Difference between revisions of "Team:SMMU-China/Design"

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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.
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AR185 is a RyR2-specific VHH antibody which is designed to inhibit RyR2 phosphorylation. It was isolated from a camelid VHH antibody library via phage display screening which is a technique received the Nobel Prize in Chemistry this year (2018). VHH, also called nanobody, derives from the variable domains of camelid heavy-chain-only antibody. Compared to the conventional antibodies, VHH has a much lower molecular weight, is a qualified candidate for intracellular antibody.  
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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.
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Revision as of 13:16, 17 October 2018

Propulsion part: AAV9
Precision guidance: BNP promoter
Warhead: AR185
References

Design

To combat heart failure, we designed a targeting device Cartin to implement RyR2-specific inhibition of phosphorylation. A heart-failure-regulatory BNP promoter and heart-tissue-specific vector AAV9 were utilized to increase the device’s specificity and biosafety. The whole project could be divided into three aspects: AAV9, BNP promoter, and AR185, respectively. Here, we will make an analogy between Cartin and homing missile to explain how the device works. The propulsion part (AAV9) is responsible for transporting the missile to the corresponding position. AAV9 can deliver the target gene sequence to the failing heart. The BNP promoter is responsible for precision guidance, which is similar to a targeting system. BNP promoter will only initiate when the heart is damaged and is a controllable switch of gene expressing. The warhead (AR185) is the center of the device that combats heart failure.

Propulsion part: AAV9

Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells and has attracted a significant amount of attention in the field of gene therapy. It has various advantages such as high transduction efficiency, low probability of immune response, and long-term gene transfer potential in slowly dividing or non-dividing cells. So far, several AAV serotypes have been discovered, and each of them has diverse tissue specificity. For example, AAV serotype 9 has a high cardiomyocyte specificity, thus has emerged as a new and promising vector for gene therapy of heart diseases. In our experiment, we verified that AAV-9 vector has a high specificity and efficiency.

Precision guidance: BNP promoter

Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a hormone secreted by cardiomyocytes in the heart ventricles. It has been regarded as an important biomarker in diagnosis of heart failure (HF), for its level is highly related to the severity of HF. The activity of BNP promoter remains low in normal hearts and elevates dramatically in that of patients with HF. Based on the qualities mentioned above, it is natural to think that we can utilize BNP promoter to control gene expression in HF gene therapy.

Warhead: AR185

AR185 is a RyR2-specific VHH antibody which is designed to inhibit RyR2 phosphorylation. It was isolated from a camelid VHH antibody library via phage display screening which is a technique received the Nobel Prize in Chemistry this year (2018). VHH, also called nanobody, derives from the variable domains of camelid heavy-chain-only antibody. Compared to the conventional antibodies, VHH has a much lower molecular weight, is a qualified candidate for intracellular antibody.

The mechanism of BNP promoter driven AR185 inhibiting RyR2 phosphorylation

References

  1. 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.
  2. 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.
  3. 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.
  4. Bennett, A, et al. "Thermal Stability as a Determinant of AAV Serotype Identity." Molecular Therapy Methods & Clinical Development6.C(2017):171.
  5. 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.
  6. Marx, S. O., et al. "PKA Phosphorylation Dissociates FKBP12.6 from the Calcium Release Channel (Ryanodine Receptor)." Cell 101.4(2000):365-376.
  7. 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.
  8. 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.
  9. Majalahti, Theresa, et al. "Cardiac BNP gene activation by angiotensin II in vivo." Molecular & Cellular Endocrinology 273.1–2(2007):59-67.
  10. 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.
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