Dilated cardiomyopathy (DCM): CRISPR-Cas9 therapy can reverse the mutation

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Using the CRISPR-Cas9 gene editing system, UT Southwestern researchers corrected mutations responsible for a common inherited heart condition called dilated cardiomyopathy (DCM) in human cells and a mouse model of the disease.

Their findings, published in Science Translational Medicine, may one day provide hope to an estimated 1 in 250 people worldwide who suffer from this condition.

“All of the disease characteristics we see because of these mutations were reversed with CRISPR-Cas9 therapy. It’s fair to say the success of this approach completely exceeded our expectations,” said Eric Olson, Ph.D., Chair and Professor of Molecular Biology at UTSW, who co-led the study with colleagues Rhonda Bassel-Duby, Ph.D., Professor of Molecular Biology, and Takahiko Nishiyama, M.D., Ph.D., a postdoctoral fellow in the Olson lab.

DCM is caused by mutations in a gene known as RNA binding motif protein 20 (RBM20), which affects the production of hundreds of proteins in cardiac muscle cells responsible for the heart’s pumping action. This disease wreaks widespread havoc throughout the heart, gradually destroying its ability to contract and causing it to become extremely enlarged and fail over time.

Treatment is limited to drugs, which can improve contractile function but don’t provide a permanent fix, or a heart transplant, which frequently isn’t an option due to a shortage of donor organs.

Seeking to attack the root cause of this disease, Drs. Olson, Bassel-Duby, Nishiyama, and their colleagues looked to CRISPR-Cas9, a popular tool for genetic research recognized with the Nobel Prize in Chemistry in 2020. Using this system, researchers can potentially correct disease-causing mutations in important genes.

Thus far, the Food and Drug Administration has approved a single clinical trial that uses this technology to try to treat sickle cell disease. However, Dr. Olson said CRISPR-Cas9 has huge potential to treat an untold number of other genetic diseases. Dr. Olson and colleagues have used CRISPR gene editing to develop a technique to halt progression of Duchenne muscular dystrophy in animal models.

To determine the feasibility of this approach for DCM, the research team used a virus to deliver CRISPR-Cas9 components to cardiac muscle cells derived from human cells carrying two different types of DCM-causing mutations.

Scientists used this gene-editing technology to swap a single nucleotide, the basic unit of DNA, to correct one type of mutation. In another set of cells, researchers replaced a piece of DNA from mutated RBM20 with a healthy segment of this gene.

After CRISPR-Cas9 treatment, the mutant cells gradually lost characteristics inherent to DCM: The protein produced by RBM20 moved to its normal place in the nucleus, and the cells began making healthy proteins.

When the researchers delivered the CRISPR-Cas9 treatment to 1-week-old mice carrying one of these mutations, the animals never developed enlarged hearts and had normal life spans. Untreated mice had symptoms mirroring those of human DCM patients.

The scientists said that several challenges remain before this therapy can be used in DCM patients. Work is needed to ensure that the effects of CRISPR-Cas9 are permanent and precise, and that the smallest dose possible is delivered. Also to be determined is whether the treatment could be used in patients whose disease is more advanced. However, Dr. Olson said he’s optimistic that this system could be used to treat a variety of other familial diseases.

“The pace of this field is really breathtaking,” he said. “I expect that if this moves forward into patients, we’re not talking within decades—we’re talking within years.”


Inherited cardiomyopathies are the leading causes of cardiac-related deaths [1,2]. Dilated cardiomyopathy (DCM) is a disease that affects approximately 1 out of 2500 persons and has been found in the last few years with accelerated frequency [3,4,5].

DCM is diagnosed according to two criteria:

(1) left ventricular enlargement, and

(2) systolic dysfunction recognizable by the reduction in the myocardial contraction force [5].

DCM patients can display a broad range of phenotypes, ranging from heart failure to arrhythmias and thromboembolic disease [4]. Approximately 50% of the cases of DCM are inherited [6].

