Anemix: A promising treatment for renal anemia
Anemia is caused by CKD which is a disease that affects around 37 million people in America, making it a massive problem that needs to be addressed urgently (Anemia in Chronic Kidney Disease — NIDDK, 2020). Around 1 in 7 people suffering from CKD suffer from anemia. Patients like Maya Hügle have to go to the hospital regularly for hemodialysis and suffer from recurring episodes of exhaustion due to the disease (Living With Anemia, 2022). Their symptoms include fatigue, splitting headaches, shortness of breath, and more. CKD anemia is caused by the lack of erythropoietin (EPO), a crucial growth factor in the formation of red blood cells, which is produced by healthy kidneys (Anemia in Chronic Kidney Disease — NIDDK, 2020). EPO regulates the production of red blood cells in the bone marrow, depending on the oxygen content of the blood. However, in CKD patients, the kidneys no longer produce sufficient EPO, leading to anemia, or less oxygen content in the blood (Mayo Clinic, 2021). To address the massive problem of anemia caused by CKD, we created Anemix, a novel cellular machine that secretes EPO within the body, providing patients with independence from the dependency on ESA injections for energy, which is often short-lasting and inconsistent throughout the day. This innovative treatment has the potential to positively impact the future of healthcare delivery by providing a safe, effective, and convenient alternative to traditional treatments for renal anemia. Our design involves two kinds of self-amplifying mRNA (Sa-mRNA) that express EPO within the human body for the long term. The expression is controlled through positive and negative feedback loops using riboswitches. Our solution provides a different approach to treating renal anemia: making traditional daily treatment injections bi-weekly or even monthly; and transforming continuous heaviness and fatigue into lightness and energy that lasts the entire day. If successful, this could be a promising solution to CKD anemia and potentially help the millions of people affected by it in America alone. By addressing the issue of CKD anemia with a novel approach, we hope to improve the quality of life for millions of people worldwide who suffer from this disease.
Anemia is a condition where the body has a low amount of red blood cells (RBC) or hemoglobin (Anemia in Chronic Kidney Disease — NIDDK, 2020). As a result of fewer RBC in the body, less oxygen is carried throughout the bloodstream to vital organs, affecting their abilities to carry out essential functions. CKD is a progressive condition affecting more than 10 percent of the world’s population, It affects more than 800 million people worldwide (Kovesdy, 2022). As the stages of CKD progresses, the chances of developing CKD anemia increase significantly. At CKD stage 1, the chances of having anemia is 8.4%, while it increases to 53.4% in stage 5 (Portolés et al., 2021). Renal anemia is caused when the peritubular cells in the kidney fail to respond to hypoxia in the blood by producing and secreting a hormone called EPO (due to CKD), which is essential for the production of new RBC. EPO binds the surface of erythroblasts inside bone marrow, activating them to mature and become RBC. This process keeps the RBC count balanced, accounting for the short RBC lifetime of 115 days. Less RBC being produced leads to anemia (Schoener & Borger, 2023). The main symptoms of anemia include exhaustion, fatigue, weakness, throbbing headaches, irregular heartbeats, shortness of breath, and more (Anemia — Symptoms and Causes, 2022). According to Maya Hugle, a renal anemic patient, from a film from Bayer, ‘It is a struggle to get up in the morning… Your body doesn’t want to do what you want to do… It stops listening to you… But the medication doesn’t last forever… [Energy] rises very slowly and drops eerily quickly. When you have energy you don’t know what to do with it. Then suddenly you’re back to where you were before. Right at the bottom. I hope that they‘ll find something that will be of use to us, the patients… so that energy can be more stable throughout the day. So that we can look after ourselves without outside help.” Current treatments for renal anemia involve the use of erythropoiesis-stimulating agents (ESAs), which are synthetic versions of erythropoietin that stimulate the production of RBC in the bone marrow. However, a major limitation of this treatment is there is a need for frequent injections, which can be more than three times a week. There are also many health side effects to current treatments. Some patients do not respond adequately to ESAs, leading to persistent anemia (UpToDate, n.d.). High doses of ESAs can also result in an increased risk of cardiovascular events, such as heart attack and stroke, particularly those at increased cardiovascular risk. Additionally, the cost of ESA can be prohibitive for some patients, especially those without adequate insurance coverage (Pergola et al., 2019). This proves the need for alternative treatments for CKD-associated anemia.
