A Study on Preventing Aortic Dissections in Marfan Patients by Base Editing Smooth Muscle Cells

Our project leverages an advanced form of CRISPR, base editing, to minimize mortalities and morbidities arising from Marfan syndrome (MFS). It was inspired by our shared fascination with genetic engineering. Biotechnology is advancing at breathtaking speeds and shows promise in saving and improving millions of lives by granting unprecedented control over our own genome. Judy herself was born with a de novo case of MFS and has always felt an inexplicable sense of curiosity toward its causes. She enjoys pondering solutions to this disorder using modern medical innovations.

Marfan syndrome is a genetic disorder that is caused by defects in the human body’s microfibrils. It affects about 1 in 5,000 people globally. The average lifespan of an untreated MFS patient is 45 years, 30 years shorter than the global average. Individuals affected by MFS exhibit more elastic connective tissue, which can affect the heart and blood vessels, respiratory system, eyes, bones, joints, and more. The most prominent complications arise from aortic root dilation, underdeveloped respiratory muscles, and ectopia lentis. Current medical interventions are mitigatory and involve long-term continuous maintenance, such as decades of avoiding strenuous physical activity, making them time and cost-intensive.

Our project aims to permanently eliminate MFS’s severest symptoms: aortic aneurysm and dissection. This treatment, when preferably implemented in pediatric patients, presents itself as a modern biotechnological solution with lifesaving potential. It could not only extend life for individuals with MFS, but it would also improve the quality of life for patients and their families.

To conduct the following research, predominantly qualitative scientific evidence was synthesized, specifically focusing on the potential effectiveness of base editors in minimizing the effects of Marfan syndrome by editing aortic smooth muscle cells to correct the FBN1 gene, resulting in a healthier extracellular matrix and overall more resilient aorta. The criteria for preliminary eligibility for inclusion in this study were: consensus (all authors of the study agree the source should be included), reliability (peer-reviewed publications from medical journals, .org domains, and authors with MDs and/or PhDs were considered more trustworthy than articles lacking these features), corroboration (information from one source confirmed by multiple others), and recency (consider including what year you considered ‘recent’)(to avoid outdated explanations). Credible databases such as PubMed and ScienceDirect were often consulted. Ultimately, of the 112 sources reviewed, 64 are included and cited in this study.

For background research, we focused on the following key terms: Marfan syndrome (signs, symptoms, etiology, diagnosis, and current interventions), the FBN1 gene, CRISPR-Cas9, and the development of base editors and prime editors.

Our proposed treatment combines recent advances in biotechnology, including next-generation sequencing panels, Sanger sequencing, adeno-associated virus 9, and base editing, to genetically edit the aortic cells of postnatal patients with MFS. The objective of this treatment is to correct vascular smooth muscle cells in the aorta to improve its structural integrity.  For the proposed treatment to be considered therapeutically effective, it must fit the following criteria: identify the target sequence precisely, perform the necessary editing without affecting non-target genes, deliver to the correct site in the body, and correct sufficient aortic cells to result in a significant reduction in dilation and aneurysm risk.

Firstly, MFS is diagnosed in a patient. If it is known to be present within a family line, it is presumed that genetic testing has already been done. The gold standard for diagnosing MFS is the Ghent nosology: the presence of both aortic root dilation and ectopia lentis results in an unequivocal diagnosis. The presence of one of these signs, coupled with a family history of the disorder or a mutation of the FBN1 gene, also results in a diagnosis. Other examinations may be employed, such as an echocardiogram, slit-lamp exam, eye pressure test, or more, all of which test for specific symptoms associated with MFS. It is noted that newborn screening is maximally beneficial, since implementing the proposed treatment in younger patients yields the most effective results.

