Ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease

Abstract

CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIIδ, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIIδ gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIIδ editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIIδ gene editing may thus represent a permanent and advanced strategy for heart disease therapy.

Fig. 1.. Genomic editing of CaMKIIδ in human iPSC-cardiomyocytes.

(A) Schematic of CaMKIIδ and its three domains. Both critical methionines (Met281 and Met282) are located in the regulatory domain. Upon oxidative stress, these methionines are oxidized, resulting in increased CaMKII activity and cardiac disease. Using CRISPR-Cas9 adenine base editing, we identified sgRNA1, which edited only c.A841G (p.M281V; sgRNA1), and sgRNA6, which edited c.A841G (p.M281V), c.A844G (p.M282V), and c.A848G (p.H283R; sgRNA6), thereby preventing CaMKII activation upon oxidative stress. (B) Sequence of a segment of exon 11 of CaMKIIδ genomic DNA encoding part of the regulatory domain of CaMKIIδ. Alignment of sgRNA1 and sgRNA6 with CaMKIIδ. PAM sequences for sgRNA1 and sgRNA6 are in purple and green, respectively. Both ATGs encoding methionines are highlighted in yellow. Adenines within the sequences of sgRNA1 (purple) and sgRNA6 (green) are numbered (starting from the PAM). (C) Percentage of adenine (A) to guanine (G) editing in human iPSCs for each adenine in sgRNA1 after base editing with ABE8e and sgRNA1, as determined by Sanger sequencing. (D) Percentage of adenine (A) to guanine (G) editing in human iPSCs for each adenine in sgRNA6 after base editing with ABE8e and sgRNA6, as determined by deep amplicon sequencing. (E) Western blot analysis of oxidized CaMKII (specific antibody), autophosphorylated CaMKII (specific antibody), total CaMKII, and GAPDH in human WT, sgRNA1, and sgRNA6 iPSC-CMs for control group and after simulated ischemia/reperfusion (IR). (F) Mean densitometric analysis for oxidized CaMKII normalized to total CaMKII in control and post-IR iPSC-CMs (n = 5 independent iPSC-CM differentiations). (G) Mean densitometric analysis for autophosphorylated CaMKII normalized to total CaMKII in control and post-IR iPSC-CMs (n = 5 independent iPSC-CM differentiations). (H) Scatter bar plot showing mean CaMKII activity in control and post-IR iPSC-CMs and in lysates of WT post-IR iPSC-CMs in presence of the CaMKII inhibitor AIP (n = 5 independent iPSC-CM differentiations). (I) Western blot analysis of ryanodine receptor type 2 (RyR2) phosphorylation at the CaMKII site (serine 2814), total RyR2, and GAPDH in control and post-IR iPSC-CMs (n = 5 independent iPSC-CM differentiations). (J) Mean densitometric analysis for phosphorylated RyR2 normalized to total RyR2 in control and post-IR iPSC-CMs (n = 5 independent iPSC-CM differentiations). (K) Mean Ca²⁺ transient amplitude for each group (based on the number of cardiomyocytes). (L) Percentage of iPSC-CMs showing arrhythmias, as measured by epifluorescence microscopy. Statistical comparisons are based on one-way analysis of variance (ANOVA) post-hoc corrected by Holm-Sidak [(F) to (H)] and [(J) and (K)] and on Fisher’s exact test (L). Data are presented either as individual data points with means ± SEM or as percent of cells (L).

Fig. 2.. CaMKIIδ base editing improves cardiac function in vivo post-IR.

