Abstract 8 CRISPR-Cas9 base editing to ablate oxidative CaMKIIδ activation as a therapeutic strategy for cardiac disease

Background:

Cardiovascular diseases are the most common cause of death worldwide, highlighting the need for new therapeutic strategies. Overactivation of the cardiac enzyme CaMKIIδ by oxidation of two methionines (Met281 and Met282) has been shown to be a main indicator and inducer of cardiac disease. We sought to develop a gene editing approach to mutate these critical methionines as a new therapeutic strategy.

Methods and Results:

CRISPR-Cas9 adenine base editing (ABE) of the CaMKIIδ gene was used to alter two ATG to GTG codons, thereby switching the methionines to valines and rendering the enzyme insensitive to oxidative activation (Fig. 1A). Several sgRNA and ABE combinations were screened to identify ABE8e combined with either of two sgRNAs (sgRNA1 and sgRNA6) with the highest adenine (A) to guanine (G) editing efficiency for the CaMKIIδ gene. This resulted in conversion of the oxidation-sensitive methionine residues 281 and 282 to valines (Fig. 1A).

Two independent iPSC lines were generated for each sgRNA and then differentiated to cardiomyocytes (iPSC-CMs). Following 5 h of ischemia and 14 h of reoxygenation (IR), Western blot analyses revealed increased CaMKII oxidation only in wildtype (WT), but not in edited iPSC-CMs where the oxidation-sensitive methionines were ablated (Fig. 1B+C). We detected increased CaMKII-dependent phosphorylation of RyR2 in WT, but not in CaMKIIδ edited iPSC-CMs (1D+E). Accordingly, WT iPSC-CMs post-IR exhibited increased diastolic Ca²⁺, decreased Ca²⁺ transient amplitude, and arrhythmias (epifluorescence microscopy, Fig. 1F-H). In contrast, iPSC-CM lines edited with sgRNA1 or sgRNA6 were protected from deleterious alterations. Since sgRNA6 was more effective than sgRNA1, we used sgRNA6 for CaMKIIδ editing in vivo (Fig. 2).

C57BL/6 mice at 12-weeks of age were subjected to cardiac injury by 45 minutes of ligation of the left anterior descending artery and subsequent reperfusion (IR). This was followed by either no treatment, injection of a control adeno-associated virus 9 (AAV9) or injection of AAV9 encoding ABE components to the injured cardiac area (n=8 for each group). Two weeks post-IR, mice administered ABE8e and sgRNA6 showed recovery of heart function compared to unedited mice, as seen by echocardiography (Fig. 2A+B) and cardiac MRI analysis. Three weeks post-IR, CaMKIIδ edited mice showed further recovery and attained the level of fractional shortening close to sham control mice (Fig. 2A+B).

Sequencing analyses revealed a high editing efficiency of 82.7±1.2% (Met281) and 85.7±0.7% (Met282) at the cDNA level in the injured area of the heart. We found increased CaMKII oxidation in hearts from control mice post-IR, but not when both methionines were ablated (Fig. 2C+D). Using bulk RNA sequencing, we detected 209 genes that were differentially expressed in CaMKIIδ edited mice post-IR, compared to mice administered control AAV9. While the upregulated genes were mainly related to cardiac function and contractility, the downregulated genes were linked to cardiac disease (Fig. 2E). Furthermore, control mice post-IR showed increased myocardial fibrosis, while CaMKIIδ edited mice were protected (trichrome staining, Fig. 2F+G).

