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| <h2 style="text-align: center">Description</h2> | | <h2 style="text-align: center">Description</h2> |
| </header> | | </header> |
| + | <p class="inner-text"> |
| + | Ca2+RTIN is the abbreviation of cardiomyocyte RyR2 targeting Intra-nanobody. It is also the name of our project. Our project focus on heart failure and want to make contribution to treatment. Meanwhile, RyR2 is one of the most important calcium channels in cardiomyocytes and we expect to achieve the treatment effect by improving Ca2+ handling. So, we add ‘2+’ after ‘Ca’.</p> |
| | | |
| + | <p class="inner-text"> |
| + | Chronic PKA phosphorylation of RyR2 has been shown to lead to cardiac dysfunction. We designed a targeting device, CaRTIN (Cardiomyocyte RyR2 Targeting Intra-Nanobody), to implement RyR2-specific inhibition of phosphorylation. Here, one of the isolated RyR2 nanobodies, AR185, inhibiting RyR2 phosphorylation in an in vitro assay was then chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of AR185 to combat post-ischemic heart failure. Adeno-associated viral gene delivery elevated AR185 protein expression in rat heart, and this administration normalized the contractile dysfunction of the failing myocardium in vivo and in vitro. Moreover, CaRTIN therapy to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca<sup>2+</sup> leak, restoring the diminished intracellular Ca<sup>2+</sup> transients and Ca<sup>2+</sup> load and reversed the phosphorylation of RyR2. To achieve controlled intra-nanobody release, a BNP promoter based platform was also accessed. Our results established a role of CaRTIN as a promising therapeutic approach for heart failure.</p> |
| <div id="to_hf" style="height: 15px"></div> | | <div id="to_hf" style="height: 15px"></div> |
| <h2 class="inner-h">1 Heart failure</h2> | | <h2 class="inner-h">1 Heart failure</h2> |
| <h3 class="inner-h">What is heart failure?</h3> | | <h3 class="inner-h">What is heart failure?</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
− | 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. | + | The primary function of the heart is to pump blood and promote blood circulation to meet the metabolic needs of different tissues throughout the body. |
| </p> | | </p> |
| <p class="inner-text"> | | <p class="inner-text"> |
− | 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. | + | Heart failure is when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Heart failure is the severe and terminal stage of various heart diseases. Patients with hypertension, coronary heart disease and diabetes are at risk of heart failure. |
| </p> | | </p> |
| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
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| <h3 class="inner-h">Clinical status and influence of heart failure</h3> | | <h3 class="inner-h">Clinical status and influence of heart failure</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
− | The global incidence of chronic heart failure is 3 percent among adults and as high as 10 percent over the age of 80, according to the study. Death rates from heart failure have increased six fold over the past 40 years, and about 60 percent of patients die within five years of being diagnosed, with a five-year survival rate similar to that of malignant tumors. Heart failure also brings huge economic burden to society. In Europe and North America, the hospitalization of heart failure accounts for 1% to 4% of the hospitalization quantity, and 46% of the discharged patients are re-admitted due to the worsening of heart failure in 2 months. The average hospitalization time is 5-10 days, accounting for a large amount of medical resources, and the cost of heart failure treatment accounts for 1% to 2% of the total medical expenditure. The United States spent about $39.3 billion on heart failure in 2010, and total spending on patients with heart failure is expected to increase by 50-100% over the next 10 years. At the same time, as the end-stage patients of heart failure lose their activity and work capacity, the side effects are also very large, resulting in a huge family burden. | + | Based on the latest statistics, heart disease has become the leading cause of death worldwide. The global incidence of chronic heart failure, the terminal stage of many heart diseases, is 3 percent among adults and as high as 10 percent over the age of 80. About 60 percent of patients die within five years of being diagnosed, with a five-year survival rate similar to that of malignant tumors. Heart failure also brings substantial economic burden to society. In Europe and North America, the hospitalization of heart failure accounts for 1% to 4% of the hospitalization quantity. The United States spent about $39.3 billion on heart failure in 2010, and total spending on patients with heart failure is expected to increase by 50-100% over the next 10 years. At the same time, as patients with severe heart failure will lose their working capacity, heart failure will also cause a considerable burden to the patient’s family. |
| </p> | | </p> |
| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
− | <img src="https://static.igem.org/mediawiki/2018/d/d9/T--SMMU-China--Description_Fig_2.jpg" style="width: 60%;"> | + | <img src="https://static.igem.org/mediawiki/2018/d/d9/T--SMMU-China--Description_Fig_2.jpg" style="width: 70%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
| <strong>Figure 2</strong> | | <strong>Figure 2</strong> |
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| <h3 class="inner-h">Current treatment methods and limitations</h3> | | <h3 class="inner-h">Current treatment methods and limitations</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
− | At present the treatment of heart failure mainly treated by drug therapy and placement equipment including beta blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and aldosterone inhibitors, diuretics, sinoatrial node ion channel inhibitors, these made tremendous contributions to the traditional therapy for the prevention and treatment of heart failure, and improve the long-term survival of patients. However, although these laws can control the disease to a certain extent, they cannot fundamentally treat heart failure. As the disease progresses, patients with heart failure will still enter the terminal stage of the disease. | + | At present, the treatments of heart failure mainly focus on drug therapies such as beta blockers, angiotensin-converting enzyme inhibitors, aldosterone inhibitors and so on. These drugs have made tremendous contributions to the control of heart failure and have improved the long-term survival of patients. However, these treatments could only control the disease to a certain extent, the prognosis of heart failure is still dismal. As the disease progresses, patients with heart failure will even enter the terminal stage of the disease. |
| </p> | | </p> |
| <p class="inner-text"> | | <p class="inner-text"> |
− | With the development of molecular biology and the research progress of human genetics, it is believed that heart failure is related to abnormal expression and regulation of some genes in cardiac cells. As a result, gene therapy is gaining attention. | + | Facing with such conditions, gene therapy has emerged as a novel and promising approach for treating heart failures in recent years. |
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| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
− | <img src="https://static.igem.org/mediawiki/2018/6/65/T--SMMU-China--Description_Fig_3.jpg" style="width: 90%;"> | + | <img src="https://static.igem.org/mediawiki/2018/6/65/T--SMMU-China--Description_Fig_3.jpg" style="width: 95%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
| <strong>Figure 3</strong> | | <strong>Figure 3</strong> |
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| </div> | | </div> |
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− | <h3 class="inner-h">What is gene therapy?</h3> | + | <h3 class="inner-h">Cardiac Muscle Contraction and Ca2+ cycling</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
− | Heart failure gene therapy is through a variety of technologies such as chemical, physical or biological, will be packed with gene carrier after importing the receptor through various methods of myocardial cells or tissues, expressed through a correct or interference in heart failure pathological process of abnormal expression of target genes, so to cure or reduce symptoms of heart failure treatment. At present, the research on heart failure gene therapy mainly focuses on calcium circulation-related gene therapy, b-adrenalin signaling system related gene therapy, adenylate cyclase 6 related gene therapy, and vascular endothelial growth factor b-related gene therapy. Despite the initial difficulties in gene delivery to the heart of large animals, genetic treatment studies in preclinical large animal models have shown encouraging results. Notably, gene therapy is considered promising for heart failure. The US Food and Drug Administration has awarded MYDICAR a "breakthrough therapy" for heart failure, its first approved gene therapy. The treatment reactivates the serca2a enzyme in the body, improving the heart's ability to pump blood. | + | Calcium ion plays a significant role in mediating cardiac muscle contraction. When cardiac muscles are excited, changes in cell membrane potential can activate the opening of L-type calcium channels on the cell membrane, and the extracellular Ca2+ ions flow into the cells. The Ca2+ influx then binds to Ryanodine receptor 2 (RyR2) which functions as a calcium channel located on sarcoplasmic reticulum (SR), and cause more Ca2+ to release into cytoplasm from SR through RyR2. The increasing Ca2+ ions then result in the contraction of cardiac muscle via a series of reactions. After each contraction, most of the calcium ions will be restored into SR and the calcium concentration in cytoplasm goes back to a low level. |
| </p> | | </p> |
− | <div style="text-align:center" class="resultimage">
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− | <img src="https://static.igem.org/mediawiki/2018/a/ab/T--SMMU-China--Description_Fig_4.jpg" style="width: 60%;">
