Introduction:
Cardiovascular disease (CVD) has been a focal point in research due to its high incidence and disability rates. With certain inherited cardiovascular diseases showing resistance to conventional treatments, researchers are exploring genetic techniques. However, obstacles like limited understanding of CVD nature, genetic technology constraints, and ethical concerns hinder the clinical application of gene therapy. Recent years have seen a growing acknowledgment of genetic factors' crucial role in human diseases, because of the advancements in life science research and the rapid progress of gene-editing technologies.
What Is Meant by Gene Editing for Cardiovascular Diseases?
Gene editing technology offers hope for curing various genetic CVDs. germline genome editing could permanently eliminate monogenic CVDs from affected families’ offspring, but its implementation raises ethical concerns. Somatic genome editing, though facing technical challenges, provides a potential avenue for treating cardiovascular disorders in already affected individuals, avoiding germline ethical issues. Despite concerns about CRISPR-Cas9’s efficacy in gene correction for the human heart, its success depends on overcoming technical challenges in Cas9-gRNA delivery, addressing off-target effects, and refining homology-directed repair rates. Initially, scientists used homologous recombination of gene knock-out in animal models. Subsequently, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) emerged as second-generation gene-editing tools, relying on nucleic acid binding proteins and endonucleases. The third-generation CRISPR/Cas9 system, functioning through protein-nucleic acid complexes, holds great promise for refractory and genetic diseases.
What Are the Therapeutic Applications of Gene Editing?
Genome editing can potentially revolutionize the treatment of monogenic diseases lacking effective therapies. Within cardiovascular medicine, heritable cardiomyopathies like hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy, Duchenne muscular dystrophy (DMD), along with heritable arrhythmic disorders, vasculopathies such as Marfan’s syndrome, and infiltrative diseases like transthyretin amyloidosis, are prime candidates for germline gene editing. Focusing on disorders with a single gene mutation causing manifestation offers the possibility of permanently correcting the disorder in future generations.
While germline genome editing might not play a significant role in disorders influenced by both genetic and environmental factors somatic genome editing holds promise for complex disorders such as coronary artery disease and atherosclerosis. Additionally, somatic genome editing may be valuable for post-natal treatment of monogenic disorders, especially considering the ongoing ethical concerns surrounding germline genome editing. Despite the controversy, with around 7,500 monogenic disorders affecting approximately 780 million people, germline editing theoretically has the potential to eliminate these diseases permanently.
Hypertrophic cardiomyopathy (HCM) is a cardiac muscle disease leading to ventricular hypertrophy arrhythmias, syncope, and heart failure, recent studies demonstrated successful CRISPR-Cas9 correction of a MYBPC3 mutation in human germ cells. The method involved injecting recombinant Cas9 protein with gRNA and ssODN DNA into human zygotes, resulting in a high percentage of embryos with a homozygous wild-type genotype. Adjustments to the timing of Cas9 injection during embryogenesis improved HDR efficiency, showcasing the potential of CRISPR-Cas9 in abolishing disease-causing mutations in human embryos and preventing transmission of HCM and other monogenic disorders. The potential of somatic genome editing in HCM arises due to the gradual development of cardiac hypertrophy, myocardial fibrosis, and symptomatic disease.
Duchenne muscular dystrophy (DMD) causing progressive skeletal muscle weakness and fatal cardiomyopathy, is a candidate for germline genome editing due to its heritability and lack of effective therapies. Cas9, gRNA, and template ssODN DNA injected into mouse zygotes with a DMD mutation demonstrated successful correction, even with mosaicism. Adjustments to injection methods further enhance results. Additionally, studies utilizing S. aureus Cas9 and gRNA in AAV vectors showed promising results in restoring the DMD reading frame and improving cardiac function in mice and dogs, indicating potential for somatic genome editing.
Nonischemic cardiomyopathies include disorders related to the phospholamban gene (PLN), and may become treatable through germline or somatic genome editing. Germline CRISPR-Cas9 editing successfully knocked out the PLN gene in a mouse model, leading to improved cardiac size and function.
Dyslipidemia, associated with atherosclerotic CVD, presents a target for somatic genome editing studies using CRISPR-Cas9 targeting PCSK9 lowered plasma and cholesterol levels showing promise for reducing cardiovascular risk.
