Supercharged protein-surfactant bioconjugates for next-generation cell therapies

细胞膜工程是一个快速发展的领域，对细胞治疗具有重要的影响，因为将外源蛋白引入细胞膜对体内定向组织修复非常有利。这是因为细胞疗法的广泛应用存在许多障碍，包括移植细胞缺乏植入，这导致有限的功能整合。几项细胞治疗研究表明，静脉或动脉内输注干细胞会导致肺部的不良积聚，从而降低全身输送的效率，增加产生致命微栓塞的可能性。即使直接植入目标器官，治疗效果所需的细胞数量也可能高得令人望而却步。因此，本研究计划描述了一类具有化学营养归巢特性的新型人工膜结合蛋白的合理设计，这将有助于克服这些限制，并在疾病和再生医学的细胞治疗中具有深远的意义。新方法涉及蛋白质-聚合物表面活性剂生物偶联物的细胞功能化，它绕过了直接细胞表面化学的需要，并提供了高度的灵活性，因为该方法可以很容易地应用于多种蛋白质，用于潜在的任何细胞类型。PI最近首创的合成方法涉及蛋白质表面的两个关键步骤的重新设计：(i)蛋白质表面正电荷密度的放大（增压）；（ii）阴离子聚合物表面活性剂链与蛋白质表面显示的阳离子位点的静电偶联。值得注意的是，通过构建这种聚合物表面活性剂电晕，可以系统地调节细胞膜亲和力，以促进生物偶联物自发插入干细胞膜，同时保留与细胞表面连接的蛋白质的天然功能。PI最近将这种方法应用于氧结合蛋白肌红蛋白，并证明了生物偶联物可以快速插入成人骨髓来源的人间充质干细胞（hMSCs）的细胞质膜。由于心血管疾病（CVD）是全球死亡的主要原因（2015年世卫组织估计每年有1800万人死亡），该研究规划的重点是（但不是唯一的）开发用于心脏干细胞治疗的细胞归巢的生物偶联物。在这里，概念上的进展集中在通过固定干细胞细胞质膜上细菌粘附蛋白CshA的纤维连接蛋白（Fn）结合域来劫持感染性细菌的归巢特性。CshA-Fn相互作用被认为是指导和固定哥氏弧菌在心脏内皮的定植，促进感染性心内膜炎发病的原因。因此，在干细胞膜上显示这种结合基序的多个拷贝的能力，已经经过进化优化，可以识别和结合心脏内皮中的特定分子靶标，具有推进心脏细胞治疗的潜力。值得注意的是，细胞膜修饰平台具有潜在的临床影响，除了CVD的细胞治疗，因为它可以很容易地应用于其他细胞类型和囊泡（例如，单核细胞，血液内皮细胞，自然杀伤细胞或外泌体），并涉及其他归巢蛋白或肽基分子（例如，整合素，抗体或其他细菌粘附素）。该研究计划描述了一种科学方法，它结合了生物物理学和再生医学的内部技术，以及大型设施中可用的尖端技术。由于该计划有很强的医学重点，申请人已聘请临床科学家合作伙伴协助医学翻译。

Cell membrane engineering is a rapidly emerging field with significant potential to impact on cell therapies, as the introduction of exogenous proteins into the cell membrane is highly advantageous for in vivo site-directed tissue repair. This is because there exist a number of barriers to the widespread uptake of cell therapies, including the lack of engraftment of transplanted cells, which results in limited functional integration. Several cell therapy studies have shown that intravenous or intra-arterial infusion of stem cells leads to undesirable accumulation in the lungs, which reduces the efficiency of systemic delivery and increases the likelihood of producing lethal microemboli. Even when implanted directly into the organ of interest, the number of cells required for therapeutic benefit can be prohibitively high. Accordingly, this research programme describes the rational design of a new class of artificial membrane binding proteins with chemotrophic homing properties that will help overcome these limitations and have far-reaching implications in cell therapies for disease and regenerative medicine.The new approach involves cell functionalisation with protein-polymer surfactant bioconjugates, which circumvents the need for direct cell-surface chemistry, and offers a high degree of flexibility, as the approach can be readily applied to a diverse range of proteins for use on potentially any cell type. The synthetic methodology recently pioneered by the PI involves the re-engineering of a protein surface in two key steps: (i) amplification of the positive charge density on the protein surface (supercharging) (ii) electrostatic coupling of anionic polymer surfactant chains to the cationic sites displayed on the protein surface. Significantly, by constructing this polymer surfactant corona, the cell membrane affinity can be systematically tuned to facilitate spontaneous insertion of the bioconjugate into a stem cell membrane, whilst retaining the native function of the protein linked to the cell surface. The PI recently applied this methodology to the oxygen-binding protein myoglobin and demonstrated that the bioconjugates rapidly inserted into the cytoplasmic membranes of adult bone-marrow derived human mesenchymal stem cells (hMSCs). The research programme has a strong (but not exclusive) focus on developing the bioconjugates for cell homing for cardiac stem cell therapy, as cardiovascular disease (CVD) is the leading cause of death globally (2015 WHO estimate is 18 million deaths p.a.). Here, the conceptual advance is centred on hijacking the homing properties of infectious bacteria by immobilising the fibronectin (Fn) binding domain of the bacterial adhesion protein CshA on the cytoplasmic membranes of stem cells. The CshA-Fn interaction has been proposed to be responsible for directing and immobilising S. gordonii colonisation at the cardiac endothelium, promoting the onset of infective endocarditis. Accordingly, the ability to display multiple copies of this binding motif, which has been evolutionarily optimised to recognise and bind specific molecular targets in the cardiac endothelium, on the membrane of stem cells has the potential to advance cardiac cell therapy.Significantly, the cell membrane modification platform has the potential to have clinical impact beyond cell therapies for CVD, as it could be readily applied to other cell types and vesicles (e.g., monocytes, blood outgrowth endothelial cells, natural killer cells or exosomes) and involve other homing protein- or peptide-based molecules (e.g., integrins, antibodies, or other bacterial adhesins). The research programme describes a scientific approach that combines in-house techniques for biophysics and regenerative medicine, as well as cutting-edge techniques available at large-scale facilities. As there is a strong medical focus within the programme, the applicant has engaged clinical scientist partners to aid with medical translation.