�� Berkeley Pharma Tech Journal of Medicine Correspondence:� Nabeen.chu@gmail.com Keywords: *TDIFNiB $BSEJBD�UJTTVF�SFHFOFSBUJPO .PE/3" .FTFODIZNBM�TUSPNBM�DFMMT *OEVDFE�DBSEJPNZPDZUFT $BSEJBD�HFOFT (FOF�UIFSBQZ Submitted�.BZ�� ������ Accepted�+VOF��� ����� Published July 19, 2022 Full Open Access Creative Commons Attribution �License 4.0 Abstract 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. Novel Methods for Inducing Cardiac Tissue Regeneration Following Ischemic Injury �ïw�§×ØÛÛä��Þëu�¬ÞèÛï×�£Ûï×áëã×èu��åä�¡ÛÛ�¤ååu�®ã×� è×äÚÞßu�and ¯×äâå×ä�§ÝëïÛä Introduction Much research has been done on various approaches to gene therapy1 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 world2, and has also been on the rise since the beginning of the COVID-19 pandemic, disproportionately a�ecting minority populations3. 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 improvement4. Since the rise of gene therapy as a potential treatment method for various illnesses in the 1990s, the �eld has been consistently researched5. 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 applications5, 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�ects6. Ironically, surgically restoring blood �ow to these regions may actually cause cell death and organ failure7. 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). Berkeley Pharma Tech Journal of Medicine | 2 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 persist8 . 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 survivability8. 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 lineages9. 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 unknown9. Previous and ongoing research has shown that the usage of mesenchymal stromal cells has proved to have signi�cant promise in treating ischemic myopathies11. Many clinical trials have shown that the application of mesenchymal stromal cells to infarcted cardiac tissue has been shown to reduce �brosis12, improve regional contractility11, and reduce arrhythmias amongst other outcomes13. 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 infarction14. 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 outcome14. 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 controls12. 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 Berkeley Pharma Tech Journal of Medicine | 3 intramyocardial tissues15. 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 manner15. Speci�c types of MSCs like umbilical MSCs, which promote vascular regeneration and cardiomyocyte protection, are accessible and easily expandable in experiments13. 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 donors13. 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 aging13. 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 vasculature16. 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 term16. 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 trial16. 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 treatment16. 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 doses16. 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 group16. There was no report of long-term ectopic tissue Berkeley Pharma Tech Journal of Medicine | 5 formation either, which showed the signi�cant therapeutic bene�ts of the administration of the hMSCs overall16. 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 MSCs13. The results of this study showed that the UC-MSC treated patients expressed no adverse reactions to the infusion treatment and showed signi�cant improvement in left ventricular ejection fraction over multiple time checkpoints post-administration13. The results of these studies show that the incorporation of MSCs as a treatment option is safe and feasible with di�erent types of MSCs, and there were no noteworthy deleterious reactions to this treatment. The results described above also showed improvement and potential in recovery and o�ers a potential usage in future medicine and therapy. Many questions are still unanswered regarding the mechanism of action of MSC treatment9. Studies have shown that injection of MSCs in a�ected heart tissues reduced infarct size and induced angiogenesis and myogenesis14. 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 cells11. This is usually caused by MSCs’ ability to expand and di�erentiate into cardiomyocyte-like cells13. 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 cardiomyocytes14. 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 tamponade11. 