Toward CRISPR Therapies for Cardiomyopathies

Genetic cardiomyopathies are a common cause of heart failure and sudden death. Despite an understanding of their underlying genetics, effective long-term therapies remain an unmet medical need. The recent advent of gene editing technologies provides a promising therapeutic opportunity for permanent correction of disease-causing mutations. Mutations in genes encoding cardiac structural proteins such as dystrophin, titin, β-myosin heavy chain represent attractive targets for therapeutic gene editing.

Although gene therapy can replace a mutant gene with a wild-type copy, this approach is limited to genes small enough to fit within viral vectors and is dependent on their continued expression. Gene editing strategies, in which a mutant gene is corrected within the context of its normal genetic milieu, allows for sustained expression of the edited gene. There are three general types of gene editing: gene disruption, reading frame restoration and precise correction. Gene disruption can inactivate dominant negative or pathogenic gain-of-function mutations and eliminate the dysfunctional protein. Reading frame restoration can enable the expression of nonfunctional genes, often by reframing or skipping of out-of-frame exons, as is common for Duchenne muscular dystrophy (DMD). The recent development of precise correction strategies, using base or prime editors, allows the editing of pathogenic mutations.

CRISPR/Cas9 gene editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing involves two components, a single guide RNA (sgRNA) complimentary to the target DNA sequence, and a CRISPR-associated endonuclease (Cas9) (1). DNA cleavage is induced by a Cas9-sgRNA ribonucleoprotein complex when the target DNA sequences pair with the sgRNA near a protospacer-adjacent motif (PAM) (Figure). Repair of the double-stranded DNA break (DSB) is mediated by nonhomologous end joining (NHEJ), which generates insertions or deletions (INDELs), or by homology-directed repair (HDR), which precisely repairs DSBs by insertion of a specific DNA sequence. Correction of genetic cardiomyopathies by gene editing would likely require NHEJ because the HDR machinery is absent in post-mitotic cells.

Figure.

A: DMD correction by CRISPR editing with DSBs.

NHEJ, which induces INDELs at the cutting site, is the main mechanism for repair of DNA DSBs. HDR inserts a precise DNA fragment. NHEJ-mediated repair introduces INDELs to restore the open reading frame either by exon skipping or reframing in a deletion of DMD exon 44.

B: DMD correction by base editing.

Base editors convert A-T to G-C or C-G to T-A base pairs without DNA DSBs. This approach can be used to disrupt splice sites, thereby causing exon skipping, as shown for DMD exon 52.

C: DMD correction by prime editing.

Prime editing can introduce specific DNA sequences to reframe exons, as shown for DMD exon 52.

Most mutations responsible for DMD involve exon deletions or duplications that disrupt the expression of the dystrophin protein, leading to progressive muscle degeneration and cardiomyopathy. CRISPR/Cas9 editing has been deployed in patient-derived induced pluripotent stem cells (iPSCs), as well as in mice and dogs with DMD, to restore dystrophin expression in cardiac and skeletal muscles (2). For example, a deletion of exon 44 of the dystrophin gene generates a premature stop codon in exon 45, causing DMD, which can be corrected either by skipping or reframing of exon 45.

Base and Prime Editing.

Base editing (BE) and prime editing (PE) are new technologies that perform precise genetic modifications without the creation of DSBs. BE enables the modification of base pairs, such as a C-G to T-A base pair in Cytosine BE (CBE) or an A-T to a G-C base pair in Adenine BE (ABE) (Figure). BE could potentially be deployed for correction of cardiomyopathies caused by specific point mutations, such as hypertrophic cardiomyopathy caused by an R403Q mutation in the MYH7 gene. Recently, a mouse model of Hutchinson-Gilford progeria syndrome caused by a LMNA gene mutation was rescued by ABE (3).

Modification of splice sites of exon junctions by BE can also be used to inactivate genes or cause exon skipping. In a primate model, ABE was used to inactivate the PCSK9 gene by modifying a splice donor site, reducing low-density lipoprotein cholesterol levels (4). Exon skipping by ABE has also restored expression of dystrophin in patient iPSC-derived cardiomyocytes and skeletal muscle of mice with a deletion of DMD exon 51 (Figure) (5). However, potential drawbacks of BE include a limited editing window, unwanted by-stander editing, and off-target editing of RNA.

The PE system consists of a Cas9 nickase fused to reverse transcriptase and a pegRNA that recognizes the target DNA sequence and contains a template that enables reverse transcriptase to specifically correct various mutations (Figure), including those in which ABEs and CBEs are ineffective. Recently, PE was used in mice with inherited human liver and eye disorders and for reframing a DMD mutation and holds much promise for inherited cardiomyopathies.

Delivery challenges.

Therapeutic gene editing can be deployed in vivo or in vitro, depending on the tissue to be targeted. For blood disorders, such as beta-thalassemia and sickle cell disease, autologous patient-derived hematopoietic stem cells have been edited ex vivo and then reinfused into patients. In contrast, genome editing for cardiomyopathies requires an efficient and safe delivery system. Adeno-associated virus (AAV) is currently the most promising viral vector for delivering CRISPR/Cas9 components to the heart. However, its packaging capacity is limited to ~4.7kb, which necessitates packaging of the most widely used Cas9 from Streptococcus pyogenes (SpCas9) and its sgRNAs in separate vectors. To overcome this challenge, several orthologs of small Cas9 have been engineered. Delivery of BEs or PEs is also limited by AAV packaging capacity. A dual-AAV system using trans-splicing inteins has been shown to be capable of reconstituting full-length BEs and PEs. Since the heart is highly vascularized, other delivery strategies, such as nanoparticle-mediated delivery might overcome the bottlenecks of AAV delivery if they could be delivered efficiently.

Potential safety concerns.

There are several potential safety concerns associated with in vivo gene editing that need to be carefully assessed. Cutting the genome with CRIPSR/Cas9 has the potential to introduce unintended INDELs and deleterious off-target mutagenesis. Although significant off-target toxicity has not been observed in animal models of cardiac gene editing, it is essential to assess the long-term effects of CRISPR/Cas9 and base editing. Because CRISPR enzymes are derived from bacteria, immunogenicity is also a significant concern that will likely need to be mitigated by immunosuppression. Finally, toxicity of high doses of AAV has been observed in clinical trials, and pre-existing immunity to AAV will also need to be assessed in potential patients.

Conclusions.

CRISPR-Cas9 therapy is developing rapidly toward clinical applications. Despite various challenges and issues of safety, the pace and potential of this field of investigation promise to revolutionize the treatment of genetic cardiomyopathies and many other genetic disorders in the foreseeable future.