We designed a cellular machine that secretes EPO within the body, making a patient move from a dependency on frequent injections for energy and ability (which is inconsistent throughout the day). We propose the use of two types of self-amplifying mRNA (Sa-mRNA) that are controlled by riboswitches. The first, codes for Erythropoietin (EPO) and nsPs, which allow for massive replication or amplification of the mRNA. The expression of the mRNA is regulated by riboswitches that respond to HIF levels in the cell, resulting in EPO production when oxygen levels are low. The second type of Sa-mRNA is controlled by riboswitches that respond to existing EPO proteins in the cell. This results in a steady rate of EPO expression in the bloodstream. Erythropoietin is produced at a constant rate during normoxia and at an amplified rate during hypoxia by the kidneys. The hormone is critical as it stimulates the production of RBC in the bone marrow to replace dead cells. In CKD patients, EPO production is reduced, leading to anemia. Therefore, our proposed approach to regulating EPO production using Sa-mRNA and riboswitches could potentially provide a promising solution for CKD anemia and benefit the millions of people affected by it. The cellular machinery will be delivered into the bloodstream where it moves into the surrounding tissue. To avoid the immune system, Sa-mRNA will be contained inside a PEGylated liposome, a small synthetic multicellular vesicle formed using a lipid bilayer. The liposomes will deliver the genetic material into host cells through endocytosis, sa-mRNA will be translated and amplified, and in the process create EPO creation in the body. To conclude, we have designed a machine that stimulates a controlled secretion of EPO into the blood, to balance oxygen levels.
Self-amplifying mRNA: Keeping the cellular machine going
Self-amplifying mRNA is a type of synthetic RNA that can be produced and delivered similarly to conventional mRNA vaccines, but it has the advantage of being self-replicative. This means that after injection, saRNA can safely enter cells and use cellular machinery to make proteins at high levels without requiring large doses or repeat injections (Strategies for Controlling the Innate Immune Activity of Conventional and Self-amplifying mRNA Therapeutics: Getting the Message Across, 2021).
The Sa-mRNA amplification uses many steps to amplify the protein:
- Once the Sa-mRNA is injected into the patient, it enters the cells near the bloodstream using liposomes, which avoids detection from the immune system.
- Ribosomes in the cytoplasm of the cells translate the RNA to make non-structural proteins (nsP1–4).
- The non-structural proteins use the Sa-mRNA molecule as a template to make complementary negative-sense sa-mRNA, which needs to be converted into positive-sense RNA by an RNA polymerase before translation can occur.
- The non-structural proteins split into four separate nsPs that form a new cellular machine. This machine uses the negative-sense Sa-mRNA as a template to make new positive-sense Sa-mRNA, which is similar to the original RNA that was injected and an enormous amount of subgenomic RNAs.
- The cycle continues and the subgenomic mRNA can be used as a template to make erythropoietin. Both RNAs contain a riboswitch that controls the expression of protein and prevents harmful side effects related to excess erythropoietin.
Riboswitches: Keeping the machine in control
Preventing over-expression of EPO, due to the amplification of the mRNA is critical to avoid many clinical complications like polycythemia (excess RBC count resulting in thickening of blood). The effects of polycythemia are dangerous including headaches, dizziness, shortness of breath, blood clotting, cardiovascular disorder, and more. To control mRNA translation we are using riboswitches, structural regulatory elements generally found in the 5′ UTR of mRNA. It regulates the expression of a downstream sequence in response to the binding of a ligand or a small molecule like cellular metabolites and proteins. The riboswitch is made up of two parts: an expression platform and an aptamer domain. The aptamer is made of RNA that structurally rearranges in response to the binding of a specific ligand. After the binding, resulting in a structural change, the expression platform is made accessible (called an activating riboswitch) or inaccessible (it is sequestered and called a deactivating riboswitch) to the ribosome, thus controlling translation. In our solution, we designed aptamers that specifically bind to the ligands including HIF-1 and EPO itself. The expression platform used is the Ribosome Binding Site (RBS), also referred to as the Kozak sequence found in eukaryotes (and Shine-Dalgarno sequence found in prokaryotes). The Kozak sequence typically involves a purine-rich segment of approximately six nucleotides located a short distance upstream of the translation start codon which differentiates them from the rest of the Sa-mRNA. As shown in the diagram, riboswitches can be designed to allow or forbid protein synthesis with the binding of a ligand (Breaker, 2018).
