Wednesday, February 5, 2025
2/5/2025
Project Summary Recent innovations by project investigators have established an important new framework for sustaining the anti-thrombotic properties of blood contacting films in order to improve the clinical performance of a variety of implanted cardiovascular devices. Notably, we have discovered highly active, orthogonal sortase mutants that recognize unique peptide sorting motifs, enabling repeated covalent assembly and disassembly of surface bound biomolecular constituents, thereby providing a means to regenerate anti-thrombotic constituents on blood contacting films after device implantation. While precedent exists for the therapeutic use of foreign proteins, recent innovations by project collaborators have also led to the development of novel computational and experimental tools for protein deimmunization to mitigate any immunogenic risk that may be associated with sortase-derived enzymes. Importantly, we have also developed new chemoenzymatic schemes leading to the discovery of a synthetic heparin that is resistant to heparanase mediated biodegradation, forming the basis of a novel family of highly durable heparin thin films. These breakthroughs offer an opportunity to extend the lifetime of a bioactive film with commensurate improvements in clinical outcomes. In this proposal we intend to: (1) Design synthetic heparins that are resistant to heparanase and characterize their capacity to limit thrombus formation as a component of a ‘rechargeable’ thin film. Chemoenzymatic and orthogonal ligation schemes will be used to generate synthetic heparins that are resistant to heparanase mediated biodegradation. In addition to characterizing the biostability of these new heparin analogues, anticoagulant activity will be determined, and pharmacokinetic properties defined. The rechargeability of thin films comprised of synthetic heparin analogues will be evaluated in vitro and after extended blood-contacting periods in vivo. (2) Design functionally deimmunized sortase variants to facilitate in situ recharging of bioactive thin films through deletion of immunogenic T cell epitopes. The cellular immunogenicity of a pentamutant sortase variant, eSrtALPETG, will be assessed and T cell epitopes driving the response mapped in naïve human peripheral blood mononuclear cells. Computational tools will be used to design functionally deimmunized candidate eSrtALPETG variants, which will be evaluated for their maintenance of transpeptidation activity and their reduction of cellular immunogenicity. Lead candidates will be assessed in humanized HLA transgenic mice. (3) Synthesize and characterize blood contacting thin films that enable selective and repetitive regeneration of multiple bioactive constituents that inhibit thrombin generation. In the first phase of these investigations, we plan to define the capacity of deimmunized sortase variants to facilitate the fabrication of rechargeable, multicomponent bioactive thin films that contain both synthetic heparin and recombinant thrombomodulin. During the second phase, we will define the ability of an anti-thrombogenic thin film containing multiple rechargeable bioactive constituents to extend the patency of a small diameter vascular graft.
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