Novel Methods for Inducing Cardiac Tissue Regeneration Following Ischemic Injury

Cardiac disease continues to be among the most prevalent causes of death worldwide. Presently, surgeries such as angioplasties, stents, and bypasses pose many risks. To improve outcomes in treating ischemia, researchers have been pursuing minimally invasive, biocompatible treatments such as gene therapies. Gene therapies are treatments that enhance or suppress target genes to alleviate illness. There are various applications for gene therapies, including, but not limited to, the treatment of cancers, diabetes, and heart disease. Gene and stem cell therapies can regenerate cardiac tissue that is damaged due to ischemia. Furthermore, gene therapies intended to evade the immune system may decrease infection risks due to the new tissue being better accepted by the body as it is created from patients’ own cells. While DNA treatments show poor results in treating cardiac illness, stem cells, such as mesenchymal stem cells and induced pluripotent cells, can differentiate into cardiomyocytes, and mRNA can be modified to express angiogenesis growth factors around the affected tissue. Although further research is needed to adapt these techniques for safe clinical use, they show potential for inducing cardiac tissue regeneration in ischemic injury. This paper conducts a review of the emerging techniques and evaluates gene therapy as a potential treatment for ischemic injury.


Introduction
Much research has been done on various approaches to gene therapy 1 to treat an ischemic injury, yet few techniques have gone into clinical trials, and these seldom show success. This paper aims to conduct a review of a few major techniques and evaluate the potential of gene therapy as a treatment of ischemic injury.
Cardiac-related illnesses and deaths have consistently been one of the top causes of death in the U.S. and around the world 2 , and has also been on the rise since the beginning of the COVID-19 pandemic, disproportionately a ecting minority populations 3 . Thus the importance of researching more e ective methods of treating and preventing ischemic injury is imperative.
The mortality of ischemic heart injury di ers by demographic, including one's age, country, and region, with lower-income countries and older people su ering higher mortality rates, with little to no improvement 4 .
Since the rise of gene therapy as a potential treatment method for various illnesses in the 1990s, the eld has been consistently researched 5 . The precision of the ability to modify speci c genes o ers great potential.
However, due to obstacles such as high cost, ethical concerns, and di culty translating into clinical applications 5 , there is still much to uncover.
Cardiac ischemic injury refers to cell damage due to diminished ow of blood, containing nutrients and oxygen, through the vessel. If ischemic injury is not caught quickly and treated in a timely manner, cardiomyocyte death can occur, with permanent e ects 6 . Ironically, surgically restoring blood ow to these regions may actually cause cell death and organ failure 7 .
Gene therapy thus presents as a favorable option to instead promote angiogenesis through growth factors such as VEGF-A or stem cell therapies to develop new paths of blood ow rather than risking further ischaemia-reperfusion injury (IRI).
We have decided to look not only into gene cascades that are often targeted by gene therapies, but various approaches to gene modi cation, including stem cell research using mesenchymal stem cells (MSC), modi ed mRNA (modRNA), and induced pluripotent stem cells (iPSC).

Mesenchymal Stem Cells to Treat Ischemic Injury
Myocardial infarction (MI), more commonly known as heart attack, and related injuries are a leading cause of death globally, and despite the applications of extensive novel cardiac surgery and grafting to lengthen and ameliorate life, long-term cardiac damage may persist 8 . The usage and application of mesenchymal stromal cells (MSCs) in heart tissue has become an increasingly popular candidate in the treatment of myocardial infarctions, as they exhibit factors that increase cardiomyocyte survivability 8 .
There is an abundance of research aimed toward understanding the implementation of MSCs in the prognosis and improved function of heart tissue, due to their desirable trait of being able to di erentiate into disparate cellular lineages 9 . It is by far one of the most promising treatments in regenerative medicine. However, it is di cult to gather results from existing clinical trials due to suboptimal reproducibility and e cacy outcomes, 10 and the exact mechanism of the cell treatment is still largely unknown 9 .