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 understood9. But transplantation of MSCs in many Berkeley Pharma Tech Journal of Medicine | 6 recent reviews and studies have proven to be safe13 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 factors17 and secretory products like angiogenic factors, mitogenic factors, antiapoptotic factors, and growth factors18. In particular MSC’s have been frequently involved in experimental models of cardiovascular diseases13 and express attributes that make them desirable for use in cardiac function modulation after myocardial infarction9 including secretions that could prevent cardiac in�ammation and aid in cardiac injury repair19. Although the underlying detailed mechanism of MSC transplantation is still mostly unknown and incomplete19, and clinical trial results are still suboptimal9, transplantation treatments show promising e�ects15 regarding improvement in myocardial function in experimental models13. Additionally, the trajectory of evidence shows that transplanted cells have a bene�cial e�ect on overall cardiac function after myocardial infarction20 and ischemic heart disease18 due to varying di�erent results. Transplanted MSCs from red bone marrow in adult rat specimens showed di�erentiation into cardiomyocytes by the presence of cardiac proteins like desmin, and also showed to form connections with native cardiomyocytes in the rat sample18. This di�erentiation into both vascular endothelial cells and smooth muscle cells promoted cardiac function via myogenic and angiogenic e�ects14. Engrafted cells harvested from a healthy human sample also showed similar results, with the stem cells strongly morphologically resembling host cardiomyocytes and also expressing desmin, myosin heavy chain, actinin, and cardiac troponin amongst others, each of which are native cardiac tissue proteins21. Echocardiographic assessment of transplanted bone marrow MSCs into post-MI tissues also showed that the cells relieved the restrictive e�ects of left ventricular function and geometry caused by MI20. Additionally, intravenous application of umbilical cord MSCs showed improved left ventricular ejection fraction and left ventricular end diastolic volume in control groups for patients with systolic heart failure or reduced ejection Berkeley Pharma Tech Journal of Medicine | 7 fraction13. Novel experiments have also shown that direct injection of MSC exosomes also reduced infarct sizes, improved cardiac function, and o�ered protection of cardiomyocytes from hypoxia8. Fig 1. MSC Differentiation Potential. Based on the diagram of differentiation potential for MSC terminal phenotypes, we can see how bone-marrow MSCs or umbilical cord derived MSCs have the potential to differentiate to different cell types including cardiomyocytes, vascular endothelial cells, and skeletal muscle cells amongst other cells. This differentiation into cardiomyocytes explains why the expansion of the cells in cardiac tissue settings is very desirable. The differentiation into cardiomyocytes has been measured by proteins like desmin and troponin, and is also noted to form connections with native cardiomyocytes within the tissue. Although much of the application of MSCs is still subject to more extensive clinical testing and development15, there is a huge promise of the already performed experiments discussed above to show safety of the clinical application and the therapeutic bene�ts of administration. The studies above have depicted the many potential and con�rmed bene�ts of administration of either bone-marrow derived MSCs or umbilical cord-derived MSCs, including reduced infarct sizes, overall improved Berkeley Pharma Tech Journal of Medicine | 8 cardiac function and LVEF, cardiomyocyte production and protection, angiogenesis, and myogenesis. Safety of administration of BMSCs and UC-MSCs were both measured with signi�cant results in cohort groups, showing that overall, administration is safe and feasible with generally little to no adverse e�ects13. 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 MSCs13. 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 infarction14. 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 miRNA23. 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 vaccine24. 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 death25. 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 stability26 and they must be engineered to be more e�cient in their expression in the cell, or be longer lasting. So far, this is achieved through four di�erent methods: altering nucleotides, mRNA capping, untranslated region manipulation, and the length of the poly A chain27. Understanding the alterations of mRNA requires an understanding of mRNA synthesis. Often, a DNA plasmid is transcribed in vitro and the resulting mRNA is used for therapy. In this process, substitutions can be made in the nucleotide pairing process. Kierzek et al. showed in 2014 the Berkeley Pharma Tech Journal of Medicine | 9 speci�c e�ects of substituting pseudouridine for uridine with the di�erent nucleic acids, in which Ψ-A pairing demonstrated greatest enhanced stability due to hydrogen bonding, at about 0.5kcal/mol. Due to the 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 TLR828. 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 intravenously29. mRNA caps are methylated guanosine groups attached to the 5’ end by a triphosphate group present in all eukaryotic mRNA30. These give identity to the mRNA strand as well as signal for degradation when removed31. 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 localization32. 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 stability33. 