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. 2025 Nov 14;46(43):4610-4613.
doi: 10.1093/eurheartj/ehaf703.

CRISPR activation to repair electrocardiogram abnormalities caused by a FLNC truncating variant in mice

Rodrigo Cañas-Alvaro  1 , Laura Lalaguna  1 , Blanca Rubio  1 , Antonella Ausiello  1 , Marina López-Olañeta  1 , Rocío F Serrano-Blanco  2 , Juan Pablo Ochoa  1 , Jose Luis de la Pompa  2   3 , Alejandro Chavez  4 , Pablo García-Pavía  1   3   5 , Enrique Lara-Pezzi  1   3
Affiliations

CRISPR activation to repair electrocardiogram abnormalities caused by a FLNC truncating variant in mice

Rodrigo Cañas-Alvaro et al. Eur Heart J. .
No abstract available

Keywords: CRISPR activation; Cardiomyopathy; Dilated cardiomyopathy; FLNC truncating variants; Gene therapy; Non-dilated left ventricular cardiomyopathy.

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Figures

Figure 1
Figure 1
CRISPR activation–based gene therapy improves cardiac function in a mouse model of cardiomyopathy caused by FLNCtv. (A) Schematic showing the design of the mutant mouse bearing a Filamin C truncating variant. Deletion of exon 15 (Ex15del) in the mouse Filamin C gene. Below, Filamin C amino acid sequence of the FLNCtv patient on which the model is based and our mouse model for Ex15del, compared with wild type in each species. Filamin C mRNA (n = 8–15/group) and protein (n = 6/group) expression were quantified in the left ventricle of wild-type (wt/wt) and heterozygous mutant mice (Ex15del/wt). Genomic DNA and RNA were extracted from left ventricle myocardial samples from Ex15del mice, and RNA was reverse-transcribed into complementary DNA. The Filamin C region flanking the exon 15 was amplified by polymerase chain reaction, and the resulting product was cloned and transformed into bacteria. The plasmid in each colony was sequenced, and the number of colonies (as % of the total) bearing a wild-type or mutant allele was quantified for genomic DNA and RNA. n = 24 colonies. (B) The amplitude and duration of the QRS complex were measured by electrocardiogram in wt/wt and Ex15del/wt mice (n = 11–28/group and time point). (C) Mice were injected intraperitoneally with 40 mg/kg flecainide at 37 weeks of age, and the incidence of arrhythmias, QRS amplitude, and duration were measured in wild-type and mutant mice electrocardiograms (n = 4–10/group). Arrhythmias were defined as the occurrence of more than three premature ventricular complexes and/or second-degree Type II atrioventricular blocks within a 10-s interval after flecainide administration. (D) Schematics showing the CRISPR activation approach comprising the dead Cas9 linked to the VP64 transcription activation domain guided to the promoter of interest and the adeno-associated virus vector design (‘Beta-adeno-associated virus’). HL-1 cells were transfected with constructs bearing different protospacer sequences and Filamin C expression was quantified by qRT-PCR (n = 6/condition). The scaffold sequence of the sgRNA (#1) was modified (#3), and the effect on Filamin C induction was assessed by quantitative real-time reverse transcription polymerase chain reaction (n = 14–16/condition). Transfection with EGFP was used as a negative control. Protospacer: 5'-GCCAGGAATGCCGCCCGGCCC-3’; Scaffold: 5'-GTTTAAGTACTCTGGAAACAGAATCTACTTAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGA-3’. (E) Exdel15/wt mice were infected with Beta-adeno-associated virus carrying the CRISPR activation machinery and the Filamin C promoter-targeting sgRNA, and the expressions of Filamin C and dCas9-VP64 were determined by quantitative real-time reverse transcription polymerase chain reaction in the left ventricle myocardium (n = 8–10/condition). Filamin C protein expression was quantified by western blot. Wild-type (wt/wt) and uninfected Exdel15/wt mice (Ex15del/wt) were used as controls. (F) Mice were infected as in (E) at 32 weeks of age, and the QRS amplitude, QRS duration, and incidence of arrhythmias were determined at 40 weeks in the absence or presence of flecainide intraperitoneal injection (n = 10–14). Data shown as group mean ± standard deviation. Dashed line indicates wild-type average values at 40 weeks or age (F). Specific P-values shown for each comparison. Samples were tested for normality and homogeneity of variance. χ2 (C and F; no expected cell count was <1 and fewer than 20% were <5), independent t-test (A and E), one-way analysis of variance (D and E), and two-way analysis of variance plus Šídák’s multiple comparisons test (B, genotype and time; C and F, genotype and treatment) were used
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References

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