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Personalized & precision medicine approaches for the treatment of cardiovascular disease

Isabela Garcia Pleijlar

 

Abbreviations

CAD, Coronary Artery Disease; CHD, Congenital Heart Disease; CVD, Cardiovascular Disease; DNA, Deoxyribonucleic acid; ESCs, Embryonic Stem Cells; FDA, Food and Drug Administration; FRESH, Freeform Reversible Embedding of Suspended Hydrogels; hERG, the human ether-a-go-go related gene; hESC-CM, human Embryonic Stem Cell-derived Cardiomyocytes; HGF, Hepatocyte Growth Factor; hiPSC-CMs, human induced Pluripotent Stem Cell-derived Cardiomyocytes; HLHS, Hypoplastic Left Heart Syndrome; iPSCs, induced Pluripotent Stem Cells; LA, Left Atrium; LPLD, Lipoprotein Lipase Deficiency; LV, Left Ventricle; LQTS, Long QT Syndrome; LQT1, Long QT Type 1; LQT2, Long QT Type 2; LQT3, Long QT Type 3; PMM, Personalize My Medicine; RA, Right Atrium; RNA, Ribonucleic Acid; RV, Right Ventricle; µCOP, Light Based Micro-Continuous Optical Printing

 

Glossary

3D Bioprinting, Action Potential, Allogeneic, Anaemia, Arteries, Atherosclerosis, Atoms, Atrium, Autologous, Automaticity, Blood, Cardiomyocytes, Cardiovascular Diseases, Cardiovascular System, Cells, Control, Coronary Arteries, Cytoplasm, Deoxyribonucleic Acid, Electrocardiogram, Electrophysiology, Embryonic Stem Cells, Gene, Gene Expression, Gene Therapy, Growth Factor, Haematopoietic Stem Cells, Heart Failure, Heart Regeneration, Hemoglobin, Hydrogel, Immunosuppressants, Induced Pluripotent Stem Cells, Lipoprotein Lipase, Morphology, Myocardial Infarction, Myocardium, Organ, Passive Diffusion, Personalized Medicine, Phase I Clinical Trial, Phase II Clinical Trial, Phase III Clinical Trial, Phase IV Clinical Trial, Plasma Membrane, Plasmids, Pluripotent, Precision Medicine, Repolarization, Ribonucleic Acid, Skin Fibroblasts, Stem Cells, Tissue, Transcription Factor, Veins, Ventricle

 

Introduction

Precision medicine and personalized medicine are two terms that are often used interchangeably. However, the meaning of these terms varies slightly. Precision medicine considers not only an individual’s genetic information, but also other factors such as lifestyle and the environment. For Personalize My Medicine (PMM), the eventual aim is to achieve the personalization of medicine, which includes but is not limited to precision medicine. Personalized medicine involves anything that tailors the treatment to the individual. It means that healthcare professionals consider all types of relevant factors, including but not limited to the genetics of an individual and their particular needs. It is the opposite of prescribing a standardized drug, treatment or procedure for everyone that is diagnosed with that specific condition. This is ultimately achieved by the precise application of biological and medical knowledge, which is used to design and develop individualized treatments for people.

Having a personalized medicine approach will mean that the chosen treatment will have a higher chance of being more effective for a particular individual. Ultimately, it should potentially lead to decreased expenses for alternative treatments, higher success therapy rates, and, consequently, decreased mortality rates. Striving for a personalized medicine approach regarding cardiovascular diseases (CVD) is worthwhile, given that CVD constitutes 32% of deaths worldwide and is currently the leading cause of death[1]. This article will give a specific focus to CVD, with insights into personalized and precision medicine models, as well as therapies that are currently being tested or are clinically available.

Levels of Organization: from cells to systems

Atoms < Cells < Tissue < Organ < System

Have you ever wondered how many cells make up your body? Scientists have concluded that approximately 37.2 trillion cells work in concert to ensure the continued functioning of the human body[2]. Cells range between 0.01-0.1mm in size, hence most cells cannot be seen by the naked human eye. However, despite their small size, they contain critical molecules within their nucleus, known as deoxyribonucleic acid (DNA). DNA contains the information needed by a cell for it to be able to carry out gene expression and synthesize proteins that are essential for the correct functioning and survival of the cell. Gene expression is the process by which the information encoded within the gene gets transcribed into ribonucleic acid (RNA) and subsequently translated into a protein.