In the first machine, riboswitches control the translation of EPO in response to the HIF ligand levels in the blood. Hypoxia-Induced Factors are proteins that play a crucial role in the body’s response to hypoxia. It is a regulatory factor that binds to certain genes and upregulates their expression in response to hypoxia, expressed in most body cells. Generalized target genes are mainly EPO, VEGF, HO-1, ADM, and Glut-1 (Cheng et al., 2017). For example, HIF stimulates the production of erythropoietin (EPO) in the kidneys. HIF also stimulates angiogenesis in certain cells (VEGF), which results in the formation of new blood vessels, to attempt increasing oxygen delivery to tissues. Additionally, HIF regulates glucose metabolism, which is important for providing energy to cells under low oxygen conditions (Ziello et al., 2007). The genes HIF-1 activates varies based on the type of cell. The aptamer domain is designed to activate translation in response to the binding of the HIF ligand, which will result in the production of EPO. In the second, deactivating riboswitches are used to maintain a constant EPO production in the blood. If EPO levels are higher than normal, there is a higher probability that the aptamer design will bind to an EPO protein. This will forbid the synthesis of new proteins, causing EPO concentrations to decrease. Similarly, if EPO levels are lower than normal, the bound EPO proteins will dissociate and it will be unlikely that new EPO will bind. Thus EPO levels will rise. To ensure the Sa-mRNA will continue its presence will not degrade, when riboswitches do not allow translation, one of two Kozak sequences will allow the translation of nsPs while the other will not be able to express EPO. This allows the nsP portion of the Sa-mRNA to interact with the ribosome replicating the Sa-mRNA (Breaker, 2018). The techniques can be separated into treatments or together. To identify which is most effective, we will have to perform lab testing.
Delivery: Transporting the machine safely and successfully
To deliver Sa-mRNA to target cells around the body, liposomes are used as a transportation method. Liposomes are vesicular nanocarriers containing at least one lipid bilayer on the outside and an aqueous solution containing delivered material on the inside. Liposomes can range in size from 20 nanometers to 1 μm. However, smaller liposomes can be more efficiently transported and fused into target cells. Liposomes used for gene delivery are usually on the smaller end of the spectrum, ranging from 20 to 200 nanometers (Balazs & Godbey, 2010). When the liposome reaches the target cell, it binds to the cell membrane and is taken into the cell through the process of endocytosis. After entering the cell, the liposome releases the genetic material into the cytoplasm of the cell, where it can be used to synthesize erythropoietin and create copies of genetic information through the ribosomes in the host cell. There are many different methods for creating liposomes, but all methods follow the same general steps (Akbarzadeh et al., 2013): (1) Drying down selected lipids from the organic solvent (2) Dissolve the lipids in an aqueous solution (3) Evaporate the solvent, creating a layer of lipid film (4) Rehydrate lipids, forming multicellular vesicles with the lipid bilayer (5) Purify liposomes and analyze the final product. To maximize the effectiveness of the liposomes and ensure they reach target cells, polyethylene glycol chains will be added to the membrane of the liposome. This process is known as PEGylation. PEGylation is a biomedical modification process of adding polyethylene glycol(PEG) onto bioactive molecules, increasing the efficacy and biocompatibility of gene delivery. Since liposomes are foreign to the human body, they can be detected and killed by liver and spleen macrophages. PEGylation of a liposome provides a shield on the surface of the liposome, preventing aggregation, opsonization, and phagocytosis, allowing liposomes to become less immunogenic (PEGylation as a Strategy for Improving Nanoparticle-based Drug and Gene Delivery, 2015). Extra protection of the liposome provided by the addition of PEG chains on their surfaces leads to prolonging blood circulation time, increasing chances of the liposome binding to host cells, and leading to increased efficacy of gene delivery.
Development: Steps to a fully functional treatment
There are many steps in creating a fully functional treatment. The Sa-mRNA and Riboswitches must be synthetically produced and packaged in liposomes. Then the treatment’s efficiency must be tested. And finally if capable enough, the treatment must go through an extensive testing process on mice and humans to prove the efficacy of the treatment.
