CAP1

A research group used a unique 'PureCap' method to produce high purity mRNA vaccines with improved synthesis of proteins that stimulate the immune system and low inflammatory side effects. A research group from Japan has developed a method to produce highly active mRNA vaccines at high purity using a unique cap to easily separate the desired capped mRNA. This 'Purecap' technique extracted up to 100% pure Cap2-type mRNA, which showed 3-4 times better production of the protein that stimulates the immune system. These results open up the possibility of purer vaccines with a lower risk of inflammation caused by impurities. Their findings were published in Nature Communications. mRNA vaccines have been used successfully as therapy against variants of the coronavirus. This has given researchers hope for their future use as a cancer vaccine. However, the purity of vaccines hinders this goal because impurities can trigger the immune system. This may cause inflammation around the injection site, a common side effect of vaccination. Impurities in mRNA vaccines are often introduced in the capping stage. During this stage, a cap structure is added that improves the translation of mRNA and protects and stabilizes it. Caps can only be added to single-stranded mRNA, so ideally a vaccine should contain 100% pure single-stranded mRNA. However, unwanted double-strands of mRNA may be present, reducing its purity. As single- and double-stranded mRNAs have different properties, they can be separated using a technique called reversed-phase high-performance liquid chromatography (RP-HPLC). This technique separates mRNAs on the basis of their hydrophobicity or hydrophilicity, i.e., their repulsion to or attraction to water. A research group led by Professor Hiroshi Abe (he, him), Project Assistant Professor Masahito Inagaki (he/him), and Project Associate Professor Naoko Abe (she, her) of the Graduate School of Science, Nagoya University, in collaboration with Tokyo Medical and Dental University, used a unique PureCap method to introduce a hydrophobic tag at the capping stage. The tagged mRNA was easily separated at the RP-HPLC stage. The tag was then easily removed by light treatment, resulting in a 98%-100%-pure vaccine. "We were very excited about the result when we saw on the chart that the RP-HPLC process had separated completely the capped and uncapped RNAs," Hiroshi Abe said. "For a coronavirus mRNA, which is 4247 bases long, we successfully used the PureCap method to produce capped mRNA with over 98% purity." The research group paid particular attention to a group of cap structures that exist in animal and plant cells, called Cap0, Cap1, and Cap2. Although Cap2 is found in animal and plant cells, the evaluation of its function has been difficult because there was no way to obtain pure capped mRNA to ensure a fair test. "The Cap structure used in mRNA vaccines has so far been limited to Cap0 and Cap1 types. However, we used our technique to manufacture Cap0, Cap1, and Cap2-type structures," Abe said. "Highly purified Cap0, Cap1, and Cap2-type mRNA synthesized using the PureCap method showed lower immunostimulatory activity compared to mRNAs synthesized using conventional techniques showing their potential use in pharmaceuticals." As viruses mostly produce Cap1 mRNA, the immune system is less stimulated by Cap2. This suggests that a vaccine that uses Cap2 would be less likely to cause unwanted side effects such as inflammation when it is injected. However, it would still be able to create viral proteins when transcribed that make the vaccine effective. The group used Purecap to create Cap2 mRNA and analyzed its protein synthesis capacity. They found that Cap2 mRNA produced 3-5 times more protein than Cap1 mRNA, which would enhance the immune response. They also showed that their Cap2-type mRNAs caused lower stimulation of the inflammatory response than mRNAs synthesized using conventional techniques. "Conventional mRNA vaccine production methods could not prepare capped mRNA with high purity, raising concerns about reduced protein synthesis and impurity-derived inflammatory reactions," Abe said. "The PureCap method solves these problems by selectively purifying only capped mRNA. Furthermore, the Cap2-type structure created using this technique is more efficient in protein synthesis and less irritating to the immune system. This technique has the potential to improve the safety and efficacy of mRNA vaccines. It is a revolutionary advance toward the practical application of mRNA medicine, as well as deepening our understanding of the fundamentals of mRNA science."

