Design of Heme Protein-Based Blood Substitutes

描述(申请人提供)：我们的长期目标是开发重组人血红蛋白(RHB)，作为制造所有基于珠蛋白的氧气输送药物的起始材料。我们已经确定了以重组人血红蛋白为基础的血液替代品的关键要求：(A)中等的O2亲和力和大的O2解离速率常数，以有效地在毛细血管中运输；(B)显著降低NO清除率，以防止高血压副作用；(C)对自动和化学诱导的氧化的抵抗力，以抑制氧化应激；以及(D)低的血红素解离率，以延长货架期。在临床前的动物研究中，我们已经使用控制这些特性的机制来设计第二代基于RHb的细胞外血液替代品，具有更有效的氧气转运和很少或没有高血压副作用。然而，显然需要设计第三代非血管活性rHb，使其具有更强的变性抵抗力、更高的表达产量和更低的生产成本。我们建议通过以下方法解决这些问题：(1)在不影响珠蛋白稳定性和对降解反应的抵抗力的情况下，优化人重组人血红蛋白与O2的结合和对NO的清除；(2)通过(A)合理和比较的突变来增加载脂蛋白的抗解折叠能力，以及(B)与新发现的红系伴侣蛋白--α-血红蛋白稳定蛋白(AHSP)共表达RHb链；以及(3)通过RHB与来自志贺氏菌和相关病原体的血红素利用基因(Hug)的共表达，促进外源添加的血红素快速整合到新合成的球蛋白中。公共卫生：我们正在开发重组人血红蛋白，以取代捐献的血液，作为所有蛋白质基氧载体的来源材料，这些载体正在开发用于治疗严重失血，并相信这一努力应该成为国家公共卫生的优先事项。一种有效的基于血红蛋白的血液替代品将缓解长期的全血短缺，在军事和民事灾难后提供紧急治疗，并允许在缺乏安全血库系统的发展中国家使用输血。

DESCRIPTION (provided by applicant): Our long-range goal is to develop recombinant human hemoglobin (rHb) as the starting material for manufacturing all globin-based O2 delivery Pharmaceuticals. We have established the key requirements for a rHb-based blood substitute: (a) moderate O2 affinity and large O2 dissociation rate constants for efficient transport in capillaries; (b) significantly reduced rates of NO scavenging to prevent hypertensive side effects; (c) resistance to auto- and chemically-induced oxidation to inhibit oxidative stress; and (d) low rates of heme dissociation to increase shelf-life. We have used the mechanisms governing these properties to design second-generation extracellular rHb-based blood substitutes with more efficient O2 transport and little or no hypertensive side effect in pre-clinical animal studies. However, there is a clear need to engineer third-generation, non-vasoactive rHbs with increased resistance to denaturation, enhanced expression yields, and reduced production costs. We propose to solve these problems by: (1) optimizing O2 binding to and NO scavenging by human rHb without compromising globin stability and resistance to degradative reactions; (2) improving the stability and enhancing production of recombinant hemoglobin in E. coli by (a) rational and comparative mutagenesis to increase the resistance of the apoprotein to unfolding and (b) co- expression of the alpha and beta rHb chains with the newly discovered erythroid chaperone, alpha hemoglobin stabilizing protein (AHSP); and (3) facilitating rapid incorporation of exogenously added heme into newly synthesized globins by co-expression of rHb with the heme utilization genes (hug) from Plesiomonas shigelloides and related pathogens. PUBLIC HEALTH: We are developing recombinant human hemoglobin to replace donated blood as the source material for all protein-based oxygen carriers that are being developed for the treatment of severe blood loss and believe that this effort should be a national public health priority. An effective hemoglobin- based blood substitute will alleviate chronic shortages of whole blood, provide emergency treatment following military and civilian disasters, and allow the use of transfusions in developing countries lacking a safe blood banking system.