Previous and ongoing research has shown that the usage of mesenchymal stromal cells has proved to have signi cant promise in treating ischemic myopathies 11 . Many clinical trials have shown that the application of mesenchymal stromal cells to infarcted cardiac tissue has been shown to reduce brosis 12 , improve regional contractility 11 , and reduce arrhythmias amongst other outcomes 13 . In clinical trials with mice, it has been demonstrated that direct application of MSCs could aid in angiogenesis and myogenesis of ischemic myocardium in murine post-acute myocardial infarction 14 . This is a signi cant e ect of MSCs, since heart failure can be caused by death of large numbers of cardiomyocytes, so the induction of angiogenesis and myogenesis can prove to be a desirable outcome 14 . Clinical studies have also shown that application of autologous bone marrow MSCs (BMSCs) correlated to reduced scar sizes, reduced infarct sizes, improved left ventricular ejection fraction (LVEF), and a signi cant increase of viable tissue in patients who underwent coronary artery bypass graft surgery (CABG) compared to controls 12 . One particular study aimed to investigate the direct e ect of bone marrow-derived MSCs on cardiac function immediately after MI by injecting the cell sample into infarcted intramyocardial tissues 15 . Although the experiment showed that the donor cells minimally remained in the myocardium after implantation, and the exact mechanism is still unknown, there was a signi cant reduction in infarct sizes, improved cardiac function, expression of pro-angiogenic factors, all occurring in a paracrine manner 15 .
Speci c types of MSCs like umbilical MSCs, which promote vascular regeneration and cardiomyocyte protection, are accessible and easily expandable in experiments 13 . However, with the varying stem cell types, there are also many di erent constraints that have hindered more innovative research into the application of MSCs. The application of bone-marrow derived MSCs in particular include invasive or unethical harvesting procedures, and decreased proliferation among varying donors 13 . But in a more promising facet, umbilical-derived MSCs demonstrate more safe and feasible qualities, including being more easily attainable, posing less ethical concerns, and undergoing less cellular aging 13 .
Safety of MSC application is an important facet of the treatment to ensure since there are several potential health concerns surrounding the treatment, including tumor formation, organ toxicity, and ectopic tissue formation in the vasculature 16 . Safety pro les of intravenous MSC application in acute MI patients are successfully present, showing signi cant results such as reduced tachycardic episodes, improved forced expiratory volumes, and no concerns for ectopic tissue creation in the long term 16 . However, one study aimed to evaluate the safety of human bone marrow-derived mesenchymal cell application in patients su ering from acute MI by using a multicenter trial 16 . In the study, the Data Safety and Monitoring Board approved the application of MSC dosage in each experimental cohort, and primary safety assessments were carried out to monitor any adverse reactions to the treatment 16 . The results of this study showed that there was no apparent evidence of increased toxicity with the application of the MSCs and there the administration was well tolerated in the cohorts at all doses 16 . There was also evidence that the arrhythmia ratio was signi cantly lower in the MSC administered patients vs. the placebo group, and also showed improved function in cardiac performance and pulmonary function in comparison with the placebo group 16 . There was no report of long-term ectopic tissue formation either, which showed the signi cant therapeutic bene ts of the administration of the hMSCs overall 16 . Another study aimed to evaluate the safety of intravenous administration of umbilical cord-derived MSCs (UC-MSCs) as well. The experimental group involved patients with heart failure and reduced ejection fraction, and were presently treated with application of intravenous infusion of allogeneic umbilical-cord derived Many questions are still unanswered regarding the mechanism of action of MSC treatment 9 . Studies have shown that injection of MSCs in a ected heart tissues reduced infarct size and induced angiogenesis and myogenesis 14 . One main mechanism of action is injection of bone marrow-derived-MSCs directly into infarcted heart tissue to promote cardiac function via expansion of the cells 11 . This is usually caused by MSCs' ability to expand and di erentiate into cardiomyocyte-like cells 13 .
One experiment conducted coronary artery ligations in mice and then implemented MSCs to note any changes in grafting in the ischemic myocardium and proliferation into cardiomyocytes 14 . This particular experiment showed results illustrating improved cardiac function after MSC implementation through the enhancement of myogenesis and angiogenesis. Other experiments involving injection of MSCs or BMCs into infarcted tissue have shown some results correlating with improved regional contractibility, and no signs of ventricular arrhythmias, worsening cardiac function, pulmonary embolisms, or cardiac tamponade 11 . There are still suboptimal results correlating MSC transplantation and therapeutic improvement of cardiac function, since the mechanisms of action are quite complex and not fully understood 9 . But transplantation of MSCs in many recent reviews and studies have proven to be safe 13 and the selective study results shown could be bene cial.
