Supercharged protein-surfactant bioconjugates for next-generation cell therapies

人工膜结合蛋白(AMBP)有可能影响过继细胞治疗的疗效，因为引入外源蛋白为治疗细胞提供额外的功能可能对定点组织修复非常有利。该方法的核心是合理设计模块化的双功能增压蛋白质-聚合物表面活性复合体，这些复合体自发地插入干细胞的质膜，以传递氧气输送和趋化归巢特性。这可以解决细胞疗法中的许多挑战，包括缺乏可行的细胞移植，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和脾等组织下沉的积聚。这些非靶点效应降低了全身给药的效率，增加了产生致命性微栓子的可能性。因此，这项研究计划描述了一类具有响应性氧气输送和化学营养归巢特性的新的双功能AMBP的合理设计，这将有助于克服这些限制，并对疾病和再生医学的细胞治疗具有深远的影响。新方法避免了对共价细胞表面化学的需要，并提供了高度的灵活性，因为该方法可以应用于潜在的任何细胞类型的广泛的蛋白质。由PI开创的AMBP方法包括两个关键步骤的AMBP的合理设计：(I)对AMBP锚进行增压以放大正表面电荷密度(Ii)将聚合物表面活性剂链静电接枝到膜锚上的阳离子位置。由此产生的聚合物表面活性剂电晕允许系统地调节细胞膜的亲和力，以促进AMBP自发地插入细胞膜，同时保留细胞结合蛋白的天然功能。PI已经成功地将这种方法应用于氧结合蛋白肌红蛋白，以及融合到细菌粘附素基序CSHA的纤维连接蛋白结合域的增压绿色荧光蛋白。在这里，AMBP迅速插入成人骨髓来源的hMSCs的细胞膜，并提供响应的氧气输送或趋化干细胞归巢到心脏组织。该研究计划的重点是开发心脏干细胞疗法的AMBP，因为心血管疾病(CVD)是全球主要的死亡原因(2021年世界卫生组织估计每年将有1790万人死亡)。这里，概念上的进展集中在开发人源化的双功能AMBP嵌合体，该嵌合体可以通过增压的肌红蛋白锚定模块(以提高体内低氧环境中的细胞生存能力)响应地向细胞输送氧气，该模块与提供心脏细胞外基质靶向的模块融合在一起。因此，展示这些AMBP的多个副本的能力具有促进心脏细胞治疗的潜力。这些AMBP可以输送氧气，并且已经经过进化优化，以识别和结合心脏内皮中的特定分子靶点。AMBP平台可能不仅对心血管疾病的细胞治疗具有临床影响，因为它可以很容易地应用于其他类型的细胞和囊泡(例如单核细胞、自然杀伤细胞、外切体或脂质纳米颗粒)，并涉及其他基于归巢的蛋白质或多肽分子(例如整合素、纳米体或其他细菌粘附素)。该研究方案描述了一种科学方法，它结合了生物物理学、合成生物学和再生医学的内部技术，以及大规模设施中可用的尖端技术。由于该计划有很强的医学重点，申请人聘请了临床科学家和工业合作伙伴来协助医学翻译。

Artificial membrane binding proteins (AMBPs) have the potential to impact on the efficacy of adoptive cell therapies, as the introduction of exogenous proteins to provide additional functionality to therapeutic cells could be highly advantageous for site-directed tissue repair. The methodology is centred on the rational design of modular bifunctional supercharged protein-polymer surfactant complexes that spontaneously insert into the plasma membrane of stem cells to impart oxygen delivery and chemotropic homing properties. This could address many of the challenges in cell therapies, including the lack of viable cell engraftment, which results in limited functional integration. Several cell therapy studies have shown that intravenous or intra-arterial infusion of stem cells leads to accumulation in tissue sinks, such as the lungs and spleen. These off-target effects reduce the efficiency of systemic delivery and increase the likelihood of producing lethal microemboli. Accordingly, this research programme describes the rational design of a new class of bifunctional AMBPs with responsive oxygen delivery and 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 circumvents the need for covalent cell-surface chemistry, and offers a high degree of flexibility, as the approach can be applied to a wide range of proteins for use on potentially any cell type. The AMBP methodology pioneered by the PI involves the rational design of an AMBP in two key steps: (i) supercharging the AMBP anchor to amplify the positive surface charge density (ii) electrostatic grafting of polymer surfactant chains to the cationic sites on the membrane anchor. The resulting polymer surfactant corona allows the cell membrane affinity to be systematically tuned to facilitate spontaneous insertion of the AMBP into the cell membrane, whilst retaining the native function of the cell-bound protein. The PI has successfully applied this methodology to the oxygen-binding protein myoglobin, as well as supercharged green fluorescent protein fused to the fibronectin binding domain of a bacterial adhesin motif CshA. Here, the AMBPs rapidly inserted into the membranes of adult bone-marrow derived hMSCs and provided either responsive oxygen delivery or chemotactic stem cell homing to cardiac tissue. The research programme has a strong (but not exclusive) focus on developing AMBPs for cardiac stem cell therapies, as cardiovascular disease (CVD) is the leading cause of death globally (2021 World Health Organisation estimate is 17.9 million deaths p.a.). Here, the conceptual advance is centred on developing humanised bifunctional AMBP chimeras that can responsively deliver oxygen to the cells via a supercharged myoglobin anchor module (to improve cell viability in hypoxic in vivo environments), fused to a module that provides cardiac extracellular matrix targeting. Accordingly, the ability to display multiple copies of these AMBPs, which can deliver oxygen and have been evolutionarily optimised to recognise and bind specific molecular targets in the cardiac endothelium, has the potential to advance cardiac cell therapy.The AMBP platform is likely to have clinical impact beyond cell therapies for CVD, as it could be readily applied to other cell types and vesicles (e.g., monocytes, natural killer cells, exosomes, or lipid nanoparticles) and involve other homing protein- or peptide-based molecules (e.g., integrins, nanobodies, or other bacterial adhesins). The research programme describes a scientific methodology that combines both in-house techniques for biophysics, synthetic biology 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 scientists and industrial partners to aid with medical translation.