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’.

1 Heart failure

What is heart failure?

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.

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.

Figure 1

Clinical status and influence of heart failure

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.

Figure 2

Current treatment methods and limitations

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.

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.

Figure 3

What is gene therapy?

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.

Figure 4

Diastole and contraction of the normal heart

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 Ca²⁺ concentration gradient enters the cell and binds to the Ca²⁺ release channel on the sarcoplasmic retina, further activating the stored Ca²⁺ release in the sarcoplasmic reticulum, causing the intracellular Ca²⁺ concentration to increase rapidly. Ca²⁺ 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 Ca²⁺ concentration can activate the Ca²⁺-Mg²⁺-ATP enzyme in the head of myosin, hydrolyze ATP to release energy, and induce myocardial contraction.

When the heart is diastolic, most of it is ingested and stored by sarcoplasmic reticulum Ca²⁺-ATP, and a small fraction is transported by cell membrane sodium-calcium exchange protein and cell membrane Ca²⁺-ATP outside the cells, resulting in a rapid decrease of cytoplasmic Ca²⁺ concentration. The flow process of Ca²⁺ 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.

Figure 5

Diastole and contraction of the heart in heart failure

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.

2 CaRTIN

What is RyR2? What is it made up of?

As we have introduced before, Calcium (Ca²⁺) is the important physiological ligand that activates the channels in cardiac muscle during excitation-contraction (EC) coupling. The heart dysfunction will happen when Ca²⁺ cycle is in a mess, which in the end leads to heart failure. That’s why we have to mention the Ca²⁺ release channels (a kind of ryanodine receptor) on the sarcoplasmic reticulum (SR) of striated muscles. They adjust and control Ca²⁺ between cytoplasm and SR as a biphasic channel such that low cytosolic [Ca²⁺] (mM) activates the channels and high cystolic [Ca²⁺] (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).

Figure 6

How does it work?

FKBP12s are regulatory subunits that stabilize RyR channel function and facilitate coupled gating between neighboring RyR channels which are packed into dense arrays in specialized regions of the SR that release intracellular stores of Ca²⁺ that trigger muscle contraction. RyRs are ligand-activated channels and One FKBP12 molecule is bound to each RyR subunit, and dissociation of FKBP12 significantly alters the biophysical properties of the channels, resulting in the appearance of subconductance states and increased P0 (resting potential of myocardium) due to an increased sensitivity to Ca²⁺-dependent activation. In addition, dissociation of FKBP12 from RyR channels inhibits coupled gating, resulting in channels that gate stochastically rather than as an ensemble. Coupled gating of arrays of RyR channels is thought to be important for efficient EC coupling that regulates muscle contraction.

The function of S2808

According to Marx’s study, PKA phosphorylation regulates the binding of FKBP12.6 to the channel both in vitro and in vivo. PKA phosphorylation of the cardiac RyR2 dissociates the regulatory subunit FKBP12.6 from the channel. Such situation will occur when catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and “leaky”, leading to arrhythmias and other pathology. Since that we aim to protect the S2808 from phosphorylation which can rectify calcium current in order to cure heart failure. However, it is difficult to find a way to lower the phosphorylation level of S2808 using traditional gene editing methods, which frustrates us more concerning the production’s long-term and repeatable effect. That’s why our instructor suggested us considering nanobody.

Figure 7

Nanobody

As introduced in Wikipedia, nanobody, which is also named as single-domain antibody (sdAb), is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, nanobodies are much smaller than common antibodies. The first nanobodies were engineered from heavy-chain antibodies found in camelids. Up till now, they have been shown to be just as specific as a regular antibody and in some cases, they are more robust. As well, they are easily isolated using the same phage panning procedure used for traditional antibodies, allowing them to be cultured in vitro in large concentrations. The smaller size and single domain make these antibodies easier to transform into bacterial cells for bulk production, making them ideal for research purposes. With sufficient documents related to support, we assured that it could be used to block biochemistry course in living cells.

Figure 8

Phage display antibody library

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.

Figure 9

Precise guidance to myocardium: recombined adeno-associated virus serotype 9 (rAAV9)

Then we came to the terminal part: transduce the “shield” into the myocardium precisely. According to a paper published in 2006, we picked recombined adeno-associated virus serotype 9 (rAAV9), an ideal virus born with specific affinity to cardiac tissue, which was proved by cell and tissue experiments later in our laboratory. Meanwhile rAAV9 has low biotoxicity, since its stable expression lasts 4 weeks according to the Western blot consequence.

Figure 10

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.