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− | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;">
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− | <strong>Figure 4</strong>
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− | </p>
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− | </div>
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− |
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− | <h3 class="inner-h">Diastole and contraction of the normal heart</h3>
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| <p class="inner-text"> | | <p class="inner-text"> |
− | The heart is made up of the heart cells, each time the heart pumps blood is made up of all the heart cells diastolic and systolic together. When myocardial cells are excited, changes in cell membrane potential can activate the opening of l-type calcium channels on the cell membrane, and the extracellular Ca<sup>2+</sup> concentration gradient enters the cell and binds to the Ca<sup>2+</sup> release channel on the sarcoplasmic retina, further activating the stored Ca<sup>2+</sup> release in the sarcoplasmic reticulum, causing the intracellular Ca<sup>2+</sup> concentration to increase rapidly. Ca<sup>2+</sup> in the cytoplasm can bind to troponin and change the position of myosin, thus exposing the site of action of actin on the actin and making the head of myosin bind to actin to form a cross bridge. Increased cytosolic Ca<sup>2+</sup> concentration can activate the Ca<sup>2+</sup>-Mg<sup>2+</sup>-ATP enzyme in the head of myosin, hydrolyze ATP to release energy, and induce myocardial contraction. | + | However, when heart failure happens, the calcium channel RyR2 on SR will be hyperphosphorylated by PKA, thereby leading to calcium leakage from SR and reduction of myocardium contractility. |
− | </p>
| + | |
− | <p class="inner-text">
| + | |
− | When the heart is diastolic, most of it is ingested and stored by sarcoplasmic reticulum Ca<sup>2+</sup>-ATP, and a small fraction is transported by cell membrane sodium-calcium exchange protein and cell membrane Ca<sup>2+</sup>-ATP outside the cells, resulting in a rapid decrease of cytoplasmic Ca<sup>2+</sup> concentration. The flow process of Ca<sup>2+</sup> accompanying the contraction and relaxation of cardiac cells is called calcium cycle, which is also the most important factor in the contraction and relaxation of cardiac cells.
| + | |
| </p> | | </p> |
| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
| <img src="https://static.igem.org/mediawiki/2018/9/96/T--SMMU-China--Description_Fig_5.jpg" style="width: 60%;"> | | <img src="https://static.igem.org/mediawiki/2018/9/96/T--SMMU-China--Description_Fig_5.jpg" style="width: 60%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
− | <strong>Figure 5</strong> | + | <strong>Figure 4</strong> |
| </p> | | </p> |
| </div> | | </div> |
− |
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− | <h3 class="inner-h">Diastole and contraction of the heart in heart failure</h3>
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− | <p class="inner-text">
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− | As mentioned before, the stability of calcium circulation is an important guarantee to maintain the normal systolic function of cardiac cells. At present, calcium circulation disorder is considered to be an important pathophysiological basis for heart failure. Under the condition of heart failure, the dysfunction of RyR2, a calcium ion release channel on the sarcoplasmic network, leads to the disturbance of calcium circulation and leads to the dysfunction of excitation-contraction coupling of cardiac cells, which in turn leads to the myocardial cell contraction.
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− | </p>
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| <div id="to_cartin" style="height: 15px"></div> | | <div id="to_cartin" style="height: 15px"></div> |
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| <img src="https://static.igem.org/mediawiki/2018/0/07/T--SMMU-China--Description_Fig_6.jpg" style="width: 60%;"> | | <img src="https://static.igem.org/mediawiki/2018/0/07/T--SMMU-China--Description_Fig_6.jpg" style="width: 60%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
− | <strong>Figure 6</strong> | + | <strong>Figure 5</strong> |
| </p> | | </p> |
| </div> | | </div> |
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| </p> | | </p> |
| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
− | <img src="https://static.igem.org/mediawiki/2018/5/57/T--SMMU-China--Description_Fig_7.jpg" style="width: 60%;"> | + | <img src="https://static.igem.org/mediawiki/2018/5/57/T--SMMU-China--Description_Fig_7.jpg" style="width: 50%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
− | <strong>Figure 7</strong> | + | <strong>Figure 6</strong> |
| </p> | | </p> |
| </div> | | </div> |
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| </p> | | </p> |
| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