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 cells23. modRNA is able to stay longer in the cell than RNA by changing its structure with pseudouridine and evading RNAase34, but is still shorter-lived than DNA, thus reducing the risk of mutation and multiple transcriptions over time25. Rather, it is described as a “pulse”35, producing a rapid episode of protein synthesis that can be better controlled than DNA therapy methods. Berkeley Pharma Tech Journal of Medicine | 10 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 expensive25. modRNA is already required in large doses36, 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 angiogenesis25. One such method is cell-penetrating peptides that have been demonstrated to carry DNA, RNA, or proteins into the cell via endocytosis37. Figure 2. The Structure of a Modified mRNA. The poly A tail increases translation and allows it to remain longer within a cell. 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 surgery38. Furthermore, modRNA coding for VEGF-A, a growth factor that promotes revascularization was demonstrated to successfully induce angiogenesis following myocardial infarction39. 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 Berkeley Pharma Tech Journal of Medicine | 11 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 characteristics29. 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 fold38. 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 delivery40. Carlsson et al. (2018)29 demonstrated the e�ects of VEGF mRNA after further replacing the pseudouridine with 1-methylpseudouridine and injecting naked mRNA in a citrate-saline bu�er in swine. The treatment resulted in e�cient and long-lasting protein expression, local e�ects, and improvement in cardiac function at 1 week and 2 months after injection29. Improvement here was de�ned as increase in muscle contractility and decrease in �brosis, and were dose-dependent. These �ndings further support and build upon Zangi et al.’s �ndings in 201339 but with greater speci�city in the injections’ e�ects, as measured through bioluminescence, and with higher e�ciency. With cardiac angiogenesis in both species of mice 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, Berkeley Pharma Tech Journal of Medicine | 12 placebo-controlled nature, splitting participants into three groups to receive either placebo or VEGF-A mRNA at various doses41. 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 humans41. 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 pericarditis42. 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 surgery36. While no paper published in a scienti�c journal has reported the results, AstraZeneca published data on the company’s website and presented it at the American Heart Association’s Scienti�c Sessions in 2021. The treated groups demonstrated the biocompatibility and safety of AZD8601, and in agreement with the phase 1 clinical trial41, there were no infections or severe adverse events. Furthermore, because the injections in this trial were epicardial injections, they better demonstrate that VEGF mRNA is safe for Berkeley Pharma Tech Journal of Medicine | 13 human use. But more research must be done to increase e�ciency of the treatment for signi�cant bene�ts in cardiac angiogenesis43. 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 medicine44. iPSCs also provide a safer alternative to potentially toxic drug treatments and can serve as excellent disease models44, 45. Though iPSCs remain the most common cell that are used in iCM derivation, cardiomyocyte-like cells have also been derived from the reprogramming of �broblasts. Derived from direct lineage reprogramming via upregulation of genetic reprogramming factors, several studies have reported the generation of functional iCMs from cardiac �broblasts in both in vitro and in vivo mouse models46, 47. Qian et al. (2012)46 examined the conversion of murine cardiac �broblasts to cardiomyocyte-like cells via the expression of three cardiac-speci�c transcription factors (Gata4, Mef2c, and Tbx5) in vitro and observed a large percentage of cells that were partially reprogrammed. They applied their �ndings to an in vivo model and observed a similar rate of conversion with an even higher level of success in fully reprogramming the �broblasts into cardiomyocyte-like cells46. Wada et al. (2013)47 also previously reported conversion using the aforementioned transcription factors to be successful for mouse �broblasts in vitro47. Following their in vitro study, they observed the generation of new cardiomyocytes from endogenous cardiac �broblasts with the aid of the same transcription factors, and various improved aspects of overall cardiac function. Following these discoveries, the possibility of directly reprogramming human cardiac �broblasts (HCFs) was also studied. While success in the Berkeley Pharma Tech Journal of Medicine | 14 conversion of HCFs to iCMs was observed, the process was observed to be more complex than the mouse model, requiring additional reprogramming factors47, 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 vitro47. However, the iCMs derived from this process “did not beat spontaneously”47. To