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

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<h2 style="text-align: center">Design</h2>
 
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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.
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<h3 class="inner-h">Propulsion part: AAV9</h3>
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The main function of the heart is to pump blood, providing power to promote blood circulation to meet the metabolic needs of tissue cells throughout the body.
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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.
 
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<h3 class="inner-h">Precision guidance: BNP promoter</h3>
 
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Heart failure is when the heart's systolic or diastolic function declines, resulting in a failure of cardiac output to meet the body's metabolic needs. Heart failure is the severe and terminal stage of various heart diseases. According to the patients at all stages, patients including hypertension, coronary heart disease and diabetes are at risk of heart failure.
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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.
 
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<strong>Figure 1</strong>
 
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<h3 class="inner-h">What is RyR2? What is it made up of?</h3>
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As we have introduced before, Calcium (Ca<sup>2+</sup>) is the important physiological ligand that activates the channels in cardiac muscle during excitation-contraction (EC) coupling. The heart dysfunction will happen when Ca<sup>2+</sup> cycle is in a mess, which in the end leads to heart failure. That’s why we have to mention the Ca<sup>2+</sup> release channels (a kind of ryanodine receptor) on the sarcoplasmic reticulum (SR) of striated muscles. They adjust and control Ca<sup>2+</sup> between cytoplasm and SR as a biphasic channel such that low cytosolic [Ca<sup>2+</sup>] (mM) activates the channels and high cystolic [Ca<sup>2+</sup>] (mM) inactivates the channels, confirming their crucial role in EC coupling. In cardiac muscle, the Calcium release channels on the SR is named as the type 2 ryanodine receptor (RyR2). It is a tetramer comprised of four 565,000 Dalton RyR2 polypeptides and four 12,000 Dalton FK-506 binding proteins (FKBP12.6).  
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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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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 15:46, 9 October 2018

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

  1. Johnson, F. L. "Pathophysiology and etiology of heart failure." Cardiology Clinics 32.1(2014):9-19.
  2. Smith, J. Gustav. "Molecular Epidemiology of Heart Failure: Translational Challenges and Opportunities." Jacc Basic to Translational Science2.6(2017):757-769.
  3. Eschenhagen, T. "Is ryanodine receptor phosphorylation key to the fight or flight response and heart failure?." Journal of Clinical Investigation120.12(2010):4197-4203. Ullrich, Nina D., H. H. Valdivia, and E. Niggli. "PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity." Journal of Molecular & Cellular Cardiology 53.1(2012):33-42.
  4. Marx, S. O., et al. "PKA Phosphorylation Dissociates FKBP12.6 from the Calcium Release Channel (Ryanodine Receptor)." Cell 101.4(2000):365-376.
  5. Menzel, S., et al. "Nanobody-Based Biologics for Modulating Purinergic Signaling in Inflammation and Immunity." Frontiers in Pharmacology9(2018):266.
  6. Wikipedia. Single-domain antibody. https://en.wikipedia.org/wiki/Single-domain_antibody.
  7. Wikipedia. Phage display. https://en.wikipedia.org/wiki/Phage_display.
  8. Ma, X., et al. "Therapeutic delivery of cyclin-A2 via recombinant adeno-associated virus serotype 9 restarts the myocardial cell cycle: an in vitro study." Molecular Medicine Reports 11.5(2015):3652-3658.
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