Cells that have the same functions combine to make up a wide variety of tissues that can be classified as being connective, epithelial, muscle, or nervous tissue.  Such tissues then combine to form organs such as the heart or lungs, that perform a specific function. When specific organs work collectively, they give rise to a system, such as the cardiovascular system. The cardiovascular system is comprised of the heart, blood vessels, and blood.

A closer look at the cardiovascular system

The heart is designed to pump blood around the body, while the vessels are responsible for  carrying blood to tissues and organs in order to supply vital nutrients and oxygenated blood, and to remove waste products. This is essential to support the functions and the survival of the tissues and organs. Oxygenated blood, which is carried by the arteries, is rich in oxygen and low in carbon dioxide. Conversely, deoxygenated blood, which is carried via the veins, is low in oxygen and high in carbon dioxide. It is important to note that within expected anatomy and physiology, oxygenated and deoxygenated blood are kept separate and do not mix between the right and left sides of the heart (Figure 1) [3].

The Heart

The heart is composed of four chambers: two upper chambers which receive blood, called atria, and two lower chambers which pump blood to the pulmonary or systemic circulation, called ventricles.  The right side of the heart is composed of the right atrium (RA), the tricuspid valve, and the right ventricle (RV). It is on this side that the deoxygenated blood arrives via the superior and inferior vena cava, before passing onto the right ventricle to be pumped to the lungs via the pulmonary artery for oxygenation. Separated from the right side by the atrial and ventricular septa, the left side of the heart is made up of the left atrium (LA), the mitral valve, and the left ventricle (LV). On this side, the oxygenated blood is collected and subsequently pumped to the rest of the body.

Of note, both sides of the heart are supplied by veins, which may seem contradicting to the aforementioned fact that veins only carry deoxygenated blood. However, pulmonary veins are an exception to this general rule, in that they are the only veins in the body that carry oxygenated blood. The pulmonary artery is also the only artery in the body that carries deoxygenated blood.

When looking at the right side of the heart in detail, the superior and inferior vena cava are the veins responsible for returning deoxygenated blood from the body to the RA (Figure 1). Approximately 70% of the blood that enters the RA, passively diffuses to the RV via the tricuspid valve. In other words, the blood leaks from the RA to the RV without requiring the atria to contract. However, when an action potential is achieved (an electric current that results in the contraction of the atrial muscle), the resultant 30% is pumped into the RV. To prevent regurgitation (backflow) of blood  into the RA and resulting inefficiency, the tricuspid valve closes. Once the pressure within the RV exceeds that of the pulmonary artery, the pulmonary valve opens and the ventricle contracts. Thereby pumping the deoxygenated blood from the RV to the lungs, where it becomes re-oxygenated[3].

The oxygenated blood subsequently returns to the left side of the heart via the pulmonary veins, filling the left atrium (LA). Once the pressure in the LA exceeds that of the LV, the mitral valve opens, and the oxygenated blood passively diffuses into the LV. Following contraction, the oxygenated blood is pumped into the LV. The mitral valve then closes, and the LV subsequently pumps the oxygenated blood into the aorta via the aortic valve. The oxygenated blood then circulates, providing the body with the oxygen and nutrients necessary via the vasculature, which is distributed throughout the tissues and organs of the body.

For blood to be successfully pumped out of the heart, the simultaneous contraction of both atria (RA and LA), followed by the delayed simultaneous contraction of both ventricles (RV and LV) has to occur.

Figure 1. Heart anatomy and blood flow The atrial and ventricular septa separate the right side of the heart from the left side of the heart, preventing the mixing of oxygenated and deoxygenated blood. The blue arrows represent the flow of deoxygenated blood in the right side of the heart. 1) deoxygenated blood returns to the right atrium via the inferior and superior vena cava, 2) the blood passively diffuses and is pumped from the RA to the RV through the tricuspid valve, and 3) the blood is pumped out from the RV to the pulmonary artery, via the pulmonary valve. The burgundy arrows represent the flow of oxygenated blood in the left side of the heart. A) The blood enters the LA via the pulmonary veins, B) the blood is then pumped from the LA to the LV through the mitral valve, and C) the blood is subsequently pumped out from the LV to the aortic arch via the aortic valve. This schematic has been adapted from the Heart Anatomy template in BioRender.