Once the diagnosis is confirmed, the specific locus of the mutation on the FBN1 gene sequence will be found via next-generation sequencing (NGS) panels and Sanger sequencing. NGS panels process massive amounts of DNA simultaneously to locate the missense mutation, efficiently identifying candidate variants. Sanger sequencing is a highly accurate but slower method, so it is used to confirm the results of NGS panels for quality control.

Next, the base editor will be assembled. Components include the following: Cas9 nick (nCas9), which nicks a single strand of DNA, single guide RNA (sgRNA), which acts as a guide to target the specific DNA locus, deaminase enzyme to remove a targeted amino group, and a protospacer adjacent motif (PAM), which allows nCas9 protein binding to occur. It should be noted that over time, it will likely be possible to draw base editors from a library of functional sgRNAs, under the assumption that more research is conducted on FBN1 gene mutations.

Lastly, the base editor is delivered. The base editor is delivered via a systemic intravenous (IV) injection into a peripheral vein, eventually travelling to the aorta by following the natural venous return to the heart. Adeno-associated viruses (AAV) will be used as a vector due to their track record in FDA-approved treatments for genetic disorders; they are non-pathogenic, exhibit long-term gene expression, and have a significant ability to remain latent. A dual-AAV system will be employed, as the base editor exceeds the capacity of a single AAV. Specifically, the most suitable vehicle is AAV9 because of its considerable transduction efficiency in the heart when injected intravenously.

Notably, this treatment does not need to correct 100% of aortic smooth vascular muscle cells to improve aortic durability significantly. Mosaicism (heterozygosity in mutant and corrected aortic cells) is somewhat tolerable. Clinical cases of MFS tend to be less severe in patients exhibiting somatic mosaicism as opposed to a mutation in all cells on the FBN1 gene sequence. Additionally, even partial correction creates a healthier extracellular matrix (ECM). The ECM provides structural support and regulates growth factors and cellular signalling; an improved ECM results in a more resilient cellular environment. This has important implications for the proposed treatment: as long as a critical mass of cells can be edited, researchers can eliminate the need to identify, deliver the base editor to, and edit all aortic cells.

The social impact of our project is threefold. Firstly, it holds the potential to save the lives of individuals with MFS by preventing aortic aneurysms; aortic dissections are a highly fatal medical emergency. Furthermore, this treatment improves the quality of life for patients because it is a one-time and permanent solution. In contrast with the status quo treatments that require continuous maintenance, it is more time-efficient and convenient. The treatment empowers severely affected patients to partake in more normal activities, allowing them to enjoy themselves and contribute to communities more fully. Also, it paves the way for using this treatment to address other symptoms of MFS or similar diseases. For example, a similar base editing treatment might apply to zonules affected by ectopia lentis.

To clinically implement this treatment, further research is needed to facilitate functional base editors. This process will use online models to stimulate FBN1 gene mutations using DNA libraries and assess the ability of various base editor iterations to correct missense mutations to restore unaffected phenotype. Next, animal model preclinical trials can be used to determine the effectiveness of the treatment in whole organisms. The treatment must follow all legal frameworks and be approved for clinical trials before it can be implemented. In the future, a similar theory can be applied to zonule cells to minimize ectopia lentils in MFS patients.

First and foremost, we are grateful for the wonderful support of the BASEF team, including Lead Delegate Caroline Mahut and Project Mentor Adrienne Hol. They worked tirelessly to coordinate competition logistics, ensure our comfort and safety throughout the trip, and give invaluable feedback on the structure of our written submissions and presentation. Additionally, we would like to acknowledge Siqi Tan and Jasmine Wang, the co-presidents of our school’s Science Fair Team. Since the start, they have provided guidance by answering our many questions and generously sharing their considerable experience, including abstract phrasing and special awards selection. Mr. Alan Kirkup, our teacher supervisor, made it possible for us to attend BASEF in the first place. We acknowledge Taelyn Kaknevicius for assisting in the creation of one of our images. Last but not least, we thank Judy’s mother and little brother for helping us construct the regional fair poster board.

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