Unraveling the Genetic Code: Harnessing Precision Editing for Improper Cargo Sorting and Membrane Trafficking Within Inherited Cardiovascular Diseases

Abstract

Background: Cardiovascular diseases affect over 80,000,000 individuals in the United States alone. Each disease has a broad range of debilitating consequences that affect not only the individual but, quite possibly, their offspring. For most cardiovascular diseases, inherited DNA sequences are influential in the genotype and phenotype (Kathiresan & Srivastava, 2012). As cardiovascular diseases remain the most common cause of death worldwide, the scientific community needs to utilize the most up-to-date technology, such as genetic editing techniques, to try and conquer these diseases. Myriad tools such as genome-wide association studies have recently been used to help identify previously unknown affected genes and variants, allowing for a further and more in-depth understanding of cardiovascular diseases (Vrablik et al., 2021). The scientific community already understands many inherited cardiovascular diseases, but the utilization of the newest technology has yet to make a significant appearance in reducing the number of affected individuals. Hypotheses: I hypothesize that each inherited cardiovascular disease can be effectively treated using a specific genetic editing technique, tailored to the unique characteristics of each condition. The approaches are outlined as follows: H1: The CRISPR/Cas9 technique would successfully treat Familial Hypertrophic Cardiomyopathy (FHCM) because by gene correction, editing the faulty DNA sequences responsible for FHCM could potentially restore the normal function of the affected proteins. H2: The Cpf1 (Cas12a) technique would successfully treat Danon Disease because Cpf1 offers extremely high precision in targeting genes, whether in gene silencing, insertion, or replacement, could offer complete restoration of the LAMP2 gene and correct the LAMP2 protein function. H3: The MAGE technique would successfully treat Pompe Disease because the MAGE technique not only offers to edit the mutated GAA gene but also could be beneficial by gene augmentation by providing increased production of the GAA gene, which could compensate for the defect and improving affected cells, in the breakdown of glycogen. H4: The Zinc Finger Nucleases (ZFNs) technique would successfully treat Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) because this technique targets with specificity and precision but it could not only correctly target the genes associated with one desmosomal protein, but the myriad involved with ARVD/C such as desmoglein (DSG), desmoplakin (DSP), and plakoglobin (JUP). H5: The Base Editing technique would successfully treat Barth Syndrome because base editing allows for specific editing of individual nucleotides which would directly affect the mutation in the TAZ gene. Methods: A multi-centric retrospective cohort review will be performed on individuals with a confirmed inherited diagnosis of Barth Syndrome, Arrhythmogenic Right Ventricular Dysplasia / Cardiomyopathy (ARVD/C), Pompe Disease, Danon Disease, and Familial Hypertrophic Cardiomyopathy (FHCM). Utilization of published articles, reports, trials, and journals between the years 2000-2023 will be used to gather information on the five above-listed inherited cardiovascular diseases and the specificity of each disease’s cargo sorting membrane trafficking, and disease function. Diagnosed individuals will be compared to others with the same diagnosis to confirm whether their disease presents the same genetically and establish a common baseline of genetic similarity. Those who have undergone treatment for the above-listed diseases will be analyzed to compare how their treatment has affected the disease and estimate whether genetic editing or engineering would be affected by such treatment. To establish a baseline of genetic editing techniques, this study will solely focus on the following approaches: multiplex automated genome engineering (MAGE), Cpf1 (Cas12a), base editing, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). As each of the above-listed techniques provides a unique approach, utilization of various journals and published articles will be analyzed to articulate the benefits and drawbacks of each genetic editing technique and conclude which approach would pose the greatest chance of influencing or curing each inherited cardiovascular disease. Results: The initial hypotheses proposed that CRISPR/Cas9 would effectively treat FHCM by correcting faulty DNA sequences, Cpf1 (Cas12a) would restore LAMP2 gene function in Danon Disease, and MAGE could change the outcomes of Pompe disease treatment by editing and augmenting the GAA gene. Additionally, ZFNs were hypothesized to provide precise correction of multiple desmosomal gene mutations in ARVD/C, while base editing was expected to correct point mutations in the TAZ gene for Barth Syndrome. The results confirmed the potential of CRISPR/Cas9 for FHCM and Cpf1 for Danon Disease, aligning with the original hypotheses. However, MAGE was found to be less viable for Pompe disease, with ZFNs emerging as a more effective approach. Base editing also proved to be the most promising treatment method for both ARVD/C and Barth Syndrome due to its precision in correcting point mutations without introducing double-stranded breaks. These findings refine the understanding of gene- editing applications for inherited cardiovascular diseases and highlight how their therapeutic potentials can facilitate change in treatments for diseases. Conclusion: This study evaluated the feasibility of various gene-editing techniques for treating inherited cardiovascular diseases, identifying the most promising approaches based on precision, efficiency, and safety. CRISPR/Cas9 demonstrated strong potential for treating FHCM due to its versatility and ability to generate targeted genomic modifications. Cpf1 (Cas12a) emerged as a superior option for Danon Disease, offering enhanced repair efficiency and reduced off-target effects compared to other nucleases. ZFNs showed the greatest promise for Pompe disease, leveraging their precision and established success in clinical applications. Base editing was identified as the most effective method for treating ARVD/C and Barth Syndrome, given its ability to precisely correct point mutations without causing double-stranded breaks, minimizing genomic instability. These findings highlight the strengths of each technique in addressing specific genetic disorders, paving the way for future advancements in gene therapy.