di�erentiate into functional, beating iCMs, “human iCMs [required] coculture with murine cardiomyocytes,” unlike their mouse counterparts47. Finally, the level of authenticity of the replicated iCMs in comparison to natural cardiomyocytes was unable to be conclusively de�ned47. Somatic �broblasts were also found to give rise to iCMs through upregulation of cardiac gene expression, which can occur both in vitro and in vivo49. Further studies involving human cardiomyocytes in vitro and in vivo are needed to understand the speci�c mechanisms required to produce an e�ective treatment based on this technology. While iCMs are a very promising �eld of cardiac gene therapy, they are, of course, not without their limitations. Horikoshi et al. (2019)44 observed that iCMs derived from iPSCs appear to lack the mature characteristics of normal adult cardiomyocytes. Based on the natural process of cardiomyocyte maturation, they cultured iPSC-derived iCMs with fatty acids, the basis for the fatty acid β-oxidation process that supplies mature cardiomyocytes with energy44. Their study revealed that a fatty acid culture can promote the maturation process of iPSC-derived iCMs in order to allow them to function as fully-matured adult iCMs44. Another limitation observed in the �eld is the applicability of iCM-based treatment to chronic heart failure, a condition for which regenerative therapy is in high demand48. While iPSCs have provided excellent platforms for disease modeling thus far, in vivo cardiac reprogramming studies have been focused on models in the acute stage of myocardial infarction (MI), so its potential to alleviate chronic heart failure has yet to be determined48. However, given the marked improvement of overall cardiac function observed in numerous studies, there seems to be a de�nite potential for chronic heart failure reversal through treatment with iCMs. Berkeley Pharma Tech Journal of Medicine | 15 iCMs themselves have not been observed to proliferate independently, a characteristic which would greatly improve the e�cacy of regenerative therapy48. Instead of relying on the proliferation capabilities of the source cells, such as iPSCs, development of iCMs that are able to endogenously proliferate would remove the necessity for repeated administration of iCMs. Certain critical factors have been identi�ed in addition to the possibility of using a third iCM derivation source in the form of induced cardiac-progenitor cells (iCPCs) that would be converted into iCMs; however, further study is needed to solidify the technology and translate it to clinical application48. Figure 3. Common Derivation Methods of iCMs. Diagram outlining two major sources, iPSCs and fibroblasts, used for the derivation of iCMs. Details the processes by which both sources can be used to create iCMs in vitro that can be inserted for treatment. Also touches on how both of the sources can be directly inserted and used to develop functional iCMs in vivo as part of treatment regimens. Berkeley Pharma Tech Journal of Medicine | 16 Gene Cascades to Target for Cardiac Regeneration The manipulation of certain cardiac genes has shown signi�cant progress in improving ischemic heart disease. Changes in the expression of certain genes in�uence cardiac cell proliferation and di�erentiation, demonstrating gene therapies’ potential e�cacy in treating cardiac disease. This section examines multiple therapies that concentrate on changing the expression of target genes. Two main techniques were investigated, one focusing on the transplantation of BMSCs with speci�c gene deletion and the other concentrating on the protein-protein interactions that in�uence a particular gene involved in cardiac cell signaling. 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 cardiomyocytes53. Injuries in the heart due to ischemic disease result in the destruction of cardiomyocytes, which releases a high concentration of iron into the extracellular space54. 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 proteins55. 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 experiments56. 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�ect56. Nonetheless, this gene therapy demonstrated unpromising results, causing scientists to quickly transition into the transplantation of either mice with bone-marrow-derived cells that are Hamp de�cient or with myeloid cells with Hamp deletions. The investigation was carried out on mice that were deliberately in�icted with myocardial infarction. Those with Hamp expression in macrophages were shown to produce high levels of hepcidin as a result of increased interleukin (IL-6) production, which is responsible for Hamp’s transcription57. Berkeley Pharma Tech Journal of Medicine | 17 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 repair56. 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 injury56. Figure 4. The Effect of the Hamp Gene on Hepcidin Production. Hamp-deficient (-/-) mice reduced hepcidin production and induced the secretion of IL4 and IL13 responsible for cardiomyocyte renewal, while mice with Hamp gene (+/+) produced high levels of hepcidin reducing the release of IL4 and IL13. 