Supercharged protein-surfactant bioconjugates for next-generation cell therapies

细胞膜工程是一个快速发展的领域，具有对细胞治疗产生重大影响的潜力，因为将外源蛋白引入细胞膜对于体内定点组织修复非常有利。这是因为细胞疗法的广泛应用存在许多障碍，包括缺乏移植细胞的植入，这导致有限的功能整合。几项细胞治疗研究表明，干细胞的静脉内或动脉内输注导致肺部不期望的积聚，这降低了全身递送的效率并增加了产生致命微栓子的可能性。即使当直接植入感兴趣的器官时，治疗益处所需的细胞数量也可能高得令人望而却步。因此，这项研究计划描述了一类新的人工膜结合蛋白的合理设计，具有化养归巢特性，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影响。新方法涉及蛋白质-聚合物表面活性剂生物缀合物的细胞功能化，这避免了直接细胞表面化学的需要，并且提供了高度的灵活性，因为该方法可以容易地应用于多种蛋白质，以用于潜在的任何细胞类型。PI最近开创的合成方法涉及两个关键步骤中的蛋白质表面的再工程：（i）蛋白质表面上正电荷密度的放大（增压）（ii）阴离子聚合物表面活性剂链与蛋白质表面上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冠，可以系统地调节细胞膜亲和力，以促进生物缀合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，并证明生物缀合物快速插入成人骨髓来源的人间充质干细胞（hMSC）的细胞质膜中。该研究计划重点关注（但不是唯一的）开发用于心脏干细胞治疗的细胞归巢生物缀合物，因为心血管疾病（CVD）是全球死亡的主要原因（2015年WHO估计每年有1800万人死亡）。在这里，概念上的进步是集中在劫持归巢特性的感染性细菌通过固定的纤维连接蛋白（Fn）结合域的细菌粘附蛋白CshA的细胞质膜上的干细胞。CshA-Fn相互作用已被认为负责引导和固定S。戈登氏菌在心脏内皮定植，促进感染性心内膜炎的发作。因此，在干细胞膜上展示该结合基序的多个拷贝的能力具有推进心脏细胞治疗的潜力，该结合基序已经进化地优化以识别和结合心脏内皮中的特异性分子靶标。重要的是，细胞膜修饰平台具有超越CVD细胞治疗的临床影响的潜力，因为它可以容易地应用于其它细胞类型和囊泡（例如，单核细胞、血液生长内皮细胞、自然杀伤细胞或外来体）并涉及其它基于归巢蛋白或肽的分子（例如，整联蛋白、抗体或其它细菌粘附素）。该研究计划描述了一种科学方法，将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相结合。由于该计划有很强的医学重点，申请人聘请了临床科学家合作伙伴来帮助医学翻译。

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.