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ENGineering extracellular matrix-based de novo proteins with high Affinity to Growth factors for Enhancing bone regeneration

Periodic Reporting for period 2 - ENGAGE (ENGineering extracellular matrix-based de novo proteins with high Affinity to Growth factors for Enhancing bone regeneration)

Reporting period: 2020-10-01 to 2021-09-30

Bone is a tissue that has the capacity to self-repair. However, in some circumstances such as big or complex fractures, or teeth missing, bone is not able to regenerate by itself. In these situations, biomaterials are being used to regenerate or substitute bone, although most of them do not induce proper bone regeneration. One strategy is the use of molecules secreted by cells, such as growth factors, that are immobilized onto the biomaterials in order to mimic the natural bone healing process. In this regard, Bone Morphogenetic Protein-2 (BMP-2) is a growth factor secreted by cells with potent bone regeneration capacities. BMP-2 binds to two different cell surface receptors (type I and type II receptors) activating a downstream signaling cascade that ends with the activation of genes related to bone regeneration. Although its use in biomaterials has been shown promising results, the main problem is that BMP-2 is rapidly degraded upon implantation. Then, huge amounts of BMP-2 have to be implanted, which has been related to side effects such as inflammation or cancer.
Therefore, alternatives to BMP-2 are important for the society because they might be used to reduce or shorten the time recovery of patients that require a biomaterial implantation after bone fracture.
The overall objective of ENGAGE project is to computationally design a synthetic protein that will substitute BMP-2 and stimulate the same activity but with higher stability. Then, less amounts of the designed protein will be required for implantation, which is expected to reduce the undesired side effects associated to BMP-2. To this end, proteins with the ability to bind type I receptors and proteins with the ability to bind type II receptors were designed during ENGAGE and were fused to obtain a single protein with the capacity to induce the BMP-2 downstream signaling cascade.
The execution of ENGAGE project has demonstrated that it is possible to computationally design proteins that bind to the BMP-2 receptors with higher affinity than the natural BMP-2. These designs will have the potential to substitute the use of BMP-2 potentially producing less side effects. In addition, the individual designs for each single receptor will have the potential to be used as antagonists in those pathological situations where these receptors are overexpressed.
Hundreds of thousands of candidates expected to bind to the type I (Alk-1 and BMPRIA) and the type II (BMPRII, ActRIIA and ActRIIB) receptors of the BMP family have been computationally designed.
The candidates have been computationally filtered to select the best among them, which have been tested in the laboratory to elucidate their affinity for their natural ligands. Those designs for each of the targets having the highest affinity were selected for cellular assays. The antagonistic effect of each of the designs was evaluated on mesenchymal stem cells, obtaining a decrease in the BMP-2 signaling in all of them. The best designs were fused in different conformations to reproduce the distance and orientation of the native BMP-2 protein, and the resulting proteins were tested on endothelial cells and mesenchymal stem cells obtaining promising activities.
Although the activity of the final designs has to be improved, the current designs have more stability than the native BMP-2 and can be produced at less costs, thereby being promising candidates for bone regeneration. For this reason, the sequences of the de novo proteins designed during ENGAGE project are susceptible for patent applications and future exploitation. There is a huge potential for exploitation of the sequences, not only for the use of the fused designs for bone regeneration, but also for the single designs as antagonistic drugs for blocking the BMP receptors in those pathological situations where they are overexpressed. Some of the sequences have been documented under a record of innovation and will be applied to a patent for potential commercialization once publications are drafted.
Some of the results obtained during ENGAGE project have been disseminated in conferences, including the Virtual RosettaCon and Virtual COVID-19 Rosetta Conference (2020 and 2021 editions), the 13th BMP Conference and the 67th Biophysical Society Annual Meeting.
Up to date, mutations of BMP-2 or short fragments derived from BMP-2 have been used to stimulate bone regeneration. However, mutations of BMP-2 are not sufficient to increase its stability while short fragments of BMP-2 do not stimulate bone regeneration at the same levels than BMP-2. To overcome these limitations, the computationally designed proteins in ENGAGE project are expected to strongly stimulate bone regeneration without eliciting side effects, as natural BMP-2 does.
The execution of ENGAGE provides an innovative alternative to the use of BMP-2 for bone regeneration with high socio-economic impact. The development of a BMP-2 substitute with high stability, less side effects and low production costs will have a strong economic impact in the field of biomaterials, where BMP-2 is being used although readmission to hospital for patients rises at 30% due to undesired side effects. In addition, the novel technology will bring to the market a new repertoire of new proteins with great technological impact, not only because of their potential to induce bone regeneration but also for other biomedical applications. If proven successful, the application of ENGAGE results could considerably increase the long-term survival rate of implants hence having a high societal impact.
A computationally designed protein (green) interacting with a natural ligand of BMP-2 (blue)