In familial dilated cardiomyopathy (familial DCM), structural or functional abnormalities develop due to a mutation, affecting the electrophysiological properties of the cardiomyocytes, e.g., calcium handling proteins, nuclear envelope proteins, or the contractile apparatus among others [1,3].

Mutations in more than 30 genes can lead to DCM, making it a highly complex and heterogeneous disease. A total of 25% of the cases of familial and 18% of sporadic DCM cases can be related to mutations (non-sense, frameshift, or essential splice site) of the sarcomeric protein Titin (TTN) [5,7,8,9,10]. Frameshift mutations in TTN alter the reading frame, leading to the premature termination of translation and thus generating truncated versions of the protein (TTNtv) [11,12].

Titin is a giant sarcomeric protein that can code up to 35,991aa (theoretical protein) (NCBI: NP_001254479) and spans half of the sarcomere [13]. The TTN gene encodes for the largest human protein and is composed of 364 exons, including a first non-coding exon. In humans, TTN is located on chromosome 2q31 [3]. TTN has a multitude of functions. It acts as a biological spring between the Z-disk and the M-line and serves as a scaffold of the sarcomere assembly.

Additionally, it is a hot spot for protein–protein interactions, a key mediator of signal transduction in cardiomyocytes, and determines the passive tension of muscle fibers [8,11,12,14,15]. TTN regions are annotated according to their position in the sarcomere visualized by immune-electron micrographs, i.e., Z-disc, I-band, A-band, and M-line [16]. Titin undergoes extensive alternative splicing generating a plethora of isoforms. N2B and N2BA are the major cardiac isoforms and comprise the four regions: Z-line, I-band, A-band, and M-line [3,17].

The clinically most relevant mutations of TTN are located in the I-band and the A-band. This often leads to early stop codons and a mutated TTN lacking the C-terminal part of the A-band and M-line [12]. Additionally, TTNtv can lead to both dominant and recessive forms of cardiac and skeletal phenotypes depending on the nature of the mutation [15,18,19].
Many efforts have been made in the last few years to associate specific genotypic alterations with the phenotypic (symptomatic) spectrum of DCM [7,10,20,21]. Mapping the mutations that lead to TTNtv and ultimately to DCM is critical to generate efficient treatments [7,8,9,11].

To date, there are only limited therapeutic alternatives for DCM. In severe cases, heart transplantation is the only option. However, transplantation is a bottleneck due to the limited availability of donor hearts. In contrast, promising approaches to the treatment of a large number of DCM patients are genome editing technologies to restore the reading frame of TTNtv [4,11].

The CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas system evolved as a powerful biotechnological tool to modify genomes in prokaryotes and eukaryotes [22,23,24]. Cas9 nucleases can generate double-strand breaks at specific sites in the genome. Cas9 is guided by short RNA guides (gRNA), derived from the CRISPR RNA array (crRNA) and trans-activating crRNA (tracrRNA) [22].

In contrast to other nucleases, such as transcription-activator-like effector nucleases (TALEN), zinc-finger nucleases (ZFN), or meganucleases, the CRISPR/Cas system is easy to apply and highly efficient, making it the method of choice for genome editing studies [25,26]. To date, CRISPR/Cas has had a strong impact on disease modeling and the understanding of biological mechanisms. A special focus is on the potential of CRISPR/Cas for in vivo genome editing [26,27,28,29,30].

A skip or deletion of in-frame mutated exons via the CRISPR/Cas9 system has long been considered a potential strategy to treat DCM and other cardiovascular diseases [25,31,32,33,34]. Recently, a variety of endonuclease-based experimental treatments were tested and established to overcome frameshift mutations in sarcomeric proteins [31,34,35,36].

These approaches can be categorized into three groups, i.e.,

(a) controlled splicing of mutated exons by inducing indel mutations in the splice acceptor–donor site,

(b) full fragment/exon removal, and

(c) restoration of the original wildtype sequence by targeting the mutated locus [31,37].