(A) Experimental design for subjecting mice to IR, injecting AAV-ABE-sgRNA6 for CaMKIIδ editing in vivo and monitoring heart function by echocardiography and cardiac magnetic resonance imaging (MRI). The AAV9 delivery system carrying the CRISPR-Cas9 base editing components with a split-intein trans-splicing system is shown. (B) Time course of fractional shortening for each group before IR as well as 24 hours, 1 week, 2 weeks, and 3 weeks post-IR (n =8 mice for each group; x axis not shown to scale). (C) Representative M-mode recordings of hearts of a sham-treated mouse, a mouse subjected to IR, a mouse subjected to IR with intracardiac injection of a control virus, and a mouse subjected to IR with intracardiac injection of AAV-ABE-sgRNA6 (IR+Edit) at 3 weeks post-IR. (D) Mean left ventricular end-diastolic diameter 3 weeks post-IR (n =8 mice for each group). (E) Mean left ventricular ejection fraction determined by cardiac MRI 4 weeks post-IR (n = 5 mice for each group). All replicates are individual mice. Statistical comparisons are based on two-way (B) and one-way [(D) and (E)] ANOVA post-hoc corrected by Holm-Sidak. Data are presented as means ± SEM.

Fig. 3.. Analysis of mouse hearts after CaMKIIδ in vivo genomic editing by administration of AAV-ABE-sgRNA6.

(A) Percentage of adenine (A) to guanine (G) editing of DNA and cDNA for each adenine along sgRNA6 in the myocardium of mice treated with AAV-ABE-sgRNA6, as determined by deep amplicon sequencing. (B) Spatial analysis of adenine (A) to guanine (G) editing efficiency at the cDNA level for each adenine along sgRNA6 in the anterior and the inferior cardiac wall of mice injected with AAV-ABE-sgRNA6 in the anterior cardiac wall, as determined by Sanger sequencing. (C) Sequencing of a TOPO-TA clone shows in vivo editing of the CaMKIIδ gene at the cDNA level. (D) Percentage of transcriptome-wide adenine (A) to inosine (I) editing in the myocardium of sham-treated, IR, IR treated with a control virus, and IR-edited mice. (E) Western blot analysis of oxidized CaMKII, autophosphorylated CaMKII, total CaMKII, and GAPDH for all groups. (F) Mean densitometric analyses for oxidized CaMKII normalized to total CaMKII for sham-treated, IR, IR treated with a control virus, and IR-edited mice. (G) Mean densitometric analyses for autophosphorylated CaMKII normalized to total CaMKII for all groups. (H) Mean CaMKII activity for all groups and for lysates of IR and IR+Control Virus mice, both in presence of the CaMKII inhibitor AIP. (I) Western blot analysis of ryanodine receptor type 2 (RyR2) phosphorylation at the CaMKII site (serine 2814), total RyR2, and GAPDH for all groups. (J) Mean densitometric analysis for phosphorylated RyR2 normalized to total RyR2 for all groups. (K) Heat map of 209 differentially expressed genes between mice subjected to IR and injected with either a control AAV9 (n = 3) or AAV-ABE-sgRNA6 for editing of the CaMKIIδ gene (n = 4). (L) Gene ontology terms associated with the 101 genes up-regulated in mouse hearts injected with AAV-ABE-sgRNA6 compared to mice receiving control AAV9. (M) Gene ontology terms associated with the 108 genes down-regulated in mouse hearts injected with AAV-ABE-sgRNA6 compared to mice injected with control AAV9. All replicates are individual mice. Statistical comparisons are based on one-way ANOVA post-hoc corrected by Holm-Sidak. Data are presented as individual data points with means ± SEM.

Fig. 4.. Genomic editing of CaMKIIδ prevents cardiac cell death and fibrosis after IR.

(A) Immunohistochemistry of TUNEL (green, arrows), Hoechst 33342 (blue, for all nuclei), and cardiac troponin (recolored in purple for colorblind accessibility) in representative heart sections of mice subjected to sham surgery, IR, IR treated with a control AAV9, and IR treated with AAV-ABE-sgRNA6 (IR+Edit; scale bar 20 μm). (B) Mean percentage of TUNEL-positive cells in each group. (C) Whole transverse cross-sections of trichrome stained hearts for each group (scale bar 500 μm). (D) Mean percentage of fibrotic cardiac tissue in each group. Replicates are individual mice. Statistical comparisons are based on one-way ANOVA post-hoc corrected by Holm-Sidak. Data are presented as individual data points with means ± SEM.