Periodic Reporting for period 4 - BIORECAR (Direct cell reprogramming therapy in myocardial regeneration through an engineered multifunctional platform integrating biochemical instructive cues) Reporting period: 2023-01-01 to 2023-12-31 Summary of the context and overall objectives of the project Myocardial infarction causes the irreversible loss of functional and contractile cardiac tissue (mainly populated by beating cells, called cardiomyocytes), and its replacement with dysfunctional fibrotic tissue (mainly populated by non-beating cells, called cardiac fibroblasts), resulting in heart failure. The only standard therapy addressing the irreversible loss of functional cardiomyocytes is heart transplantation, which however is limited by donor loss and the need for life-long immunosuppression. An ideal cardiac regenerative medicine strategy should replace lost cardiomyocytes and recover myocardial functional contractility. Many advanced therapies under investigation, such as cell therapies and tissue engineering approaches, have achieved limited success [1]. Therefore, the regeneration of a damaged heart still remains a major clinical challenge with a deep social and economic impact."Direct reprogramming” strategy, aimed at the direct conversion of cells populating the cardiac scar (i.e. cardiac fibroblasts) into beating cells (i.e. cardiomyocytes) has recently emerged as a new intriguing possibility for myocardial regeneration. Despite the excitement on the potentiality of direct cell reprogramming on cardiac regeneration, the approach is still rather immature and inefficient to prospect a short-term clinical translation [1].BIORECAR project addresses the current limitations of direct cell reprogramming by a novel combined multidisciplinary and multifunctional approach, involving a multidisciplinary team (Figure 1), integrating tools from nanomedicine, biomaterials science and tissue engineering, with the aim to achieve a significant advancement in the knowledge of direct cell reprogramming in the perspective of a future clinical application. In BIORECAR, direct reprogramming is achieved by the release of small RNA molecules, called “microRNAs” (miRNAs) to the cells populating cardiac scar. A combination of four miRNAs, called “miRcombo” is used as able to modify the behaviour of cardiac fibroblasts inducing their reprogramming into cardiomyocytes. The overall project aim (Figure 2) is achieved through:Objective 1: The design of polymer nanoparticles, loaded with miRcombo, for a targeted and efficient direct reprogramming of human cardiac fibroblasts into cardiomyocytes;Objective 2: The design of a multifunctional injectable hydrogel releasing nanoparticles and increasing direct reprogramming efficiency, for a minimally invasive approach in cardiac regeneration;Objective 3: The development of biomimetic in vitro models of human cardiac fibrotic tissue for the testing and validation of BIORECAR approach, before the final in vivo trials in mouse model (Figure 3).Objective 4: Preclinical validation of BIORECAR approach in mouse modelBeing heart failure the leading cause of mortality and morbidity in the industrialized world, any new knowledge on its effective treatment achieved in BIORECAR will have a deep social and economic impact. Additionally, BIORECAR approach may extend beyond post-myocardial infarction therapy to treat other forms of aquired or inherited heart disease, typically associated with fibrosis, further increasing the project impact. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Progresses have been made concerning the main project objectives, as detailed below:Objective 1: Design of polymeric nanoparticle for direct reprogrammingThe possibility for miRcombo-mediated direct cardiac reprogramming of human adult cardiac fibroblasts into cardiomyocytes was initially demonstrated [2]. Novel lipid-based nanoparticles were developed, able to efficiently encapsulate and release miRNAs to human cardiac fibroblasts for direct reprogramming [3]. "Bioartificial" polymer-lipid nanoparticles were formulated and patented, showing a high encapsulation ability, biocompatibility and efficient transfection of human adult cardiac fibroblasts. They represent promising candidates for in situ treatment [4], as demonstrated by in vitro tests using human cardiac fibrotic tissue models and by in vivo trials in mouse models. Objective 2: Injectable hydrogel maximising direct reprogramming efficiency.MiRcombo-mediated direct reprogramming of human cardiac fibroblasts was enhanced when cells were cultured in biomimetic 3D hydrogels compared to 2D cultures [5].Injectable hydrogels were prepared by blending a biocompatible biodegradable polymer with tailored stability and a biomimetic polymer favouring cell attachment and transdifferentiaion into cardiomyocyte-like cells.In detail self-healing injectable hydrogels were designed and widely characterized.Objective 3: In vitro models of human cardiac fibrotic tissue.In vitro models of human infarcted tissue were prepared by tissue engineering, exploiting "bioartificial" scaffolds, i.e. scaffolds combining synthetic and natural polymers [6, 7].We also collaborated to the design of a beating human cardiac scar on a chip model for the validation of BIORECAR approach [8].Objective 4: Preliminary validation in mouse model.In vivo tests at BIOEMTECH using Wild Type mouse model showed promising biodistribution and efficacy of BIORECAR nanoparticles.Tests are in progress at Humanitas, using a transgenic mouse model. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) For each project objective, progresses beyond the state of the art and expected results are the following:Objective 1-Demonstration of miRcombo ability to trigger human cardiac fibroblasts reprogramming into cardiomyocytes [2,3,5].-Design of lipoplexes for efficient miRcombo encapsulation and release to human cardiac fibroblasts, with enhanced efficacy respect to commercial agents [3,4]. -Design of new “bioartificial” polymer-lipid nanoparticles with: high miRNA loading, high cell transfection efficiency, adequate stability for in vivo treatment, surface functionalized with ligands for miRNA release to target cardiac fibroblasts [4; patented]. Objective 2- Evidence of higher direct reprogramming efficiency for cells cultured in a biomimetic 3D hydrogel [5].- Innovative design of injectable multi-functional hydrogels for miRNA-loaded nanoparticle release.Objective 3- Novel in vitro models mimicking human cardiac fibrotic tissue for preclinical testing of BIORECAR approach [6]. Objective 4- Promising in vivo biodistribution and efficacy for functionalized nanoparticles.In the future, in vivo safety and dose-dependent efficiency will be analysed.[1] Paoletti et al. Cells 2018; https://doi.org/10.3390/cells7090114[2] Paoletti et al. Front. Bioeng. Biotechnol. 2020; https://doi.org/10.3389/fbioe.2020.00529[3] Nicoletti and Paoletti et al. Nanomedicine. 2022; https://doi.org/10.1016/j.nano.2022.102589[4] Lee et al. J. Control Release 2019; https://doi.org/10.1016/j.jconrel.2019.10.007[5] Paoletti et al. Cells 2022; https://doi.org/10.3390/cells11050800[6] Spedicati et al. Front. Bioeng. Biotechnol. 2022; https://doi.org/10.3389/fbioe.2022.983872[7] Rocco et al. ACS Biomater Sci Eng 2023; https://doi.org/10.1021/acsbiomaterials.3c00483[8] Paoletti and Visone et al. Adv Health. Mat. 2023; https://doi.org/10.1002/adhm.202301481https://www.facebook.com/BiorecarProjecthttps://twitter.com/biorecar Figure 1 - BIORECAR group Figure 2 - BIORECAR objectives Figure 4 - BIORECAR logo Figure 5 - BIORECAR team at TERMIS-EU Conference 2022 Figure 6 - BIORECAR team at Biofabrication 2022 Conference Figure 3 - In vitro model of pathological cardiac tissue for testing BIORECAR approach