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 injuries54. Further Berkeley Pharma Tech Journal of Medicine | 18 investigation on hepcidin is required to understand how the production of cardiac hepcidin a�ects certain tissues in heart cells before diving into particular therapies53. 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 repair56. 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 lungs58. 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 proliferation59. 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 downstream59. 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 Wnt60. 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 tissue59. Nonetheless, the mechanism of CDMG on particular Wnt pathways is still not well-understood. There are a wide variety of Wnt proteins and related signaling pathways that a�ect distinct cellular lineages and their functions. Therefore, further studies regarding what speci�c Wnt proteins CDMG structures inhibit and what speci�c genes are a�ected by these molecules could potentially result in new gene therapies, and ultimately improvements in medicine. In addition to the newly discovered molecules, secreted frizzled-related proteins (Sfrp) inhibit canonical Wnt signaling pathways responsible for cell Berkeley Pharma Tech Journal of Medicine | 19 proliferation, while activating non-canonical Wnt pathways that lead to di�erentiation61. This research was accomplished by culturing cardiac progenitor cells (CPC) from mice. The binding of Sfrp2 to Wnt6 reduced CPC proliferation while fostering di�erentiation. Inhibition of Wnt by Sfrp causes an activation of non-canonical Wnt/Planar Cell Polarity (PCP) pathway through c-Jun N-terminal kinase (JNK)58. This activation encourages the expression of cardiac transcription factors and CPC di�erentiation suggesting that the regulation of Wnt proteins is a possible avenue through which clinicians can address ischemic heart injuries61. A signi�cant limitation of this �nding is the lack of comprehension of the particular pathways that the inhibition of Wnt6 a�ects, and what factors or genes are involved. Figure 5. The Role of the Wnt Pathway in β-catenin Production. Sfrp2 binds to Wnt6 proteins to activate non-canonical Wnt pathway that results in CPC differentiation and leads to a reduction of β-catenin responsible for proliferation. The �nding of pathways related to cardiac cell induction and regeneration has demonstrated a possibility of manipulating these processes to address Berkeley Pharma Tech Journal of Medicine | 20 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. Conclusion Mesenchymal stromal cells have been a popular and desirable candidate in treating function post-myocardial infarction, as they have shown promising e�ects in clinical trials. The administration of MSCs have shown signi�cant therapeutic bene�ts contributing to patients su�ering from myocardial infarction and other pulmonary and cardiac myopathies. Although more extensive and larger-scale clinical trials need to be implemented to exactly understand the mechanism and exact bene�ts of MSC administration, the existing studies have shown results indicating high e�cacy. Studies have shown that through both UC-MSCs, patients treated with infusion of MSCs have shown outcomes which include increased myogenesis and angiogenesis, improved left ventricular ejection reaction, reduced infarct sizes in heart tissue, cardiomyocyte proliferation, and generally improved cardiac function. The di�erence in MSC types and their correlating bene�ts and consequences are also discussed, as well as the safety and feasibility of their administration onto experimental cohorts. These studies also showed little to no adverse e�ects to any of the treatments, and little signi�cant 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. 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 Berkeley Pharma Tech Journal of Medicine | 21 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 Berkeley Pharma Tech Journal of Medicine | 22 these pathways and possible molecules provides an insight into future research directions that could potentially result in novel gene therapies. A B C D E Berkeley Pharma Tech Journal of Medicine | 23 Figure 6. Overview of Amount of Research Articles Pertaining to Each Therapeutic Approach. To obtain a broad overview of how cardiac ischemic injury has been approached in research, a PubMed search of the key terms “gene therapy + cardiac regeneration + ischemic injury” was conducted on May 6, 2022. The first one hundred relevant articles were cataloged and analyzed for the specific research topic that was covered. Panel (A) shows the number of research articles that appeared by the topic overall, and panels (B)-(F) show the number of articles per topic by year of publication. “Genetic Research without Specified Method” refers to work done with specific genes that did not mention the MSCs, modRNA, or iCMs. “Other 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. 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