Genome Editing in Cardiovascular Biology
Genome editing has emerged as a powerful tool in research and is entering the stage of therapeutic applications. In the cardiovascular field, its role in basic and translational research is well established. However, biological and technical barriers currently hamper the therapeutic potential of genome editing for cardiovascular diseases. This viewpoint discusses possible routes for promoting therapeutic use of genome editing in the cardiovascular system.
Genome editing has rapidly emerged as a powerful tool in basic and translational research. Zinc finger nucleases and TALENs (transcription activator-like effector nucleases) catalyzed the field initially. With the development of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system, the field is expanding even more rapidly because of its efficacy, specificity, and ease of use (Figure [A]). Generally, genome editing tools induce site-specific DNA double-strand breaks at a specific genomic site, resulting in the activation of the nonhomologous end-joining (NHEJ) and homologous recombination (HR) cellular endogenous double-strand break repair machinery (Figure [B]). Recent advances of the CRISPR technology also allow for RNA recognition, making it possible to cleave RNA, enhance or inhibit translation, support isolation of specific RNA:protein complexes, or induce specific post-transcriptional modifications.1 Genome editing tools can also be used to control gene expression on the transcriptional level. Deactivation of the catalytic site of CRISPR/Cas9 (dCas9) results in specific binding to the DNA without inducing double-strand breaks. Fusion of dCas9 to DNA-binding domains, such as activator or repressor domains, results in transcriptional activation or inhibition of a specific gene, respectively.2 Despite the major impact genome editing already has on basic and translational research, regulatory processes have delayed therapeutic applications from reaching the clinic. Nevertheless, effective new therapies are anticipated in the near future for various disease conditions, especially if major issues around safety and toxicity are resolved.
Genome Editing in Preclinical Research
The use of genome editing has facilitated basic and translational research across multiple disciplines, including the cardiovascular field. Genome editing already plays a key role in the generation of new genetically modified animal models. In addition, the combination of genome editing and induced pluripotent stem cell (iPSC) technology now makes it possible to elucidate complex pathophysiological mechanisms directly in human cellular models (Figure [C]). Using genome editing to derive isogenic iPSC lines may become the standard approach to characterize disease-causing mechanisms of pathogenic mutations underlying cardiomyopathies, to investigate variants of unknown significance, or to establish disease-modifying variants.3 In addition, high-throughput CRISPR-mediated large-scale functional screens with gene knockout, loss-of-function, and gain-of-function approaches will help to further elucidate molecular mechanisms of cardiovascular diseases. However, the ability to translate insights gained from iPSC-based cellular models into therapeutic strategies still needs to be proven. Further improvements in maturation of iPSC-derived cardiomyocytes (iPSC-CMs) and other derivatives, as well as the generation of organoids or 3-dimensional structures, are needed to fully realize the utility of this technique.4
Therapeutic Potential of Genome Editing in Cardiovascular Diseases
Genome editing comprises great potential for innovating therapies. Although its important role in basic and translational research is evident, the barriers to therapeutic applications remain significant. In principle, there will be 2 main routes for therapeutic applications of genome editing in cardiovascular diseases: ex vivo and in vivo approaches (Figure [C]).
Ex Vivo Approaches for Genome Editing
Because culturing primary human cardiomyocytes is extremely difficult, the major ex vivo approach for therapeutically applied genome editing in cardiac diseases is to correct iPSCs derived from patients with disease-causing mutations. Studies have shown that iPSC lines generated by CRISPR and TALEN rarely have identifiable off-target mutations.5 These corrected iPSCs can then be differentiated into iPSC-cardiac progenitors or iPSC-CMs and delivered to the patient’s heart. Extensive preclinical studies have already been performed to evaluate ways of delivering embryonic stem cell–derived cardiomyocytes (ESC-CM) or iPSC-CMs to the diseased heart, by injecting cells via the intracoronary or intramyocardial route, or as tissue-engineered constructs.6 However, only modest functional improvements have been achieved to date. In addition, several safety concerns exist using ESC-CM or iPSC-CMs in terms of immunogenicity, tumorigenicity, and the risk of arrhythmia because of insufficient electric coupling.7,8 Here, genome editing might be also applied to introduce inducible suicide genes into the genome of the iPSCs or ESC. In the rare case of tumor formation arising from the injected cells, these cells could be targeted directly for elimination.9 A potential downside of the suicide gene approach is that it would also eliminate any cells that might be contributing to positive effects. Moreover, genome editing using ex vivo therapeutic approaches can only be tested once the major obstacles for ESC or iPSC-CM therapy are resolved.
