GENE REPLACEMENT THERAPY IN HEMATOPOIETIC STEM CELLS

(改编自申请人的摘要)该计划的总体目标是为通过造血干细胞的基因修饰纠正骨髓源性细胞遗传病奠定坚实的实验基础。目前的目标集中在使用重组逆转录病毒、慢病毒和腺相关病毒(AAV)载体来实现有效地将功能基因转移到原始造血干细胞，同时保持其最大的造血潜力。其具体目标是1)检测细胞周期、纤维连接蛋白介导的黏附和细胞因子调节因子对造血细胞功能和病毒转导的关系；2)确定AAV和慢病毒衍生的重组病毒载体是否可用于小鼠和人类造血细胞高效而稳定的基因转移；3)确定使用病毒介导的基因转移来纠正两种遗传性血液疾病(X连锁慢性肉芽肿病和范可尼贫血C组)的最佳策略；以及4)评估非消融性预处理方案和使用蛋白质标记基因或耐药基因进行转导细胞的选择。实验方法包括使用体外培养系统，NOD/SCID鼠-人异种移植，以及先前通过基因打靶方法产生的X-CGD和FANC的小鼠模型。这些目标的落实将由4个项目和3个核心单位分担。这一建议来自一组研究人员，他们在干细胞生物学、逆转录病毒、慢病毒和AAV介导的基因转移、分子遗传学、病毒学、骨髓移植和新生儿学方面具有不同但互补的经验。这些目标的实现将允许将这项基础工作转化为开发有效的病毒介导的遗传性血液疾病基因转移的临床方案。在更广泛的层面上，这些研究应该提供对造血干细胞的生物学行为的洞察，以及体外操纵它们的能力。GANT=P01HL31992-17建议项目(摘自申请者摘要)本项目申请的长期目标是评估急性肺损伤的基本机制和开发新的治疗方法。高浓度氧气和败血症肺损伤是将被评估的主要模型。拟议方案由四个项目和三个核心单位组成。项目1将评估小分子催化抗氧化剂在治疗高氧血症和内毒素+脓毒症引发的肺损伤中的疗效。该项目还将开发新的抗氧化剂模拟物，并探索它们与血红素加氧酶(HO)的抗氧化性的关系。项目2将检验这一假说，即外源性凝血功能激活和纤维蛋白代谢紊乱是高氧性和败血症肺损伤的中心因素。将使用两种新的抗凝血药来测试特定阻断外源性凝血启动步骤在减少炎症和急性肺损伤方面的效果，这两种抗凝剂可以阻断组织因子(TF)的功能，而且不会导致出血。项目3将评估细胞外超氧化物歧化酶(EC-SOD)在急性肺损伤中的调节和功能，并确定该酶的C末端“肝素结合”结构域在确定其分布和功能方面的影响。项目4将评估代谢途径的控制和肺细胞糖酵解在调节对急性损伤的反应中的上调。该项目将测试这样一种假设，即适应肺中的氧化应激需要提高己糖激酶(HK)的表达，己糖激酶(HK)是肺中糖酵解的一个限速步骤。该计划项目的总体原理是使用跨学科方法来确定急性肺损伤所涉及的细胞通路和细胞适应性反应，并测试新的药物治疗策略，这些策略可以扩展到治疗患有ARDS和脓毒症的人类。

(Adapted from the applicant's abstract) The overall goal of this program is to develop a strong experimental foundation for the correction of inherited disease of bone marrow-derived cells by genetic modification of hematopoietic stem cells. Current objectives are focused on the use of recombinant retroviral, lentiviral, and adeno-associated virus (AAV) vectors to achieve efficient transfer of functional genes into primitive hematopoietic stem cells while maintaining their maximal hematopoietic potential. The specific aims are to 1) examine the relationship between cell cycle, fibronectin-mediated adhesion, and cytokine regulators on hematopoietic cell function and viral transduction; 2) determine whether recombinant viral vectors derived from AAV and lentivirus can be used for efficient and stable gene transfer in murine and human hematopoietic cells; 3) identify optimal strategies using viral-mediated gene transfer to correct the phenotype in two inherited blood disorders, X-linked chronic granulomatous disease (X-CGD) and Fanconi anemia group C (FanC); and 4) evaluate non- ablative conditioning regimens and selection of transduced cells using maker proteins or drug resistance genes. Experimental approaches include the use of in vitro culture systems, NOD/SCID mouse-human xenografts, and murine models of X-CGD and FanC previously generated by gene targeting approaches. The implementation of these aims will be shared among 4 projects and 3 core units. This proposal draws from a group of investigators with diverse but complementary experience in stem cell biology, retrovirus-, lentivirus-, and AAV-mediated gene transfer, molecular genetics, virology, bone marrow transplantation, and neonatology. Achievement of these goals will permit the translation of this basic work to the development of clinical protocols for effective viral-mediated gene transfer of genetic blood diseases. On a broader level, these studies should provide insight into the biologic behavior of hematopoietic stem cells and the ability to manipulate them ex vivo. GANT=P01HL31992-17 PROPOSED PROGRAM (Adapted from Applicant's Abstract) The long-term objectives of this Program Project application are to evaluate basic mechanisms and develop new treatments for acute lung injury. High concentrations of oxygen and septic lung injury are the primary models that will be evaluated. The proposed program consists of four projects and three core units. Project 1 will evaluate the efficacy of small molecular weight catalytic antioxidants in the treatment of both hyperoxic and LPS + sepsis-initiated lung injury. This project will also develop new antioxidant mimetics and explore their relationships with the antioxidant properties of heme oxygenase (HO). Project 2 will test the hypothesis that activation of extrinsic coagulation and disordered fibrin turnover are central elements in hyperoxic and septic lung injury. The efficacy of specific blockade of the initiating steps of extrinsic coagulation in reducing inflammation and acute lung injury will be tested using two new anticoagulant drugs that block tissue factor (TF) function and do not cause bleeding. Project 3 will evaluate the regulation and function of the extracellular superoxide dismutase (EC-SOD) in acute lung injury and determine the impact of cleavage of the C-terminal "heparin binding" domain of this enzyme in determining its distribution and function. Project 4 will evaluate control of metabolic pathways and upregulation of lung cell glycolysis in modulating responses to acute injury. This project will test the hypothesis that adaptation to oxidant stress in the lung requires elevated expression of hexokinase (HK), a rate limiting step in glycolysis in the lung. The overall rationale for the Program Project is to use an interdisciplinary approach to define the cellular pathways and cellular adaptive responses involved in acute lung injury and to test new strategies for pharmacologic therapy that can be extended to the treatment of humans with ARDS and sepsis.