Making the treatment: Sa-mRNA is synthetically engineered in labs through a process called in vitro transcription (IVT). The steps of IVT are as follows(Yang & Ma, 2016):
- The process starts with the design of a DNA template, including the sequence for desirable RNA. The DNA template has two strands: the non-coding strand and the coding strand. The coding strand contains codons and is responsible for producing complementary RNA (A to U or T, and C to G). The non-coding strand contains anticodons and provides a template for producing RNA.
- The second step is amplifying, which creates millions of copies of the DNA template for further use. This is done using the polymerase chain reaction (PCR). During this process, a polymerase enzyme is added, and it starts copying the DNA.
- The DNA template is transcribed into RNA when the RNA polymerase enzyme reads the coding strand and adds complementary nucleotides to the template, creating the desired RNA.
- Impurities such as DNA and polymerase residue are filtered through purification.
- The quality of the RNA molecule is assessed using a variety of different techniques (e.g. gel electrophoresis).
Single-stranded DNA or RNA can be used to create the aptamer domain in the Sa-mRNA (a highly complicated segment of the machine) by an iterative selection process called SELEX (systematic evolution of ligands by exponential enrichment). In the process, the HIF ligand is exposed to a pool of 1014–1015 oligonucleotide strands or aptamers (ONTs). The ONTs that do not bind are discarded and the bound ONTs are kept. This selection procedure is repeated 6–15 times and the best binding aptamers or ONTs to the ligand are used to create the riboswitch. At the end of the process, the remaining aptamers will be the best at binding with the HIF-1 ligand. These aptamers are included with the Sa-mRNA and are multiplied using traditional PCR machines. (Kong & Byun, 2013).
Lab testing: Our next step as a group would be to test our treatment in bacteria. For this, the Kozak sequence will be replaced with a Shine-Dalgarno sequence and a GFP coding sequence will be added downstream to the EPO coding sequence connected using a linker sequence. By inserting the machinery into E.Coli cells, the oxygen levels of the environment is varied, and the amount of visible colour is measured (GFP is a glowing protein). Through this process the two types of machinery we propose can be tested and through an iterative process the best treatment can be created.
FDA/Clinical Approvals: In order for the treatment to enter the mainstream, there are many processes that need to be fulfilled. The duration of clinical trials can vary significantly depending on several factors, such as the type of therapy being tested, the size of the trial, the number of study sites, and the length of the trial (Ledesma, 2020). In order for the treatment to enter the mainstream, there are many processes that need to be fulfilled. The duration of clinical trials can vary significantly depending on several factors, such as the type of therapy being tested, the size of the trial, the number of study sites, and the length of the trial (Ledesma, 2020). The first step is preclinical research where the treatment undergoes laboratory and animal testing to answer basic questions about safety. Then clinical research where drugs are tested on people to make sure they are safe and effective. Finally, the FDA review teams thoroughly examine all of the submitted data related to the drug or device and make a decision to approve or not to approve it. The average cost of the entire process exceeds $20 million.
The cost of a treatment depends on its dosage. Determining the appropriate dosage of the Sa-mRNA would require extensive testing and evaluation. The optimal dosage would depend on several factors, including the severity of the anemia, the patient’s age and overall health, and their response to treatment. In general, dosages of therapeutic mRNA are typically determined through preclinical studies in animals and early-phase clinical trials in humans. During these studies, researchers evaluate the safety and results of dosages to determine the optimal dosage (National Center for Advancing Translational Sciences, n.d.). Ultimately, the amount of the drug plays a key role in the estimation of costs. To estimate costs, we have compared our solution to other products in the market. Currently, few innovations have been made with Sa-mRNA as it is a relatively new area of study. However, mRNA therapeutics (vaccines currently) in general are estimated to cost between $20 to $40 per dose (Jain et al., 2021). In comparison to the current cost of anemia treatment, which involves the injection of ESA, our solution has the potential to be more cost-effective. ESAs, $1,731 per patient receiving monthly administration to $10,018 per patient receiving thrice-weekly administration (The Drug Development Process, 2018).
Creation of Aptamer: $75. The SELEX random library technique has an average cost of $75 (Cortez et al, 2022).