Conclusion:

We ablated the oxidative activation sites of CaMKIIδ using CRISPR-Cas9 adenine base editing, conferring cardioprotection in human iPSC-CMs and adult mice subjected to IR. In vivo editing of the CaMKIIδ gene offers a new and permanent approach for heart disease therapy.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
CRISPR-Cas9 base editing to ablate oxidative CaMKIIδ activation as a therapeutic strategy for cardiac disease
S. Lebek¹, F. Chemello², X. Caravia², W. Tan², H. Li², K. Chen³, L. Xu³, N. Liu², R. Bassel-Duby², E. Olson²
¹Klinik und Poliklinik für Innere Med. II, Kardiologie, Universitätsklinikum Regensburg, Regensburg; ²Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, US; ³Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas, US;
Background: Cardiovascular diseases are the most common cause of death worldwide, highlighting the need for new therapeutic strategies. Overactivation of the cardiac enzyme CaMKIIδ by oxidation of two methionines (Met281 and Met282) has been shown to be a main indicator and inducer of cardiac disease. We sought to develop a gene editing approach to mutate these critical methionines as a new therapeutic strategy. Methods and Results: CRISPR-Cas9 adenine base editing (ABE) of the CaMKIIδ gene was used to alter two ATG to GTG codons, thereby switching the methionines to valines and rendering the enzyme insensitive to oxidative activation (Fig. 1A). Several sgRNA and ABE combinations were screened to identify ABE8e combined with either of two sgRNAs (sgRNA1 and sgRNA6) with the highest adenine (A) to guanine (G) editing efficiency for the CaMKIIδ gene. This resulted in conversion of the oxidation-sensitive methionine residues 281 and 282 to valines (Fig. 1A). Two independent iPSC lines were generated for each sgRNA and then differentiated to cardiomyocytes (iPSC-CMs). Following 5 h of ischemia and 14 h of reoxygenation (IR), Western blot analyses revealed increased CaMKII oxidation only in wildtype (WT), but not in edited iPSC-CMs where the oxidation-sensitive methionines were ablated (Fig. 1B+C). We detected increased CaMKII-dependent phosphorylation of RyR2 in WT, but not in CaMKIIδ edited iPSC-CMs (1D+E). Accordingly, WT iPSC-CMs post-IR exhibited increased diastolic Ca²⁺, decreased Ca²⁺ transient amplitude, and arrhythmias (epifluorescence microscopy, Fig. 1F-H). In contrast, iPSC-CM lines edited with sgRNA1 or sgRNA6 were protected from deleterious alterations. Since sgRNA6 was more effective than sgRNA1, we used sgRNA6 for CaMKIIδ editing in vivo (Fig. 2). C57BL/6 mice at 12-weeks of age were subjected to cardiac injury by 45 minutes of ligation of the left anterior descending artery and subsequent reperfusion (IR). This was followed by either no treatment, injection of a control adeno-associated virus 9 (AAV9) or injection of AAV9 encoding ABE components to the injured cardiac area (n=8 for each group). Two weeks post-IR, mice administered ABE8e and sgRNA6 showed recovery of heart function compared to unedited mice, as seen by echocardiography (Fig. 2A+B) and cardiac MRI analysis. Three weeks post-IR, CaMKIIδ edited mice showed further recovery and attained the level of fractional shortening close to sham control mice (Fig. 2A+B). Sequencing analyses revealed a high editing efficiency of 82.7±1.2% (Met281) and 85.7±0.7% (Met282) at the cDNA level in the injured area of the heart. We found increased CaMKII oxidation in hearts from control mice post-IR, but not when both methionines were ablated (Fig. 2C+D). Using bulk RNA sequencing, we detected 209 genes that were differentially expressed in CaMKIIδ edited mice post-IR, compared to mice administered control AAV9. While the upregulated genes were mainly related to cardiac function and contractility, the downregulated genes were linked to cardiac disease (Fig. 2E). Furthermore, control mice post-IR showed increased myocardial fibrosis, while CaMKIIδ edited mice were protected (trichrome staining, Fig. 2F+G). Conclusion: We ablated the oxidative activation sites of CaMKIIδ using CRISPR-Cas9 adenine base editing, conferring cardioprotection in human iPSC-CMs and adult mice subjected to IR. In vivo editing of the CaMKIIδ gene offers a new and permanent approach for heart disease therapy.
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