MSCs have been used as a therapeutic application for many degenerative diseases, mainly due to their various di erentiation potential , paracrine factors 17 and secretory products like angiogenic factors, mitogenic factors, antiapoptotic factors, and growth factors 18 . In particular MSC's have been frequently involved in experimental models of cardiovascular diseases 13 and express attributes that make them desirable for use in cardiac function modulation after myocardial infarction 9 including secretions that could prevent cardiac in ammation and aid in cardiac injury repair 19 . Although the underlying detailed mechanism of MSC transplantation is still mostly unknown and incomplete 19 , and clinical trial results are still suboptimal 9 , transplantation treatments show promising e ects 15   showing that overall, administration is safe and feasible with generally little to no adverse e ects 13 . Although these studies presented signi cant outcomes, it has been suggested that more extensive and larger clinical trials are needed to fully understand the clinical bene ts of administration of certain MSCs 13 . Due to the many measured bene cial implications of MSCs, including overall cardiac function, we can conclude that MSC transplantation can be a new therapeutic strategy for treating patients su ering from myocardial infarction 14 .

Modifed RNA as a Method of Gene Therapy
DNA modi cation comes with many risks, including immunological response, and lack of speci city regarding its locus of action. Consequently, RNA therapy is becoming a further researched eld, including clinical applications for modRNA and miRNA 23 .
Modi ed mRNA (modRNA) is becoming a prevalent research topic in many di erent elds, such as diabetes, ischemic injury, and the mechanism of a COVID-19 vaccine 24 . In this method of gene therapy, mRNA is injected into the heart to enhance target protein translation, which in this case is cardiomyocytes and angiogenesis to repair the heart after massive cell death 25 . However, mRNA is short lived and cannot provide lasting e ects for cardiomyocyte regeneration. They often trigger an immune response, and are also broken down by RNAases. Thus the degree of protein expression is dependent on mRNA stability 26  increased stability and because pseudouridines are found naturally in the body, these modRNA are able to avoid detection by the immune system, speci cally by TLR3, TLR7, and TLR8 28 . Furthermore, it was found that VEGF mRNA whose uridines were completely replaced by pseudouridine, did not trigger the immune system and remained locally in the target tissue of cynomolgus monkeys or rats while promoting angiogenesis. In this experiment, the mRNA was injected intradermally or intravenously 29 . mRNA caps are methylated guanosine groups attached to the 5' end by a triphosphate group present in all eukaryotic mRNA 30 . These give identity to the mRNA strand as well as signal for degradation when removed 31 .
Thus the synthetic mRNA must also be capped adequately to increase translation of protein.
The untranslated region of RNA, called the UTR, appears on both the 5' and 3' ends of the mRNA strand, and plays a role in mRNA translation e ciency and localization 32 . Thus UTR of modRNA must also be manipulated adequately to optimize translation rate and half-life. For example, the addition of AU-rich elements (ARE) weakened protein expression, and the more stable UTR was able to increase transcription stability 33 . Synthesizing UTR speci c to the needs of the modRNA is imperative to its e ective protein synthesis, and also allows for more precise control of the therapy. mRNA is modi ed by extending the polyA tail to increase its translation.
This modRNA is administered through intramyocardial injection to a ect a large area of cardiomyocyte and non-cardiomyocyte cells 23 . modRNA is able to stay longer in the cell than RNA by changing its structure with pseudouridine and evading RNAase 34 , but is still shorter-lived than DNA, thus reducing the risk of mutation and multiple transcriptions over time 25 .
Rather, it is described as a "pulse" 35 , producing a rapid episode of protein synthesis that can be better controlled than DNA therapy methods.