AUSTIN, Texas, April 14, 2023 /PRNewswire/ -- The main results of the Magnetic Resonance Imaging Evaluation of Mineralocorticoid Receptor Antagonism in Diabetic Atherosclerosis (MAGMA) trial, a National Heart, Lung, and Blood Institute (NHLBI) sponsored trial were presented. Although the results of the trial are not final, they offer valuable information for patients using the generic drug Spironolactone. This drug therapy activates white cells and reduces tissue damage as well as reduces proteins that would normally be considered inflammatory. The results from this study will be presented at the National Kidney Foundation's (NKF) NKF Spring Clinical Meetings during the "late-breaking presentations" at the largest multidisciplinary gathering of kidney care professionals in North America from April 11-15 in Austin, Texas. My message to patients is to consider using Spironolactone as their generic drug choice if they can. QUOTE FROM RESEARCHER "My message to patients is to consider using Spironolactone as their generic drug choice if they can for appropriate indications. We want to consider using this drug in high-risk patients with diabetes and atherosclerosis, because the benefit of the drug might extend not only to preventing heart failure and kidney disease progression but potentially other benefits," explained Sanjay Rajagopalan, MD, FACC, FAHA, Chief of the Division of Cardiovascular Medicine and Chief Academic and Scientific Officer of UH Harrington Heart & Vascular Institute; Herman K. Hellerstein, MD, Chair in Cardiovascular Research and a professor at the Case Western Reserve University School of Medicine. "Having a drug that works on these systems in a connective manner is a big benefit to a patient, and this drug happens to be one of those." Highlights of the interim results include: Results: Seventy-nine patients were randomized at 4 sites in US and Canada to Spironolactone (n=37) vs placebo (n=42) for 12 months, after a 2-week single-blind placebo lead-in period and 4-week dose escalation phase. Mean age was 64±8 years; HbA1c 7.3±1.3%; eGFR 44 ml/min/1.73m2; baseline systolic BP 132 ±19 mmHg. The results demonstrated a marked reduction in thoracic plaque volume (Percent change in wall volume) response to Spironolactone at the end of 12 months compared to placebo. These changes were accompanied by changes in left ventricular mass and fibrosis. Spironolactone treatment did not change clinic systolic blood pressure but did cause a statistically significant change in 24-hour mean systolic and diastolic blood pressures compared to baseline and versus placebo respectively. Spironolactone also reduced LV wall mass and a measure of LV fibrosis (native T1 values). Plasma proteomic analysis revealed significant downregulation of a number of pathways involved in immune activation/inflammation, leukocyte activation, proliferation and pathways involved in cytokine stimulation. The top 2 molecules downregulated were DDR2, and oxidized LDL receptor-1 (LOX-1) involved in collagen synthesis and inflammation respectively. Additional top downregulated proteins included known Aldosterone targets such as Sodium/potassium-transporting ATPase subunit beta-3 and Adenylyl cyclase-associated protein 1. Data are mean ± SD. Change was defined as the value at 12 months minus the value at baseline. †p < 0.05; †††p < 0.001 Thousands of kidney care professionals will attend the Spring Clinical Meetings in-person and virtually. Hundreds of the latest studies and kidney care advances will be unveiled. NKF Spring Clinical Meetings For the past 31 years, nephrology healthcare professionals from across the country have come to NKF's Spring Clinical Meetings to learn about the newest developments related to all aspects of nephrology practice; network with colleagues; and present their research findings. The NKF Spring Clinical Meetings are designed for meaningful change in the multidisciplinary healthcare teams' skills, performance, and patient health outcomes. It is the only conference of its kind that focuses on translating science into practice for the entire healthcare team. About Kidney Disease In the United States, more than 37 million adults are estimated to have kidney disease, also known as chronic kidney disease (CKD)—and approximately 90 percent don't know they have it. About 1 in 3 adults in the U.S. are at risk for kidney disease. Risk factors for kidney disease include: diabetes, high blood pressure, heart disease, obesity, and family history. People of Black or African American, Hispanic or Latino, American Indian or Alaska Native, Asian American, or Native Hawaiian or Other Pacific Islander descent are at increased risk for developing the disease. Black or African American people are about four times as likely as White people to have kidney failure. Hispanics experience kidney failure at about double the rate of White people. NKF Professional Membership Healthcare professionals can join NKF to receive access to tools and resources for both patients and professionals, discounts on professional education, and access to a network of thousands of individuals who treat patients with kidney disease. Media Contacts:

A team of researchers has finally identified the molecular mechanism by which a key protein regulates LDL cholesterol. In a crucial step towards understanding the mechanisms involved in cardiovascular disease and certain cancers, a Canadian led research team has succeeded in a world first: they've found the molecular mechanism by which the protein PCSK9 degrades the receptor of low density lipoproteins, the richest cholesterol particles in the bloodstream. The discovery by Nabil G. Seidah, director of the biochemical neuroendocrinology research unit at the Montreal Clinical Research Institute and a medical professor at Université de Montréal, is published in the January issue of Molecular Metabolism. His work was done in collaboration with Carole Fruchart Gaillard and colleagues at the Université de Paris-Saclay's Department of Drugs and Technologies for Health, as well as with scientists in the pharmacy department of the University of Pisa, in Italy. Low density lipoproteins, or LDL, can accumulate in the blood and lead to atherosclerosis and heart disease. The level of LDL and the cholesterol associated with it (LDLc), is directly modulated by the ability of LDL receptors (LDLR) to collect LDL from the bloodstream and internalize it, mainly into the cells of the liver. The surface LDLR drives LDL into the cell where it is captured, and the LDLR returns to the surface for another round of capture. Rare cases linked to the PCSK9 protein Most cases of familial hypercholesterolemia are related to LDLR dysfunction. But rarer cases have been linked to the PCSK9 protein, which Seidah's laboratory's discovered in 2003. PCSK9 is also present in the bloodstream where it associates with LDLR and promotes its degradation by liver cells, preventing it from returning to the surface to capture LDL. Some hypercholesterolemic patients have a "super PCSK9" that enhances the degradation of the LDLR. In recent years, highly effective treatments have been available to patients that inhibit the function (called monoclonal antibody) or reduce the level (called RNAi) of PCSK9 in the bloodstream, resulting in larger amounts of LDLR that ensure a decrease in LDLc of more than 60 per cent compared to conventional statins. Now the work of Seidah and his team lifts the veil on the previously misunderstood mechanism by which PCSK9 drags the LDLR towards the lysosomes, where cells degrade the PCSK9-LDLR complex. A complex of three partner proteins In their lab, Seidah and his team conducted structural analyses that revealed the formation of a complex of three PCSK9 partner proteins, including the LDLR, CAP1 and HLA-C. A key protein in the immune system, HLA-C was found to play a critical role: it directs the entire complex to the lysosomes. HLA-C allows the recognition of the "self," and also stimulates the anti-tumor activity of T lymphocytes. PCSK9, for its part, helps protect against the growth of tumours and associated metastasis by increasing the level of HLA-C on the cell surface. Ultimately, the hope is that inhibitors can be developed that would prevent the interaction of PCSK9 and HLA-C and block the function of PCSK9 on LDLR and HLA-C. That breakthrough could then be applied in clinical practice to treat cardiovascular pathologies as well various types of cancer and metastases in patients.