Replication of Aptamer: $9 (Mahony et al., 2004). PCR technology can replicate DNA effectively and inexpensively.
Stages to FDA Approval: The cost of obtaining clinical approvals for a new medical innovation can be significant, estimated to be $20+ million. This includes costs associated with conducting clinical trials, seeking regulatory approval, and providing medical care to patients.
Distribution: The cost of distributing the Sa-mRNA treatment would depend on factors such as the manufacturing process and the scale of production
Conclusion: The effects and next steps
Our Sa-mRNA solution is a groundbreaking scientific research project that has significant relevance in today’s medical world. Renal anemia is a growing concern globally, affecting millions of people, and the Sa-mRNA solution aims to cure this disease. The solution provides patients with independence from the dependency on ESA injections for energy, which is often short-lasting and inconsistent throughout the day. This innovative treatment has the potential to positively impact the future of healthcare delivery by providing a safe, effective, and convenient alternative to traditional treatments for renal anemia. The Sa-mRNA technology used in this treatment is versatile and can be retrofitted to provide a solution to other endocrine or protein imbalance diseases, such as diabetes and thyroiditis. Thus, if clinically successful, it can spark more research into Sa-mRNA technology, creating more cures for various diseases and possible jobs and career pathways in the field. The affordability of the innovation is a crucial factor in its success. Sa-mRNA treatment has the potential to reduce healthcare costs by providing a long-term solution for renal anemia. Unlike ESA injections, which can be costly and require frequent administration, Sa-mRNA treatment is a one-time procedure. Moreover, the manufacturing and production of the Sa-mRNA constructs and their associated delivery systems could create job opportunities for bioprocess engineers, quality control personnel, and production managers, making it an affordable cost of innovation for the healthcare system and the consumers. The development of a new treatment requires a skilled workforce with specialized training. To implement the Sa-mRNA treatment, there is a need for molecular biologists, genetic engineers, and biochemists to design and test the Sa-mRNA constructs and the riboswitches used to control their expression. Cell biologists are also required to study the effects of the Sa-mRNA constructs on cellular processes and functions. Clinical research scientists, regulatory affairs specialists, and medical professionals are also necessary to conduct clinical trials, seek regulatory approval, and provide medical care to patients. In conclusion, the Sa-mRNA solution is an innovative medical technology with significant scientific and practical significance. It has the potential to positively impact the future of healthcare delivery, reduce healthcare costs, and create job opportunities across various fields. The technology requires a skilled workforce with specialized training to implement it successfully, making it a promising solution to various diseases in the healthcare field.
Team Members: Aditya Mahes, Chenggao Li, Arjun Mahes
Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S. W., Zarghami, N., Hanifehpour, Y., Samiei, M., Kouhi, M., & Nejati-Koshki, K. (2013, February 22). Liposome: classification, preparation, and applications — Discover Nano. SpringerLink. https://doi.org/10.1186/1556-276X-8-102
Anemia in Chronic Kidney Disease — NIDDK. (2020, September 1). National Institute of Diabetes and Digestive and Kidney Diseases. https://www.niddk.nih.gov/health-information/kidney-disease/anemia
Anemia — Symptoms and causes. (2022, February 11). Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/anemia/symptoms-causes/syc-20351360
Breaker, R. R. (2018, November). Riboswitches and Translation Control. PubMed Central (PMC). https://doi.org/10.1101/cshperspect.a032797
Côrtes et al. (2022, June). Selection and Identification of a DNA Aptamer for Multidrug-Resistant Acinetobacter baumannii Using an In-House Cell-SELEX Methodology. Frontier.
