Written by:
Erwin De Genst,
Senior Scientist, Biologics Engineering, R&D, AstraZeneca
Kylie Foo,
Assistant Professor, Karolinska Institutet
Heart failure (HF) is a life-threatening disease that occurs when the heart cannot pump enough blood around the body. It affects 64 million people worldwide,1 and is a leading cause of death and hospitalisation. The need for a therapy capable of addressing the root cause of cardiac dysfunction in HF remains high.
One of the characteristic signs of heart failure is defective calcium signalling. Calcium plays an integral role in the contraction and relaxation of the heart muscle.
During each heartbeat, intracellular calcium rises, leading to contraction of the muscle cell. This is followed by a rapid decrease in calcium, which leads to relaxation, preparing the cells to produce the next heartbeat.
The movement of calcium is facilitated by calcium-handling proteins on the surface of and inside cardiac cells which undergo complex dynamic transitions when they interact with other proteins.
Faulty interactions between calcium-handling proteins disrupt normal calcium signalling, impairing contraction and relaxation of the heart muscle. This can potentially lead to dangerous heart arrythmias and structural changes, which may ultimately result in heart failure and death.
Enhancing calcium cycling – and heart function - by blocking protein inhibitors
Sarco/endoplasmic reticulum calcium ATPase, known as ‘SERCA’ is a key protein involved in calcium cycling. It transports calcium from the cytosol to the sarcoplasmic reticulum where calcium is stored in cardiac muscles. The activity of SERCA is regulated by its interaction with phospholamban or ‘PLN’, a protein that inhibits calcium cycling in the heart. By binding to SERCA, PLN controls the speed of calcium removal from the cytoplasm and the amount of calcium stored within the cell.
Patients with heart failure tend to have reduced levels of SERCA, and comparatively high levels of PLN, leading to too few active SERCA molecules. This results in slow heart muscle relaxation and weak contractions. Enhancing calcium cycling by blocking or disrupting PLN could, therefore, improve the ability of the heart to pump blood.
VHH intrabodies shown to disrupt the action of PLN
Previously, attempts were made to disrupt the activity of PLN using small molecule drugs. However, this approach proved unsuccessful because PLN is flexible and exists in a dynamic equilibrium between different structural forms and lacks clearly defined, accessible ‘pockets’ in its structure, which drug molecules usually need in order to be effective.
However, in our latest research, published in Nature Communications, we have found a way to disrupt PLN ‒ using VHH intrabodies ‒ which are derived from the antigen recognition domain of naturally occurring camelid heavy chain antibodies.2
We identified potent VHHs from a synthetic library that bind to PLN and prevent PLN monomers from binding to SERCA, enhancing its activity. By linking two identical copies of the VHHs together, we showed that these dimeric VHHs increase the activity of SERCA even further by increasing the levels of inactive PLN pentamers.
We then used chemically modified messenger RNA – synthetic messenger molecules with enhanced stability inside cells – to validate the effectiveness of the VHHs against PLN in vitro, using isolated live heart cells.
Findings showed that the VHH intrabody helped normal calcium cycling to resume, restoring proper pumping functionality to the heart in a mouse with heart failure.
AAV-delivered VHH intrabodies could lead to novel heart failure treatment
Delivering drugs using adeno-associated viruses (AAVs) was previously shown to be effective for the in vivo delivery of extracellular biotherapeutics, and so these platforms presented an opportunity to evaluate the delivery of intrabodies.
We used heart-targeted AAV vectors engineered to express VHHs under the control of a cardiomyocyte-specific promoter. AAV vectors for heart-targeted intrabody delivery enable tissue selective expression using systemic administration, minimising the risk of damage to non-relevant tissues. Injection into preclinical models effectively expressed the intrabodies specifically in the heart, with no detection in the liver or any other muscle tissue.
This research shows that VHH intrabodies could open the door to potential new treatments for heart failure, particularly when combined with emerging virus-based or nucleic acid ‒ based drug delivery technologies ‒ and may ultimately bring us a step closer to stopping hearts from failing.
“This work shows the utility of mRNA approaches, normally viewed as a 'gain of function platform', to quickly screen and identify nanobody and other 'loss of function' therapeutic candidates” said Kenneth R. Chien, Professor of Cardiovascular Research at Karolinska Institutet.
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