Somatic genome editing’s potential extends to addressing age-related clonal hematopoiesis (ARCH), associated with increased cardiovascular risk. Inactivating TET2 and DNMT3A genes in mouse bone marrow cells via lentivirus vectors revealed their impact on cardiac function and inflammation, suggesting a potential therapeutic avenue. Transthyretin cardiac amyloidosis, characterized by abnormal protein deposition, may benefit from somatic genome editing using nonviral vectors, as demonstrated by lipid nanoparticle delivery of Cas9 and gRNA targeting the T tr gene in mice.
Inherited arrhythmic disorders, including PRKAG2 syndrome and long QT syndrome (LQTS), show promise for genome editing. In a mouse model, postnatal correction of PRKAG2 syndrome via AAV9 vector administration resulted in improved cardiac function. CRISPR interference effectively modulated gene expression in LQTS-induced cardiomyocytes in vitro.
Marfan syndrome, associated with a mutation in the FBN1 gene, may be addressable through base editing techniques. Experiments corrected pathogenic mutations in human cells and embryos, suggesting a potential application for correcting gene mutations in the human germline.
What Is CRISPR-Cas9 Technology?
CRISPR-Cas gene editing tools comprise Cas nuclease guided by a single-guide RNA (sgRNA) to specific genome regions, inducing a double-stranded break (DSB) proximal to the protospacer adjacent motif (PAM). this DSB activates non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways, with NHEJ, often used for gene-specific knockouts and HDR for precise edits, forming the basis for patient-specific or transgenic models.
In cardiac research, classical CRISPR-Cas9 has facilitated the engineering of isogenic lines of human induced pluripotent stem cells (hiPCS). This technology enables the study of monogenic diseases, such as arrhythmogenic cardiomyopathy (ACM) and lethal arrhythmias, by generating models with mutations in genes like PKP2 and PLN. CRISPR-Cas9 mediated in vivo genome editing, combined with adeno-associated virus (AAV)9 delivery, has shown promise in improving cardiac function in mutant mice, offering potential therapeutic insights.
Genome editing of hiPSCs has deepened the understanding of diseases like dilated cardiomyopathy (DCM), where CRISPR-based knock-in of patient-specific mutations in RBM20 led to splicing defects. Patient-specific hiPSC-derived cardiomyocytes carrying mutations in TNNT2 and MYBPC3 exhibited impaired contractile function and engineered heart muscles (EHMs) recapitulated disease phenotypes. Additionally, CRISPR has been employed to generate gene-specific reporter lines, enabling the study of hiPSC-derived cardiomyocyte-specific properties.
While CRISPR-Cas9 has been successful in modeling genetic cardiomyopathies, its translational capacity faces limitations due to DBS lesions’ potential undesired effects. Base editing and prime editing emerge as safer, more efficient, and precise technologies, especially for postmitotic systems, circumventing challenges associated with DSB induction.
What Are Base and Prime Editing Techniques?
Base Editing:
It is a precise genome editing method, that introduces point mutations without inducing double-stranded breaks (DSBs). Cytosine base editors (CBEs) and adenine base editors (ABEs) convert specific base pairs, allowing for the restoration of transition mutations. These base editors combine a single-stranded DNA deaminase with Cas9 nickase, facilitating deamination in the correct ‘activity window’ upon sgRNA hybridization. Engineered versions, like ABEmax and BE4max, feature improved activity and broader editing windows.
Prime Editing:
It offers an alternative, utilizing HNH nuclease-inactivated nCas9 fused with engineered reverse transcriptase domains. Directed by prime editing guide RNAs (pegRNAs), this system primes reverse transcription and incorporates desired edits during DNA repair. PEmax editors and engineered pegRNAs are optimized versions, showcasing versatility but facing challenges in predicting editing efficiency. While prime editing presents a promising alternative, ongoing research aims to refine its efficiency and predictability in different target sites and cell types.
Conclusion:
Gene editing technologies, including CRISPR-Cas9, and base and prime editing, have emerged as powerful tools in advancing the understanding and potential treatment options for cardiovascular diseases. These technologies enable precise manipulation of genetic information, allowing researchers to study the intricate genetic factors contributing to various cardiac conditions.