The role of the coronary arteries

The coronary arteries supply oxygenated blood to the heart muscle (myocardium). Without oxygen, the cells cannot meet their metabolic needs, leading to ischaemia (cell death through the lack of sufficient oxygen). This is clearly very serious as it can in turn, lead to heart failure. There are two main factors that influence the supply of oxygen to the heart muscle; the level of oxygen in the blood and the rate at which the blood is flowing through the coronary arteries[3].

People with anaemia or lung disease can suffer from lowered blood oxygen levels. Anaemia is a condition that is defined as having low levels of haemoglobin (Hb). Hb is a molecule that is found within red blood cells and integral in the effective transportation of oxygen throughout the body. It is this molecule that allows the oxygen to reach the many different tissues of the body by diffusing through the plasma membrane of the cells. If there is less Hb available to carry oxygen then oxygen supply to the tissue is significantly reduced[3]. Whilst coronary blood flow is constant under most conditions it can increase during exercise. When diseased, such as is the case in coronary artery disease (CAD), the lumen of the coronary arteries narrows due to the presence of plaque build-up, this can be seen in Figure 2. This narrowing, commonly due to plaque, is termed atherosclerosis. In extreme cases, the coronary artery can be completely blocked leading to a myocardial infarction (heart attack). Consequently, it will lead to the scarring or loss of cardiomyocytes (heart cells). This means that as it is not able to meet the metabolic needs of the cells, the myocardium becomes damaged or dies[3-4].

Figure 2. Coronary artery disease leading to plaque build up A schematic adapted from the template of Coronary Artery Disease in Biorender. It portrays how atherosclerotic plaques can lead to Coronary Artery Disease (CAD) by narrowing the arteries and consequently decreasing blood flow to the myocardium. 

Precision & personalized medicine approaches

The Zebrafish model is one of the most influential models in heart regeneration. This is due to the zebrafish’ ability to regenerate its heart after injury. As demonstrated by Poss et al in 2002, resection of the zebrafish’ heart ventricle results in full regeneration within two months of the injury. Given that mammals cannot regenerate their own myocardium upon injury, findings on how the zebra fish regenerate their heart could pose as ground-breaking therapies without the need to use stem cells[5]. However, the Zebrafish model’s findings are still far from being applicable to humans. Hence, stem cells are currently being investigated as potential therapies[6-7].

Stem cell based therapies

Stem cells have the unique ability to self-replicate indefinitely and to differentiate into any cell lineage. This means that the stem cell population will replenish itself and be able to specialize into any type of body cell. The principal function of stem cells is to develop and regenerate organs and tissue. Therefore, stem cell based therapies involve injecting stem cells into a damaged tissue or organ of the patient to stimulate regeneration. Stem cells include embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs)[8].

If the cells being injected originate from the patient themselves, the procedure is known as an autologous transplant (Figure 3). If the stem cells are derived from a donor, this is called an allogeneic transplant. Allogeneic transplants have a higher risk of triggering an immune response in the recipient as the transplanted cells may be recognized as ‘non-self’. This results in the immune system attacking the organ or cells since it may consider them a foreign invader and therefore a potential danger. To date, the only stem cell-based therapy that has been approved by the Food and Drug Administration (FDA) is haematopoietic stem cell transplant[9]. Most outcomes observed in humans include improved heart muscle function, or reduced scar tissue. Scar tissue in the heart can be seen after a person suffers with a heart attack, since the tissue becomes damaged due to the lack of oxygen. Hence, improving how well the heart muscle contracts, and reducing the amount of non-functional scar tissue ultimately improves the ability of the heart to properly pump blood.

Safety Study of Autologous Umbilical Cord Blood Cells for Treatment of Hypoplastic Left Heart Syndrome

In 2013, a phase I clinical trial aimed to assess how safe and practical the autologous transplantation of stem cells is in patients suffering with hypoplastic left heart syndrome (HLHS) (ClinicalTrials.gov Identifier: NCT01883076). HLHS is a type of congenital heart defect (CHD), in which the structures located on the left side of the heart are underdeveloped in newborns. CHDs are conditions with which an individual may be born with. Usually, it involves abnormalities in the structure of the heart that may result in reduced functioning or overwork of one side of the heart.

