Design
In most countries, heart problems are the leading cause of morbidity and mortality. Heart failure is the end stage of most heart diseases, with approximately 38 million heart failure patients worldwide. Despite the use of drugs, implanted cardiac assist devices and surgical treatments, many patients' conditions will still irreversibly deteriorate, eventually being difficult to control and rescue. Therefore, we designed a system similar to homing missiles to fight heart failure. The system is divided into three parts. The propulsion part (AAV9) is responsible for transporting the missile to the corresponding position. AAV9 can deliver the target gene sequence to the heart. The BNP promoter is responsible for precision guidance. BNP promoter will only start when the heart is damaged and is a controllable switch of gene expressing. The warhead (AR185) contains explosives that act to detonate and eliminate part of negative effects of heart disease.
Propulsion part: AAV9
Adeno-associated virus is a member of the parvovirus family. The virus has no envelope and the genome is single-stranded DNA. There is no report on human pathogenesis. At the same time, adeno-associated virus is an ideal gene therapy vector, which can efficiently transport genes to target organs and has a low probability of inducing an immune response in the case of stable expression. There are more than 150 clinical trials using AAV as a vector to target various genetic diseases. 1,6,8,9 serotypes of adeno-associated virus show better myocardial tissue phagocytosis, most early studies used AAV1 as a carrier. With the development of technology, AAV9 has become a new and promising vector for gene therapy of heart disease. The experimental results that have been reported and ours have shown that the AAV9 vector can effectively transfect in the heart and is fully qualified for propulsion task.
Precision guidance: BNP promoter
BNP is a quantitative marker of heart failure. It is a hormone mainly synthesized and secreted by the ventricle. It can specifically respond to changes in ventricular function and has high sensitivity. Therefore, we use a BNP promoter in our project. With this promoter, the target gene delivered by the AAV vector only expresses when the heart is damaged and the BNP concentration is significantly increased. In vivo studies of BNP stress-responsive regions revealed that -408/+100 bp hBNP and -534/+4 bp rBNP promoter fragments are able to increase luciferase reporter activity after acute MI and AngII infusion, respectively. In both cases, the level of the reporter activation was consistent with upregulation of endogenous BNP. The BNP and BNP promoter (-408/+100 bp hBNP) are used as a system with feedback regulation to achieve the function of a missile-guided system, making our warheads accurately hit the target and explode, reducing the possibility of accidental injury.
Warhead: AR185
A large number of animal experiments support that PKA catalyze the hyperphosphorylation of RyR2-S2808 in ischemic cardiomyopathy, result in calcium leakage, destroying calcium homeostasis and promoting heart failure. Based on these, we have consulted with our primary PI, Professor Shi Hu, who is a leading scientist in the field of Antibody Engineering. He introduced a novel nanobody termed AR185 which has been recently developed by his group and showed specifically inhibition of RyR2 phosphorylation In vitro.
A few studies have mutated the serine at the S2808 site in mouse RyR2 to alanine, so that the 2808 site cannot bind to the phosphate residue, Mutation blocked RyR2 phosphorylation at this site, and did not cause dysfunction of RyR2 meanwhile phosphorylation level of RyR2 Protein is reduced, FKBP12.6 dissociation is reduced, and calcium leakage is also obviously reduced. We believe that AR185 can also have a similar effect to the mutation of RyR2, and it can eliminate the negative effects of hyperphosphorylation, just like the warhead bomb out the target. Since it is difficult to establish a model of hyperphosphorylation of RyR2 in vitro, we plan to express antibodies and evaluate the effect in animals.
References
- Weiwei, C, et al. "Outline of the report on cardiovascular diseases in China, 2014. " European Heart Journal Supplements Journal of the European Society of Cardiology 18. Suppl F (2016): F2.
- Yancy, Clyde W., et al. "2016 ACC/AHA/HFSA Focused Update on New Pharmacological Therapy for Heart Failure: An Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure." Journal of the American College of Cardiology 68.13(2016):1476-1488.
- Hulot, Jean Sebastien, K. Ishikawa, and R. J. Hajjar. "Gene therapy for the treatment of heart failure: promise postponed." European Heart Journal 37.21(2016):1651-1658.
- Bennett, A, et al. "Thermal Stability as a Determinant of AAV Serotype Identity." Molecular Therapy Methods & Clinical Development6.C(2017):171.
- Zincarelli, Carmela, et al. "Analysis of AAV Serotypes 1-9 Mediated Gene Expression and Tropism in Mice After Systemic Injection." Molecular Therapy the Journal of the American Society of Gene Therapy 16.6(2008):1073-80.
- Marx, S. O., et al. "PKA Phosphorylation Dissociates FKBP12.6 from the Calcium Release Channel (Ryanodine Receptor)." Cell 101.4(2000):365-376.
- Wehrens, X. H., et al. "Enhancing calstabin binding to ryanodine receptors improves cardiac and skeletal muscle function in heart failure. " Journal of Cardiac Failure 11.9(2005):9607-9612.
- Wehrens, Xander H. T., et al. "Ryanodine receptor/calcium release channel PKA phosphorylation: A critical mediator of heart failure progression." Proceedings of the National Academy of Sciences of the United States of America 103.3(2006):511-518.
- Majalahti, Theresa, et al. "Cardiac BNP gene activation by angiotensin II in vivo." Molecular & Cellular Endocrinology 273.1–2(2007):59-67.
- He, Q., et al. "Inducible regulation of human brain natriuretic peptide promoter in transgenic mice." American Journal of Physiology Heart & Circulatory Physiology 280.1(2001):H368.
