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Improvement of the clinical applicability of tissue-engineered vascular grafts, as new regenerative therapy for children with congenital cardiovascular malformations

Final Report Summary - LIVAGRAFT (Improvement of the clinical applicability of tissue-engineered vascular grafts, as new regenerative therapy for children with congenital cardiovascular malformations)

1.1 Project objectives
Currently available artificial graft prostheses for congenital disorders lack the capacity of growth and often require complex and reconstructive surgical interventions. As a consequence, repetitive and high-risk surgical interventions during childhood are inescapable, associated with increased morbidity and mortality. This not only raises enormous costs for the society, but compromises the quality of life of the young patients. Therefore, manufacturing Living Vascular Grafts is the ultimate goal for both cardiovascular researchers and clinicians. Tissue engineering is an opportunity to create prostheses that are vital, growing, adaptive, and autologous and show optimal functionality. Such in-vitro tissue-engineered vascular grafts (TEVGs) have demonstrated functionality and growth as pulmonary replacement in lambs. However, the classical tissue engineering approach, using autologous cells, necessitates invasive cell harvesting from the patient, time consuming cell expansion, and production of a patient-specific graft. As such, the living cellular component of the grafts inherently limits clinical applicability and an accelular equivalent would provide an attractive alternative for the living tissue-engineered vascular grafts.
Therefore, the main goal of this project was to improve the clinical applicability of the tissue-engineered pulmonary grafts before the start of the envisioned first clinical study in Europe. By guardedly removal of the cells from the TEVGs, after the cells have completed their in-vitro duty of tissue- /matrix production, a new product is created with large, off-the-shelf availability as new regenerative therapy for children with congenital cardiovascular malformations. Moreover, in order to receive permission to enter clinical trials, the in-vivo recellularization capacity of these accelular tissue-engineered pulmonary grafts was evaluated in a pre-clinical animal model to demonstrate their potential to grow with the recipient.

1.2 Work performed and main results
1.2.1 The in-vitro establishment and evaluation of the off-the-shelf TEVGs
The first aim was the in-vitro establishment, evaluation, and production of the off-the-shelf TEVGs. In order to define the fixed protocol for culture and decellularization of TEVGs, firstly TEVGs were cultured in the laboratory according to standard protocols of living TEVGs. To find the best decellularization protocol, the time of incubation and further washing steps were optimized. The proof of cell removal and significant reduction of DNA remnants was demonstrated by histological analyses and quantitative DNA measurements. Additionally, a lyophilization step of the decellularized TEVGs was introduced, because it significantly simplifies the storage and (gas) sterilization of the final product. Moreover, it stops the hydraulic degradation of the remaining polymers in the product, therewith stabilizing the mechanical properties of the graft after production. Additionally, these protocols for culturing, decellularization, and lyopholization of TEVGs were transferred to Good Manufacturing Practice (GMP) protocols in close collaboration with the Swiss TransMed LifeMatrix project. The latter in order to directly apply for the first European clinical trial with the improved TEVGs when they proof their expected excellence compared to current available prostheses.
To enable the large amount of grafts needed for all analyses and animal trials, the production of the TEVGs was performed by the team of the LifeMatrix project and is currently constantly running in the GMP clean room facilities of the Center for Therapy Development of the Institute for Regenerative Medicine. Moreover, in close collaboration with the Swiss TransMed LifeMatrix project, analyses are performed to demonstrate that the final product is compliant to regulations for use in humans in both EU-countries (EMA) and Switzerland (SwissMed).
1.2.1 Pre-clinical evaluation of the off-the-shelf TEVGs
In order to receive permission to enter clinical trials also with the newly developed off-the-shelf TEVGs, pre-clinical evaluation had to demonstrate their expected in-vivo recellularization capacity, thereby proofing their potential to grow and adapt with the recipient similar to the cellular TEVGs. Therefore, GMP compliant ``off-the-shelf`` human cell-derived TEVGs were implanted as pulmonary-artery replacement in a translational GLP-compliant animal model to demonstrate the essential characteristics for the ideal vascular graft such as durability, absence of thrombogenicity, resistance to infections, lack of immunogenicity, and the potential for growth (financed by the Swiss Transmed LifeMatrix project). During 20 weeks follow-up, the flow-pattern in the TEVGs remained smooth without turbulences or any signs of thrombus-formation, kinking, dilation or graft-dehiscence. Post-mortem analysis displayed appropriate integration into the pulmonary-artery, significant endogenous cell infiltration, substantial remodelling, and partially to completely endothelialization over time. Moreover, the potential for growth was indicated by regular diameter enlargement of the implants.

1.3 Impact and implications
This study demonstrates for the first time the preclinical efficacy of non-xenogenic "off-the-shelf" vascular prostheses with regenerative capacity that have large potential for clinical translation. Moreover, we proved the in-vivo recellularization and remodelling capacity of the novel off-the-shelf TEVGs with similar outcome compared to their cellular precursor TEVGs. Importantly, indications for their potential to grow and adapt with the recipient were observed already during mid-term follow-up. The close collaboration with the Swiss TransMed LifeMatrix project that has facilitated the costly preclinical studies also ensures that the final product will be compliant to regulations for use in humans in both EU-countries (EMA) and Switzerland (SwissMed). Moreover, as the Swiss TransMed LifeMatrix project continues, the long-term (50 weeks) follow-up of the preclinical study will be completed in the coming months. Importantly, this collaborative project has led to the successful development and preclinical evaluation of a new product platform, named LifeMatrix, which provides off-the-shelf non-xenogenic matrices with regenerative capacity for several applications in cardiovascular repair. Interestingly, the clinical translation of this novel concept has been selected for funding by the recently founded Wyss Translational Center Zurich. Therefore, upon completion of the proposed additional short-term safety study, the host institute can apply for the first-in-man clinical trial with this new medical device.

The further preclinical and eventually clinical research is of great relevance to European society, as it comprises new therapies for one of the most life-threatening diseases, cardiovascular disease, and in this case more particular those of a very vulnerable group, the youngest European citizens. The realisation of a new production processes enabling off-the-shelf and unlimited availability of the non-xenogenic product in a large variety of sizes will speed up the clinical translation of the invention. At the same time product innovation, with a safer and more reliable consumer product, aims to meet people's needs and improve their quality of life, by lowering risks and bettering health and welfare.

Changing disease patterns and thereby decreasing the pressure on the sustainability of EU health systems by aiming to prevent health problems and disabilities from an early age and support healthy ageing, links to the Commission's overall strategic objective of Prosperity, ensuring a competitive and sustainable future for Europe (White paper Together for Health: A Strategic Approach for the EU 2008-2013). The transfer of the obtained research results into new products and their potential raise of intellectual property might help to boost the competitiveness of European biotechnology and medical technology industry. As research-based SMEs are the main economic drivers of healthcare, biotechnology and medical technologies, strong EU-based biomedical research will enhance competitiveness of the European pharmaceutical and healthcare industries. In turn, increased industrial competitiveness and high quality products would protect European jobs and therefore promote social and economic cohesion. This collaborative research has led to innovative ideas and excellence research in Europe and will extensively contribute to the knowledge in the field of tissue engineering of vascular grafts worldwide.

To conclude, in the near future, the clinical translation of the novel off-the-shelf TEVGs could eliminate the need for multiple reoperations as the vascular grafts with proven regenerative capacity may potentially adapt to the somatic growth of the patient. Consequently, the improved clinical applicability of the grafts developed in this project could lower health costs and changes the lives and wellbeing of many young patients.