Currently, due to the temporary nature of modRNA, treatments may need to be administered multiple times to demonstrate e ectiveness, which not only causes trauma on the cardiac tissue but also will become expensive 25 .
modRNA is already required in large doses 36 , thus production or administrative costs must be reduced to make this treatment clinically feasible. Furthermore, injecting these genes into the intracardiac tissue is invasive and can be risky for patients who have already su ered injury to the heart. Further research must be done on less invasive gene and drug delivery methods that are still able to target speci c cells and promote angiogenesis 25 .
One such method is cell-penetrating peptides that have been demonstrated to carry DNA, RNA, or proteins into the cell via endocytosis 37 . Despite these intricacies of modifying mRNA, it has been demonstrated that modRNAs can deliver relatively quick local responses both in vitro and in vivo. Mouse hearts were able to retain the e ects of a cardiac injection of modRNA for 24 hours after surgery 38 . Furthermore, modRNA coding for VEGF-A, a growth factor that promotes revascularization was demonstrated to successfully induce angiogenesis following myocardial infarction 39 . The modRNA injected into the site of injury promoted heart progenitor cells to di erentiate into vascular cells rather than muscle cells.
In this experiment, the mice injected with VEGF-A DNA and VEGF-A RNA both formed vessels, but the mice who received the RNA injection presented with vasculature that was less permeable and more similar in shape to control hearts. The vessels of VEGF-A DNA injected mice showed edema, and had higher mortality rates. These di erences, likely due to the di erences in acting speed of RNA and DNA, display the potential of modRNA in the eld of cardiology. Moreover, because VEGF-A, a commonly used and researched growth factor, can cause increased vessel permeability at long exposure times, the shorter "pulses" of RNA may be more e ective in treating ischemic heart injury.
VEGF-A's results are much more promising with mRNA than DNA largely due to its rapid and local characteristics 29 . This e ect can be further enhanced by moving away from lipid carriers, sometimes called nanoparticles, that are often used to encapsulate proteins or genetic material. Removing this layer prevents the mRNA from entering circulation and traveling away from the target tissue, and the naked delivery of the modRNA increased protein was found to increase translation 53-226 fold 38 . However, it must be noted that while for ischemic, local injury, removing the nanoparticles improves translation, for drugs that are aimed for general circulation, such as mRNA vaccines, the lipid nanoparticles are an essential part of drug delivery 40 . and swine, modRNA appears to be a strong candidate for carrying out cardiac regeneration after an ischemic injury. However, these studies will have to be carried out over longer periods to ensure whether the modRNA must be reinjected, and for any side e ects.
While clinical trials of VEGF modRNA for cardiac regeneration are scarce, its safety and e ectiveness were observed in treating patients with type II diabetes mellitus. The study was conducted in a randomized, double-blind, placebo-controlled nature, splitting participants into three groups to receive either placebo or VEGF-A mRNA at various doses 41 . The results re ect that of Carlsson et al. 29 that demonstrated dose-dependent results. VEGF-A mRNA was able to successfully promote basal skin blood ow at injection sites of the forearm, and the results were dose-dependent. At 7-14 days after injection, vasodilation and neovascularization were induced, demonstrating the potential for VEGF-modi ed mRNA applications in humans 41 .
Observed adverse events were of onsite reactions, which occurred in all participants, but with only mild severity. The e ects of cardiac modRNA injection in humans as of yet unknown, though the gene therapy method is considered safe to be researched further in the context of cardiac ischemic injury. However, this clinical trial only had male participants, thus women must also be tested to ensure comparable results, and whether there are sex di erences in dosing.
After the safety and biocompatibility of VEGF modi ed mRNA were con rmed, it was applied in the cardiac eld for patients undergoing Coronary artery bypass grafting (CABG) by AstraZeneca in a study called EPICCURE. This is a very common surgical procedure that aims to revascularize the heart by attaching grafts bypassing the clogged artery.