In the trial, stem cells were obtained from the umbilical cord blood of the patients upon birth and injected directly into the heart muscle of the RV to improve functionality (intramyocardial delivery) (Figure 3). Hence, to qualify for the trial, the patients had to be previously scheduled to undergo a stage II Glenn surgery, which is the second of three open heart surgeries that can be performed to treat HLHS. That is to avoid the patient from having to undergo a separate open heart surgery for the sole purpose of injecting the stem cells within the heart muscle. After eight years of monitoring the patients, the clinical trial resumed in 2021 concluding that it was safe and feasible to deliver umbilical cord blood-derived stem cells into the RV myocardium. This poses the first step to test the efficacy of stem cells in subsequent trials, as a concomitant therapy for HLHS patients [10].

An actively recruiting clinical trial that started in 2013 aims to obtain and process stem cells from the umbilical cord blood of newborns with HLHS to use as an autologous source of cells in future clinical trials (ClinicalTrials.gov Identifier: NCT01856049)[11]. If subsequent clinical trials can prove the viability and effectiveness of such stem cell based therapies, it may become possible to apply them as an additional therapy for managing the disease, though not as a main therapy. This is because the stem cells are not being injected to regenerate the left side of the heart, but to aid and strengthen the right side of the heart and to maximize the ventricle’s potential.

Figure 3. Stem Cell Therapeutics An original schematic created in Biorender using information from[8-11]. Light blue figures represent an autologous transplant, whereas the cells sourcing from a separate donor (dark blue) represent an allogeneic transplant.

3D Bioprinting

The majority of patients with CHDs require surgery to reconstruct the abnormalities in their heart structure. In more severe cases, a heart transplant may also be necessary. Much time would be saved if it were possible to create a heart using a 3-D bioprinter by engineering cardiac tissue using the patient’s own cells as it would mean that the patient would have a shorter wait to find a donor match, ultimately decreasing mortality rates.

The first 3D bioprint of an adult heart was constructed by Feinberg et al through the use of Freeform Reversible Embedding of Suspended Hydrogels (FRESH). This 3D model is made out of alginate and not stem cells, a material that gives the engineered heart the elasticity of cardiac tissue as portrayed in Figure 4. Although the correct shape and elasticity of cardiac tissue can be achieved using alginate, the tissue itself does not have the properties that are required for a heart to beat on its own. Therefore, the alginate heart cannot be transplanted into patients. However, it does demonstrate that it is possible to print a full size adult heart that is constructed from biological materials. The alginate heart can also aid in the training and planning of surgical procedures as it allows doctors to practice their surgery on a replica heart that has the same structure as the patient’s heart, as the 3D bioprint is based on high resolution images of the patient’s own heart [12]. This helps the doctor to improve their chances of performing a successful surgery and decreases the risk of surgical complications.

Despite the lack of functionality in the bioengineered organs, cardiac tissue engineering is advancing and it is showing significant results. Successfully engineering cardiac tissue could provide an alternative to a full-scale heart transplant, reducing the chances of post-operative organ rejection or the need for lifelong immunosuppressants. Of note, immunosuppressors are drugs that inactivate your immune system and essentially avoid rejection of the organ.

However, there are several factors that affect the functionality of the tissue. These include the choice of cell (such as iPSCs or hESCs), automaticity (whether the cells can generate spontaneous action potentials like cardiac cells), the stiffness of the model (which is dependant on the chosen material and collagen concentration used), and the proteins expressed and timing of expression during development. All of these are needed because tissue is not only made out of cells, but it also contains proteins and lipids amongst others, which affect the overall function of the organ. Given that the composition of tissue is complex, scientists have not been able to recreate the native cardiac environment in its full form. This means that the morphology of the cells (shape and organization), the electrophysiology (such as the resting membrane potential which is when an action potential has not yet occurred), mechanics (contractile force and conduction velocity; meaning how strong the muscle contracts and how fast the action potential travels through the myocardium), and gene expression (leading to specific proteins getting expressed) is not exactly the same. However, advances have shown that by combining some of these specific characteristics the model can still closely resemble the native cardiac environment[4].

