Development of a Gal-free Calcification Resistant Porcine Pericardial Heart Valve: Establishment of Biological Tissue Equivalence.

Heart valves control the normal flow of blood through the lungs and body. These valves may be damaged because of birth defects, old age, or infection. This damage may require heart valves to be replaced with artificial valves to improve the quality of life of heart valve disease patients or to save their lives. There are about 300,000 heart valve replacements worldwide each year. Replacement valves are either mechanical, made of carbon and metal, or biological, made of non-living tissue generally obtained from pigs or cows. Patients and doctors tend to prefer biological heart valves (BHVs) because they generally do not require blood thinners, which are needed with mechanical valves. In younger patients (<60 years) and in children, BHVs wear out more rapidly, sometimes within 5 years. BHVs fail because they build up bone-like deposits of calcium, which weaken the valve, leading to tears, or obstructed blood flow as the calcium deposits block the opening of the valve. Scientists and commercial valve companies have long sought to produce BHVs, which do not calcify, because these could be used in younger patients without the need for blood thinners. So far, calcification-blocking treatments have been able to reduce valve calcification when tested in animals, but have not been able stop calcification in patients or reliably allow the use of BHVs in younger adults. We identified a type of rejection that makes calcification worse in BHV material. This rejection is unique to humans as the human immune system reacts with a substance, called Gal present on BHVs. To block this rejection reaction we have genetically altered pigs that can be used to make BHVs called Gal knockout pigs. The Gal knockout pigs are healthy and normal and their Gal-free tissue has reduced calcification. Before using Gal knockout tissue in patients, we need to be certain the genetic change in Gal knockout pigs has not had a damaging effect on the tissues used to make these BHVs. We have compared the tissues in current standard and Gal knockout BHVs and used simple tests, such as measuring how hard it is to stretch and tear the tissue, to see if the mechanical properties of Gal knockout tissue remain strong and unchanged. We have also made BHVs using both current standard and Gal knockout tissue, and tested them in a laboratory machine that mimics their function in the heart. Both types of BHVs performed similarly in these tests. This current project is to compare how well the Gal knockout tissue works as a BHV in the standard industry animal model. This test is the only way of determining if the valve works well inside of the body and can function to control the normal flow of blood in the heart. Successfully performing this test, which is required by International standards, is a major step forward to making a new BHV, which reduces calcification and be usable in younger patients. Such a new BHV would greatly increase the quality of life for patients. If successful, we hope to advance a new Gal knockout heart valve for a clinical test in man.

Of the 300,000/yr replacement heart valves (RHVs) implanted, there are two types, mechanical (MHVs) requiring lifetime anticoagulation, and bioprosthetic (BHVs), made from bovine or porcine valves or pericardium, which generally do not. Patients under 60 are chiefly treated with MHVs to avoid accelerated age-dependent BHV calcification/degeneration seen in younger patients. Trends in RHV surgery include increasing use of BHV's (from 40 to 60% of total) and the use of transcatheter valves (>200,000 to date) whose leaflets are made of bovine or porcine pericardium.
Universal human anti-Gal antibody, binding to the Gal antigen on commercial BHVs, promotes calcification. GTKO pig tissue does not contain Gal and is not subject to immune mediated calcification. A GTKO BHV may potentially reduce calcification, improve durability, and make BHV's available to younger patients.
To progress development of such a BHV we compared the physical, hydrodynamic and durability performance of pericardium from GTKO and wild type (WT) pigs in an in-house constructed BHV, detecting no significant differences between the two.
This application now compares the biological properties of valves made from GTKO and WT pericardium. Objective 1 is to improve the durability of our in-house BHV to have predictable performance for in vivo testing. Manufacturing improvements in our in-house valves have already improved durability. Objective 2 is to compare the biological properties of GTKO and WT pericardial BHVs using the industry standard mitral valve replacement in juvenile sheep with 90-day survival.
Primary endpoints are gross valve pathology, histological analysis of valves for evidence of cardiac thrombus deposition, pannus formation, inflammation, calcification or structural valve deterioration and quantitative calcium determination using atomic spectroscopy on valves at explant.
This testing is the last step to de-risk commercial adoption of GTKO materials for future BHV development.

The primary beneficiaries of this research are academic researchers, heart valve industry leaders and policy regulators involved in regulating genetically modified (GM) animals and animal products. Approximately 300,000 heart valve replacements are performed annually worldwide. Patients and physicians prefer bioprosthetic heart valves (BHVs) as they generally do not require lifetime anticoagulation, which is required for mechanical heart valves. BHVs, however, show premature age-dependent tissue calcification in younger patients, which is the principle pathology leading to structural valve deterioration (SVD). Our research indicates that universally present human antibody to the dominant xenogeneic antigen, galactose alpha 1,3 galactose (Gal), enhances calcification of fixed Gal-positive pericardium from standard pigs, currently used in commercial BHVs, but does not effect pericardium from pigs with an engineered mutation in the GGTA-1 galactosyltransferase gene (GTKO) which eliminates Gal expression. Growing evidence suggests that this immune mediated tissue calcification may disproportionately contribute to BHV calcification and age-dependent SVD in children and younger patients, who have more active immune systems.
Guided by regulators and ISO testing standards, we developed BHV using Gal-free (GTKO) and standard pig pericardium to compare the physical and biological equivalence of these tissues to determine if the GGTA-1 mutation has compromised the suitability of GTKO tissue for use in BHVs. Physical and mechanical testing between these tissues showed no evidence of compromised GTKO tissue integrity. In this current project, we use the industry standard in vivo model to compare the biological compatibility and physiological performance of these tissues.
Showing physical and biological equivalence of GTKO tissue would impact academic researchers as GTKO tissue is a platform technology with the potential to provide unlimited amounts of extracellular matrix and organ scaffolds which, being Gal free, may substitute for the use of cadaveric materials currently in use. Industry leaders and researchers will have a clear benefit from this research as it will allay general concerns about using GM tissues in heart valves and therefore facilitate use of this technology. Policy regulators may also benefit, as our work towards the first clinical use of GM tissue will stimulate policy makers to refine the oversight regulatory environment governing GM animals and potentially lead to broader acceptance of this strategy. We believe advancing the clinical development of fixed bioprosthetic GTKO heart valve will demonstrate the clinical utility of this technology and help to ease the regulatory hurdles for other applications.
A durable BHV, resistant to premature calcification, would greatly improve therapeutic options for younger patients, by avoiding the haemorrhagic/thromboembolic complications of anticoagulation (approximately 0.6-0.8% per patient per year), needed with mechanical valves, as well as providing more durability in both older and younger patients. A greatly enhanced quality and quantity of life would be achieved for patients.