Epidemiology of chronic kidney disease: an update 2022. (2022, March 18). Epidemiology of Chronic Kidney Disease: An Update 2022 — ScienceDirect. https://doi.org/10.1016/j.kisu.2021.11.003
Examination of Clinical Trial Costs and Barriers for Drug Development. (n.d.). ASPE. https://aspe.hhs.gov/reports/examination-clinical-trial-costs-barriers-drug-development-0
Folliard, T., Mertins, B., Steel, H., Prescott, T. P., Newport, T., Jones, C. W., Wadhams, G., Bayer, T., Armitage, J. P., Papachristodoulou, A., & Rothschild, L. J. (2017, July 4). Ribo-attenuators: novel elements for reliable and modular riboswitch engineering — Scientific Reports. Nature. https://doi.org/10.1038/s41598-017-04093-x
Hayat, A., Haria, D., & Salifu, M. O. (2008, February 2). Erythropoietin stimulating agents in the management of anemia of chronic kidney disease. PubMed Central (PMC). https://doi.org/10.2147/ppa.s2356\
Jain, S., Venkataraman, A., Wechsler, M. E., & Peppas, N. A. (2021, October 9). Messenger RNA-based vaccines: Past, present, and future directions in the context of the COVID-19 pandemic. PubMed Central (PMC). https://doi.org/10.1016/j.addr.2021.114000
Kong, H. Y., & Byun, J. (n.d.). Nucleic Acid Aptamers: New Methods for Selection, Stabilization, and Application in Biomedical Science. PubMed Central (PMC). https://doi.org/10.4062/biomolther.2013.085
Kovesdy, C. P. (2022, March 18). Epidemiology of chronic kidney disease: an update 2022. PubMed Central (PMC). https://doi.org/10.1016/j.kisu.2021.11.003
Ledesma, P. (2020, January 2). How Much Does a Clinical Trial Cost? — Sofpromed. Sofpromed. https://www.sofpromed.com/how-much-does-a-clinical-trial-cost
Living with Anemia. (2022, November 16). Living With Anemia | Bayer Global. https://www.bayer.com/en/news-stories/living-with-anemia
Mahony, J. B., Petrich, A., Louie, L., Song, X., Chong, S., Smieja, M., Chernesky, M., Loeb, M., & Richardson, S. (n.d.). Performance and Cost Evaluation of One Commercial and Six In-House Conventional and Real-Time Reverse Transcription-PCR Assays for Detection of Severe Acute Respiratory Syndrome Coronavirus. PubMed Central (PMC). https://doi.org/10.1128/JCM.42.4.1471-1476.2004
NIH 3D. (n.d.). NIH 3D. https://3d.nih.gov/
PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. (2015, October 9). PEGylation as a Strategy for Improving Nanoparticle-based Drug and Gene Delivery — ScienceDirect. https://doi.org/10.1016/j.addr.2015.09.012
Pergola, P. E., Pecoits-Filho, R., Winkelmayer, W. C., Spinowitz, B., Rochette, S., Thompson-Leduc, P., Lefebvre, P., Shafai, G., Bozas, A., Sanon, M., & Krasa, H. B. (2019, April 9). Economic Burden and Health-Related Quality of Life Associated with Current Treatments for Anaemia in Patients with CKD not on Dialysis: A Systematic Review. PubMed Central (PMC). https://doi.org/10.1007/s41669-019-0132-5
Portolés, J., Martín, L., Broseta, J. J., & Cases, A. (2021, March 26). Anemia in Chronic Kidney Disease: From Pathophysiology and Current Treatments, to Future Agents. PubMed Central (PMC). https://doi.org/10.3389/fmed.2021.642296
Schoener, B., & Borger, J. (2023, March 11). Erythropoietin Stimulating Agents — StatPearls — NCBI Bookshelf. Erythropoietin Stimulating Agents — StatPearls — NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK536997/
Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: Getting the message across. (2021, July 26). Strategies for Controlling the Innate Immune Activity of Conventional and Self-amplifying mRNA Therapeutics: Getting the Message Across — ScienceDirect. https://doi.org/10.1016/j.addr.2021.113900
The Drug Development Process. (2018, January 4). The Drug Development Process | FDA. https://www.fda.gov/patients/learn-about-drug-and-device-approvals/drug-development-process
What Are Clinical Trials and Studies? (2023, March 22). National Institute on Aging. https://www.nia.nih.gov/health/what-are-clinical-trials-and-studies
Yang, X., & Ma, C. (2016, September 20). In Vitro Transcription Assays and Their Application in Drug Discovery. PubMed Central (PMC). https://doi.org/10.3791/54256
Ziello, J. E., Jovin, I. S., & Huang, Y. (2007, December 1). Hypoxia-Inducible Factor (HIF)-1 Regulatory Pathway and its Potential for Therapeutic Intervention in Malignancy and Ischemia. PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140184/