Liu et al aimed to create scaffolds to replace damaged tissue after a heart attack. They used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and printed them on a GelMa hydrogel. First, they took the human ESCs and directed the differentiation of the cells into the cardiac lineage creating hESC-CMs. Such cells were then printed onto the GelMa hydrogel via light based Micro-Continuous Optical Printing (µCOP), a specific technique used for 3D bioprinting using biological materials. (Figure 4).  The results demonstrated that the cells contracted within the range of immature cardiomyocytes. Despite results not exerting mature cardiomyocyte characteristics, this model can be used as a powerful tool to test pharmacotherapies as well as to further investigate how cardiac tissue matures[13].

Figure 4. 3D Bioprinting An original schematic created through Biorender summarizing the aforementioned experiments: bioengineered alginate heart and hESC-CMs tissue printed on the Gelma Hydrogel[4,13].

 
Gene therapy

The application of gene therapy is also being investigated to treat cardiovascular disease. Gene therapy is achieved by transduction, which is the process by which foreign DNA is inserted into cells in a target tissue or organ, with the aim of achieving successful expression of the transferred gene by the cells. Consequently, it should result in the synthesis of the wanted or previously lacking RNA or protein. In order to achieve transduction, vectors are utilized as a means to transport and insert the DNA. The most common vectors used in gene therapy are plasmids, adenovirus and attenuated viruses that have been modified so that they cannot cause disease to the cells[14].

Glybera was the first successful gene therapy drug to be approved. It was developed to treat lipoprotein lipase deficiency (LPLD), an autosomal inherited disease that results in high triglyceride levels in the blood. High triglyceride levels are directly correlated to atherosclerosis, which is a condition that causes plaque build-up inside of the vessels. Atherosclerosis can lead to lack of blood flow and therefore oxygen supply (ischaemia) due to narrowing or blockage of the vessel if the plaque breaks. Though Glybera is no longer available for clinical use, it served as the first step for gene therapy to become a reality rather than just an idea[15].

At present, there are two parallel phase III clinical trials that are actively recruiting patients in China. The aim of the randomized, double blinded, placebo-controlled trial is to test the safety and efficacy of inserting recombinant human hepatocyte growth factor (HGF) to treat lower critical limb ischaemia. (ClinicalTrials.gov Identifier: NCT04275323 and NCT04274049). Lower critical limb ischaemia is the blockage of the arteries found in the lower limbs (legs). In this trial, NL003 is the plasmid that is being used to insert the coding sequence that will express HGF 728 and HGF 723. The plasmid can be thought as the messenger containing the coding sequence. In turn, the coding sequence contains the instructions that are needed by the cell´s machinery to be able to produce the proteins HGF 728 and HGF 723. Currently, there are no drugs available for clinical use to treat critical limb ischaemia, so if successful, a phase IV clinical trial will be conducted in which the drug will be reviewed by the FDA.[16, 17]

Conclusion

Precision and personalized healthcare is the ultimate goal for achieving improved outcomes for patients. In cardiovascular science, animal models such as the zebrafish model are being used to learn more about how the heart may be able to regenerate. The use of stem cells to achieve regeneration of the heart and to potentially individualize treatments for patients is being investigated. A Phase 1 Clinical Trial concluded that it is safe to inject umbilical cord blood stem cells obtained from the patient into the patient’s own myocardium. In addition, an ongoing Phase II Clinical Trial is aiming to isolate stem cells from the umbilical cord blood of newborns to assess the effectiveness of using the patient’s own stem cells as a treatment for HLHS.

Scientists are also trying to print organs and tissue made from biological materials such as proteins and cells. Despite the many limitations that scientists encounter due to the complexity of the tissue, they have been successful in developing innovations such as an alginate 3D heart that can improve a patient’s surgery outcomes by individualizing the 3D bioprint to each patient’s heart structure. Moreover, scientists can now print tissues that can serve as a mean to test for different pharmacotherapies. Looking to the future, another type of approach that is being investigated by scientists is gene therapy. Two parallel clinical trials (Phase III) are actively recruiting patients to analyze the efficacy and safety of genetically inserting HGF against a placebo drug. Essentially, scientists hope to see the effects of the drug in a larger sample size.