In Vivo Approaches for Genome Editing
In vivo genome editing entails correcting disease-causing variants in primary cells in situ. Recently, a smaller Cas9 protein from Staphylococcus aureus has been described that makes it feasible to deliver CRISPR/Cas9 using adeno-associated virus vectors.10 Adeno-associated virus vectors are already approved for cardiac gene therapy and have been tested in clinical trials. Genome editing has safety advantages over integrating viral-based gene therapy methods because it avoids generating large number of random insertions into the genome.
Proof-of-concept preclinical mouse studies have also been performed with in vivo genome editing. Using CRISPR/Cas9-mediated NHEJ, the exon in the dystrophin gene carrying a mutation leading to Duchenne muscular dystrophy could be excised, resulting in a significant restoration of muscular function.11 Furthermore, in vivo CRISPR/Cas9-directed HR was sufficient to treat mice with an urea cycle disorder in the enzyme ornithine transcarbamylase, proving the feasibility of an in vivo genome editing approach that uses HR.12
However, in vivo therapeutic genome editing within the cardiovascular system faces several major hurdles. First, despite improvements in algorithms predicting the highest specificity, off-target effects can still occur on rare occasions. Subsequently, NHEJ repair mechanisms may lead to cellular dysfunction or give rise to tumor formations. Second, induction of double-strand breaks in the healthy allele in the setting of a heterozygous mutation may result in worsening of the phenotype. Third, the cellular repair mechanisms depend on cell cycle phase. While NHEJ can lead to DNA repair during the G1, S, and G2 phases, natural HR is limited to the late S and G2 phases when the sister chromatid is present.13 Because the vast majority of cardiomyocytes in the postnatal heart are postmitotic, it will take new discoveries to apply HR-mediated editing to postmitotic cells. Nevertheless, NHEJ-mediated exon skipping may be technically applicable especially for mutations in TTN (titin) and MYBPC3 (myosin-binding protein C3).14,15 Fourth, unlike skeletal muscle satellite cells that have huge regenerative potential, the inherent regenerative capacity of an adult heart is highly limited. Thus, the rate of cardiomyocytes successfully being edited has to be relatively high to mediate cardiac improvements. Taken together, these physiological and technical restrictions currently pose significant challenges for in vivo genome editing as a treatment for cardiomyopathies.
Beside cardiomyocytes, endothelial cells and smooth muscle cells might become targetable by in vivo genome editing in cardiovascular diseases. One possible scenario is the correction of validated genomic variants in endothelial cells, as identified in genome-wide association studies.16,17 Additional disease-causing or disease-modifying variants will be discovered with increasing availability of whole exome and whole genome sequencing. However, not all of these variants will be in genes specifically expressed in endothelial or smooth muscle cells, so cell-specific delivery of CRISPR/Cas9 will be a major hurdle for clinical use.
Cardiac fibroblasts are playing a key role in cardiac homeostasis and most disease conditions in the heart, but the characterization and the therapeutic approaches are limited by the heterogeneity of the cardiac fibroblast population. Ongoing research will further elucidate the contributions of subpopulations and specific gene dysregulations in cardiac diseases, making these cells a valuable source also for gene editing. Again, if cell type–specific delivery can be performed, genome editing can come into play to target distinct subtypes of cardiac fibroblasts to either enhance regeneration or slow down disease progression.
Although conceptually genome editing might be applied to germ cells or embryos to correct disease-causing variants, the ethical issues surrounding such an approach remain unresolved, clouding the prospects of its implementation in the near future. Moreover, preimplantation genetic diagnosis with selective embryo implantation, a currently accepted practice (although not covered by insurance), would achieve many of the same goals as those under editing of germ cells or embryos.
Genome editing has already established itself as a powerful tool for the generation of new cellular and animal models to investigate pathophysiological mechanisms in various diseases. The therapeutic potential of genome editing for cardiovascular disease is currently hampered by biological (notably the postmitotic nature of cardiomyocytes) and technical barriers. Cardiac fibroblasts, endothelial, and smooth muscle cells might become potential sources of targeted in vivo genome editing in cardiovascular diseases. However, major scientific and technical advances are needed before genome editing can be applied clinically in cardiovascular diseases.
We thank Amy Thomas for her help with preparation of the figure. Because of space limitations, we are unable to include all the important papers relevant to cardiovascular genome editing; we apologize to those investigators whose significant contributions are not mentioned here.
Sources of Funding
This publication was supported, in part, by research grants from the German Research Foundation (T.S.), American Heart Association 13EIA14420025, National Institutes of Health (NIH) HL130020, NIH R01 HL128170, and NIH R01 HL126527 (J.C.W.) and the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research (M.P.).
M.P. is a consultant for and has equity in CRISPR Therapeutics.
The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
- © 2017 American Heart Association, Inc.
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