− | <img src="https://static.igem.org/mediawiki/2018/5/57/T--SMMU-China--Description_Fig_8.jpg" style="width: 60%;"> | + | <img src="https://static.igem.org/mediawiki/2018/5/57/T--SMMU-China--Description_Fig_8.jpg" style="width: 40%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
− | <strong>Figure 8</strong> | + | <strong>Figure 7</strong> |
| </p> | | </p> |
| </div> | | </div> |
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| <h3 class="inner-h">Phage display antibody library</h3> | | <h3 class="inner-h">Phage display antibody library</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
− | After utilizing S2808 to stimulate camels’ immune system and isolating the mRNA coding for heavy-chain antibodies, we got a gene library of camelids-S2808 nanobodies containing several million clones by reverse transcription and polymerase chain reaction. As for the screening techniques we chose phage display, a technique inserting the gene encoding S2808’s nanobody into a phage coat protein gene, causing the phage to "display" the nanobody on its outside while containing the gene for the protein on its inside. We transfected the phages into engineering bacteria and gained amplification products. Followed with multiple rounds of antigen affinity adsorption-elution-amplification, we finally got the S2808’s specific nanobody clones. | + | To obtain antibodies with high affinity to RyR2, we adopt the phage display technique, one of the Nobel Prize technology (Chemistry, 2018). Phage display is a laboratory technique for the study of protein–protein interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In our study, a library of variable domains of camellidae heavy chain-only antibodies (VHH) was constructed. A vast majority of VHH clones were inserted into phagemid and expressed on the surface of the phages. In order to select binders to RyR2, bio-panning was performed with immobilized RyR2 protein. To obtain antibodies that functionally inhibit of RyR2 phosphorylation, antibody fragments isolated in the previous step were tested for its effect in an ELISA based RyR2 phosphorylation assay. Finally, AR185 and a negative control AR117 were obtained for further investigation. |
| </p> | | </p> |
| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
| <img src="https://static.igem.org/mediawiki/2018/6/64/T--SMMU-China--Description_Fig_9.jpg" style="width: 60%;"> | | <img src="https://static.igem.org/mediawiki/2018/6/64/T--SMMU-China--Description_Fig_9.jpg" style="width: 60%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
− | <strong>Figure 9</strong> | + | <strong>Figure 8</strong> |
| </p> | | </p> |
| </div> | | </div> |
| + | |
| + | <h3 class="inner-h">BNP Promoter:Heart Failure Inducible</h3> |
| + | <p class="inner-text"> |
| + | To ensure biosafety and avoid side effects in normal cells, a heart failure inducible BNP promoter was constructed to the upstream of our therapeutic gene. This promoter derives from the approximal region (-408/+100bp) of the human brain natriuretic peptide (hBNP) promoter, which is reported to have the response to AngⅡ, mechanical strain, and other heart-failure-related factors. According to previous reports, its activity remained low under basal conditions and elevated during heart failure. Based on these qualities, we chose to utilize this promoter as a switch to initiate and terminate gene expression. |
| + | </p> |
| | | |
| <h3 class="inner-h">Precise guidance to myocardium: recombined adeno-associated virus serotype 9 (rAAV9)</h3> | | <h3 class="inner-h">Precise guidance to myocardium: recombined adeno-associated virus serotype 9 (rAAV9)</h3> |
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| </p> | | </p> |
| <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
− | <img src="https://static.igem.org/mediawiki/2018/7/7a/T--SMMU-China--Description_Fig_10.jpg" style="width: 60%;"> | + | <img src="https://static.igem.org/mediawiki/2018/7/7a/T--SMMU-China--Description_Fig_10.jpg" style="width: 70%;"> |
| <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
− | <strong>Figure 10</strong> | + | <strong>Figure 9</strong> |
| </p> | | </p> |
| </div> | | </div> |
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| <li>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.</li> | | <li>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.</li> |
| </ol> | | </ol> |
− | </section>
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| </article> | | </article> |
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| <h2>Wanna contact us?</h2> | | <h2>Wanna contact us?</h2> |
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| <h3>E-Mail</h3> | | <h3>E-Mail</h3> |
− | <p><a href="mailto:hus@smmu.edu.cn">hus@smmu.edu.cn</a></p> | + | <p><a href="mailto:igem-smmu@smmu.edu.cn">igem-smmu@smmu.edu.cn</a></p> |
| </li> | | </li> |
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| <h3>Phone</h3> | | <h3>Phone</h3> |
− | <p>+86 13701692043</p> | + | <p>+86 021-81870925</p> |
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− | <li>© Untitled. All rights reserved.</li> | + | <li>© 2018. SMMU. All rights reserved. Thanks to: <a href="http://html5up.net">HTML5 UP</a></li> |
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