However, there are still many risks, especially for those who have a history of renal disease or stroke. Adverse events include infection, atrial brillation, myocardial dysfunction, which are at higher rates for those who are undergoing dialysis or have other underlying cardiac conditions such as peripheral artery disease or pericarditis 42 . AZD861, a VEGF mRNA drug in citrate-bu ered saline, will be administered immediately after CABG before reperfusion at various doses to determine whether angiogenesis will be promoted, and if it will improve outcomes of the surgery 36

Induced Cardiomyocytes for Cardiac Regeneration
Induced cardiomyocytes (iCMs) are cardiomyocyte-like cells that are derived from the reprogramming of other cells, the most common being induced pluripotent stem cells (iPSCs) 44 . Culturing iPSCs with speci c media allows them to be di erentiated into the target cell, in this case, iCMs. iPSCs provide a solution to the long-standing di culty of the lack of regenerative abilities of cardiomyocytes; their ability to proliferate allows treatment to surpass the stagnant number of normal cardiomyocytes in the body, a revolutionary possibility for regenerative medicine 44 . iPSCs also provide a safer alternative to potentially toxic drug treatments and can serve as excellent disease models 44,45 . Following these discoveries, the possibility of directly reprogramming human cardiac broblasts (HCFs) was also studied. While success in the conversion of HCFs to iCMs was observed, the process was observed to be more complex than the mouse model, requiring additional reprogramming factors 47,48 . The three transcription factors used in the in vitro and in vivo rodent models were not su cient; the addition of Mesp1 and Myocd allowed for the generation of human iCMs in vitro 47 . However, the iCMs derived from this process "did not beat spontaneously" 47   Recent studies have shown that hepcidin, an iron-regulating protein primarily in the liver, is also present in low amounts in the heart, to regulate iron homeostasis and ferritin content in cardiomyocytes 53 . Injuries in the heart due to ischemic disease result in the destruction of cardiomyocytes, which releases a high concentration of iron into the extracellular space 54 .
The increased iron content outside cardiomyocytes is associated with myocardial brosis and with the formation of reactive oxygen species (ROS) 53 . These conditions cause an intracellular iron de ciency that leads to the overexpression of hepcidin proteins 55 . In e orts to understand how hepcidin a ects ischemic heart disease and to study the possibilities of related therapies, the gene coding for hepcidin, Hamp, was manipulated in deletion experiments 56 .
Despite the lack of knowledge on how cardiac hepcidin functions and a ects heart injuries, researchers rst carried out direct gene deletions of Hamp in mice to prevent the production of hepcidin and study its e ect 56  This new study on the e ect of hepcidin on heart injuries revealed that this protein is related to the secretion of IL-4 and IL-13 which are each important for inducing cardiac repair 56 . Hamp de cient mice with myocardial infarction displayed a signi cant reduction in infarct size and tissue brosis as well as increased cardiomyocyte renewal. This result was related to the lack of hepcidin that led to improved receptor functions, such as C-C motif receptor 2 (CCR2)+, in macrophages. This improvement enhanced the secretion of IL-4 and IL-13 from the macrophage increasing cardiac healing as well as cardiomyocyte regeneration after a heart injury 56 . While this research demonstrates a new nding regarding the relationship between hepcidin and IL-4/IL-13, the mechanisms of how hepcidin in uences the function of certain receptors and interleukins are still not widely understood. There is limited research on the potential of using this gene therapy due to the researchers' e ort to rst better understand how cardiac hepcidin works in the heart and related injuries 54 . Further investigation on hepcidin is required to understand how the production of cardiac hepcidin a ects certain tissues in heart cells before diving into particular therapies 53 . The study of this protein has great potential of being utilized for improved cardiac function in ischemic heart diseases, particularly the Hamp-de cient BMSCs that induced cardiac repair 56 .
Although not yet implemented into paradigms of gene therapy, the ongoing in-depth study of protein-protein interactions has led to the possibility of controlling the expression of speci c genes, essential for signaling pathways involving cell proliferation. The detailed understanding of Wnt protein/β-catenin pathways, responsible for cell proliferation and growth, has led to signi cant improvements in addressing diseases in organs such as the lungs 58 . In attempts to regulate this pathway for cardiac cell regeneration, recent studies have revealed protein interactions with Wnt units that could inhibit injury-induced cardiomyocyte proliferation 59 .