 

Clinical Trial Links

 

Useful Links

 

References

[1] Cardiovascular diseases [Internet]. World Health Organization. World Health Organization; [cited 2023Jan4]. Available from: https://www.who.int/health-topics/cardiovascular-diseases#tab+tab_1 

[2] Zimmer C. How many cells are in your body? [Internet]. Science. National Geographic; 2021 [cited 2023Jan4]. Available from: https://www.nationalgeographic.com/science/article/how-many-cells-are-in-your-body 

[3] Lilly LS. Chapter 1: Normal Cardiac Structure and Function. In: Pathophysiology of heart disease: A collaborative project of medical students and faculty. Philadelphia: Wolters Kluwer; 2020. 

[4] Kato B, Wisser G, Agrawal DK, Wood T, Thankam FG. 3D bioprinting of cardiac tissue: Current challenges and perspectives. Journal of Materials Science: Materials in Medicine. 2021;32(5).

[5] Beffagna G. Zebrafish as a smart model to understand regeneration after heart injury: How fish could help humans. Frontiers in Cardiovascular Medicine. 2019;6.cu

[6] Bacakova L, Zarubova J, Travnickova M, Musilkova J, Pajorova J, Slepicka P, et al. Stem Cells: Their source, potency and use in regenerative therapies with focus on adipose-derived Stem Cells – a review. Biotechnology Advances. 2018;36(4):1111–26. 

[7] Wehman B, Siddiqui OT, Mishra R, Sharma S, Kaushal S. Stem cell therapy for CHD: Towards translation. Cardiology in the Young. 2015;25(S2):58–66. 

[8] Aly RM. Current state of stem cell-based therapies: An overview. Stem Cell Investigation. 2020;7:8–. 

[9] U.S. Food and Drug Administration. FDA. [Internet]; [cited 2023Jan4]. Available from: https://www.fda.gov/vaccines-blood-biologics/consumers-biologics/important-patient-and-consumer-information-about-regenerative-medicine-therapies 

[10] Safety Study of autologous umbilical cord blood cells for treatment of hypoplastic left heart syndrome – full text view [Internet]. Full Text View – ClinicalTrials.gov. 2013 [cited 2023Jan4]. Available from: https://clinicaltrials.gov/ct2/show/NCT01883076 

[11] Umbilical cord blood collection and processing for hypoplastic left heart syndrome patients – full text view [Internet]. Full Text View – ClinicalTrials.gov. 2013 [cited 2023Jan4]. Available from: https://clinicaltrials.gov/ct2/show/NCT01856049?term=Stem%2Bcell&cond=Hypoplastic%2BLeft%2BHeart%2BSyndrome&draw=2&rank=9 

[12] Mirdamadi E, Tashman JW, Shiwarski DJ, Palchesko RN, Feinberg AW. Fresh 3D bioprinting a full-size model of the human heart [Internet]. ACS Publications. ACS Biomaterials Science&Engineering; 2020 [cited 2023Jan4]. Available from: https://pubs.acs.org/doi/10.1021/acsbiomaterials.0c01133 

[13] Liu J, He J, Liu J, Ma X, Chen Q, Lawrence N, et al. Rapid 3D bioprinting of in vitro cardiac tissue models using human embryonic stem cell-derived cardiomyocytes. Bioprinting. 2019;13. 

[14] Korpela H, Järveläinen N, Siimes S, Lampela J, Airaksinen J, Valli K, et al. Gene therapy for ischaemic heart disease and heart failure. Journal of Internal Medicine. 2021;290(3):567–82. 

[15] Ylä-Herttuala S, Baker AH. Cardiovascular gene therapy: Past, present, and future. Molecular Therapy. 2017;25(5):1095–106. 

[16] Safety and efficacy study using gene therapy for critical limb ischemia (NL003-CLI-III-1) – full text view [Internet]. Full Text View – ClinicalTrials.gov. 2020 [cited 2023Jan4]. Available from: https://clinicaltrials.gov/ct2/show/NCT04275323 

[17] Safety and efficacy study using gene therapy for critical limb ischemia (NL003-CLI-III-2) – full text view [Internet]. Full Text View – ClinicalTrials.gov. 2020 [cited 2023Jan4]. Available from: https://clinicaltrials.gov/ct2/show/NCT04274049 

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