Krämer, Chiara
AU - Clauder-Münster, Sandra
AU - Perlas, Emerald
AU - Backs, Johannes
AU - Gotthardt, Michael
AU - Dieterich, Christoph
AU - van den Hoogenhof, Maarten M.G.
AU - Grimm, Dirk
AU - Steinmetz, Lars M.
N1 - Funding Information: We sincerely acknowledge the Laboratory Animal Resources (LAR) facility at EMBL for supporting the mouse experiments. Specifically, we thank Ernesto de la Cueva Bueno who supported ethics approval for the mouse experiments, Frank Diego Montoya Castillo who performed AAV injections and organ harvesting, and Isabel Clara Rollan Delgado who performed the zygotic injections for generation of the Rbm20 knock-in mice. Guide RNAs and donor sequences for the mouse line generation were designed by Anya Grozhik. The students Anastasiia Korosteleva, Maral Azodi and Stephanie Wendel performed supporting experiments related to this study. Further support was received from the Genomics Core Facility at EMBL, headed by Vladimir Benes, for RNA and DNA sequencing. We are very grateful for the expertise of Laura Villacorta, Mireia Osuna Lopez and Hilal Ozgur in generating WGS and bulk RNA sequencing libraries, as well as performing the Illumina sequencing. AAV9 and lentivirus were prepared by the Genetic and Viral Engineering Facility at EMBL Rome headed by Jim Sawitzke. Moreover, we acknowledge Carmen Judis (MDC Berlin) for supporting the TTN VAGE analysis. This work was mainly funded by the Bundesministerium für Bildung und Forschung (BMBF), the Else Kröner-Fresenius-Stiftung (EKFS), the CRC 1550 “Molecular Circuits of Heart Disease” and the Leducq Foundation. M.G. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 101031265. Funding Information: We sincerely acknowledge the Laboratory Animal Resources (LAR) facility at EMBL for supporting the mouse experiments. Specifically, we thank Ernesto de la Cueva Bueno who supported ethics approval for the mouse experiments, Frank Diego Montoya Castillo who performed AAV injections and organ harvesting, and Isabel Clara Rollan Delgado who performed the zygotic injections for generation of the Rbm20 knock-in mice. Guide RNAs and donor sequences for the mouse line generation were designed by Anya Grozhik. The students Anastasiia Korosteleva, Maral Azodi and Stephanie Wendel performed supporting experiments related to this study. Further support was received from the Genomics Core Facility at EMBL, headed by Vladimir Benes, for RNA and DNA sequencing. We are very grateful for the expertise of Laura Villacorta, Mireia Osuna Lopez and Hilal Ozgur in generating WGS and bulk RNA sequencing libraries, as well as performing the Illumina sequencing. AAV9 and lentivirus were prepared by the Genetic and Viral Engineering Facility at EMBL Rome headed by Jim Sawitzke. Moreover, we acknowledge Carmen Judis (MDC Berlin) for supporting the TTN VAGE analysis. This work was mainly funded by the Bundesministerium für Bildung und Forschung (BMBF), the Else Kröner-Fresenius-Stiftung (EKFS), the CRC 1550 “Molecular Circuits of Heart Disease” and the Leducq Foundation. M.G. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 101031265. Publisher Copyright: © 2023, The Author(s).
PY - 2023/12
Y1 - 2023/12
N2 - Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employ a combination of the viral vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.
AB - Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employ a combination of the viral vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.
UR - http://www.scopus.com/inward/record.url?scp=85162821581&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/s41467-023-39352-1
DO - https://doi.org/10.1038/s41467-023-39352-1
M3 - Article
C2 - 37349314
SN - 2041-1723
VL - 14
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 3714
ER -