In a study on heart regeneration and injury repair, scientists discovered novel small molecules called cardiomogens (CDMG 1 and 2) that have the ability to inhibit Wnt expression and cause β-catenin reduction downstream 59 . The investigation was carried out using embryonic zebra sh with surgically-induced heart injuries. CDMGs inhibited Wnt by speci cally targeting β-catenin and Tcf/Lef-mediated transcription that is needed to initiate the expression of particular genes speci ed by Wnt 60 . The reduction in β-catenin accelerates the proliferation of damaged heart cells, leading to improved heart function. CDMG1, particularly, was e ective in healing heart injuries by increased formation of cardiomyocytes and reduction in brotic scar tissue 59   The nding of pathways related to cardiac cell induction and regeneration has demonstrated a possibility of manipulating these processes to address heart injury. In the case of hepcidin proteins, Hamp de ciency has shown to be associated with iron-homeostasis and the secretion IL-4/IL-13 that help with cardiomyocyte renewal. CDMG molecules and Sfrp proteins were proven to be related to the inhibition of Wnt pathways that reduce cell proliferation but result in di erentiation. However, for both cases, the pathways resulting in improved heart function are still not well-understood and just provide a glance of future research directions. There is, thus, signi cant room for elaboration; further studies may implicate more speci c cell or gene therapies for treating ischemic heart diseases. worsening of conditions has been recorded. The exact mechanism of their bene cial e ect is still unknown and needs to be more extensively studied, but these studies have shown promising therapeutic bene ts.

Conclusion
Modi ed mRNA also demonstrates great potential to be used towards cardiac regeneration, especially expressing VEGF, as it has expressed no adverse reactions when used clinically, and successfully promoted angiogenesis. Developments have been made in the puri cation and evading the immune system through RNA caps, pseudo nucleotides, and the poly A tail. However, more research must be done speci cally on the ischemic injury and cardiac regeneration model for it to be used speci cally in response to a myocardial infarction. Before this, an understanding of the e ect of VEGF on humans, and increasing e ciency of the treatment is imperative, as there is very little data on optimal dosage concentration or repetitions that carry out the desired e ects. Lastly, for modi ed mRNA to be a viable clinical option, the shelf life and sustainable storage of the puri ed mRNA in bu er must be researched to ensure minimal degradation over time. Thus, modifying mRNA shows great potential as a gene therapy mechanism, though more research must be done to adapt it to be used for human cardiac regeneration.
iCMs derived from various sources have been employed in numerous treatment approaches to cardiac illness, and continue to provide many research opportunities in the eld of regenerative medicine. As observed by the evolution of iCM research over time, studies have made their way from animal models to the use of human cell sources for experimentation, suggesting that application of iCM-based treatment in clinical trials is not far o . iCMs provide a treatment option that has the potential to draw on a abundantly-sourced treatment platform, avoid the negative side-e ects of toxic pharmacological treatments, and signi cantly expand the scope of regenerative medicine. Continued research into iCMs seems to be a very promising path to treating ischemic injury induced by cardiac dysfunction. Particular genes and protein interactions related to the heart have resulted in a greater insight into cardiac regeneration to address heart injuries. Cardiac hepcidin protein reduction through Hamp gene de ciency was shown to be associated with iron-homeostasis and the secretion of IL4/IL13 from macrophages inducing cardiomyocyte regeneration. The inhibition of pathways related to Wnt proteins using CDMG and Sfrp molecules also revealed that cardiac progenitor cell di erentiation is a possible metric with which to gauge improvement in heart injuries. However, these studies are recent research interests that have only been implemented in animal models.
The particular genes, reactions, and factors involved in these pathways are still not well-understood. While there is a long way to go for a greater comprehension of how they work in the human heart, the identi cation of these pathways and possible molecules provides an insight into future research directions that could potentially result in novel gene therapies. refers to therapies used to treat cardiac ischemic injury that did not involve genetics, such as cell therapy or drug therapy. "Multiple Therapies" refers to articles that address more than one form of therapy.
Research in the eld of gene therapy to alleviate cardiac ischemic injury has been studied for decades and will continue to be studied in coming years. As the current trends in the research were reviewed, all therapies covered in this article still seem to be relevant to the eld. Overall, the most popularly researched therapy seems to be the direct interaction with speci c genes, above any other methods mentioned. However, as mentioned, the others do not fall far behind. As the eld continues to expand, it is hopeful that research will make the transition from models to clinical trials and continue to add to each of these therapies for treatment.
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