Approaches (a) and (b) lead to an incomplete protein; however, they have the potential to completely or partially restore its functionality [35,36]. In contrast, approach (c) restores the wildtype form of the protein [31]. In a recent study using patient-derived iPSC with a frameshift mutation in the A-band, the authors corrected the mutation by restoring the wildtype sequence using a Cas9 plus a gRNA targeting the mutated TTN allele and a single-stranded oligo as the donor for homology-directed repair. Furthermore, the iPSC-derived cardiomyocytes derived from the corrected cell line showed wildtype functionality as assessed by determining their force of contraction using engineered heart muscle (EHM) [38,39].

Besides endonuclease-based approaches, RNA-based therapeutics and splice-switching approaches have been tested to correct Titin, Dystrophin, and other sarcomeric protein frameshift mutations [40]. In Duchenne muscular dystrophy (DMD), mutations are concentrated in hotspots (exons 45 to 55 and exons 2 to 10). This allows a focus on specific gene locations to cover most of the mutations.

In contrast, the mutations in TTN associated with severe DCM phenotypes are located in a wide range of sites and regions [10,11,15,40,41]. Recently, Gramlich et al. showed the beneficial potential of the TTN reframing strategy using antisense oligonucleotide (AON-) mediated exon skipping by correcting in vitro an autosomal dominant mutation in the giant TTN exon 326 [8].

Approaches relying on splice site mutations and genomic deletions always have to consider exon symmetry (exons whose number of base pairs is a multiple of 3 are symmetric). In order to repair the frameshift in an allele coding for a truncated version of a TTNtv protein (“reframing”), it is essential to select symmetric exons. The deletion of symmetric exons will not affect the reading frame of the wildtype protein but would restore a shortened version of the original open reading [42]. Aiming to contribute to the development of therapies to treat familial DCM, we evaluated the TTN structure from a therapeutic perspective.

Figure 2. (ac) Overview of the exon deletion approach to restore functional Titin. (a) Schematic representation of the canonical transcription and translation of TTN. (b) The functional protein is truncated by a point mutation (frameshift). TTNtv leads to (b’) a shifted reading frame with a premature stop codon and ultimately to a protein with (b”) a non-sense C-terminus. Exemplarily presented with a frameshift mutation in exon 231 of TTN that leads to a truncated and partially non-sense version of the protein. The red color indicates a non-sense amino acid sequence different from TTN that originates from the frameshift mutation. The frameshift causes, at the same time, the introduction of a stop codon on the transcript level, which in turn results in a truncated protein variant. (c) Symmetric exon deletion to restore TTN functionality. Symmetric exons with mutations are deleted with a system combining Cas9 plus two gRNAs, targeting intronic sites up- and down-stream of the candidate exon. TTN expression results in a minimally shorter but putatively functional protein. Schematic representation of the deletion of a symmetric exon to remove the frameshift mutation located in exon 231. Double strand breaks in TTN are repaired after CRISPR/Cas9 editing without the need for a homologous template by non-homologous end joining (NHEJ).
Figure 3. Flowchart of the in vitro testing and phenotyping of the exon deletion system to restore TTN functionality in DCM patients. (a) Validation of the system will be performed using DCM patient-specific iPSC-derived cardiomyocytes. (b) AAV expression vector containing, e.g., Cas9 ortholog, Campylobacter jejuni Cas9 plus two gRNA to target sequences up- and downstream of the mutated target exon. The vector will be packaged in AAVs with tropism for cardiomyocytes (CM). (c) After the successful delivery of the CRISPR/Cas system with two gRNAs by infection, detailed evaluation of the safety and efficacy of the system will be performed by in vitro phenotyping of the treated iPSC-derived CM.

reference link : https://www.mdpi.com/2073-4425/13/6/1093


More information: Takahiko Nishiyama et al, Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy, Science Translational Medicine (2022). DOI: 10.1126/scitranslmed.ade1633

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