Basic Research on Hematopoietic Adult Human Stem Cells

成人造血干细胞基础研究

• 批准号: 7732638
• 负责人: Harry L Malech
• 金额: $ 32.6万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732638
• 关键词: Adhesions; Affect; Allogenic; Animals; Apoptosis; Autologous; Basic Science; Biochemical; Biology; Blood; Blood Cells; Bone Marrow; Busulfan; CD34 gene; CXCR4 gene; Carmustine; Cell Therapy; Cells; Chronic Granulomatous Disease; Cleaved cell; Clinical; Dipeptides; Dipeptidyl-Peptidase IV; Disease; Endopeptidases; Engraftment; Failure; Genes; Goals; Growth; Harvest; Hematopoietic; Hematopoietic stem cells; Homologous Transplantation; Human; Immune; Immune system; Immunologic Deficiency Syndromes; Infection; Inherited; Ligands; Link; Marrow; Methods; Modeling; Numbers; Outcome; Patients; Peptide Hydrolases; Process; Protein Overexpression; Rate; Role; SCID Mice; Stem cells; Stromal Cell-Derived Factor 1; Therapeutic; Transferase; Transplantation; Treatment Protocols; Umbilical Cord Blood; Xenograft Model; base; bone; chemokine receptor; conditioning; diprotin A; gene correction; gene therapy; gene transfer vector; human adult stem cell; human stem cells; improved; in vivo; inhibitor/antagonist; migration; mutant; neutrophil; nonhuman primate; peripheral blood; progenitor; receptor; release of sequestered calcium ion into cytoplasm; response; success; therapeutic gene; tool; trafficking

This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy or for use in allogeneic transplantation in the treatment of inherited diseases affecting cells of the immune system. This project also studies CD34 positive hematopoietic adult human stem cells from patients with inherited immune deficiencies with the ultimate goal of developing hematopoietic stem cell based therapies for these disorders. We have developed new methods and materials which improve our ability to get new genes into human blood stem cells. We are also exploring the potential of busulfan as a more stem cell specific and immune system sparing conditioning regimen for transplant so we need to understand better the effect of such agents on human CD34 positive hematopoietic adult human stem cells. Evidence from human and animal studies of gene therapy suggest that providing an in vivo growth or survival advantage to genetically corrected blood cells can improve the outcome of gene therapy by increasing the percent of corrected cells in the body. One approach to this is to co-express the therapeutic gene (such as the corrective gene for X linked CGD) with a gene that allows for selective enrichment. In studies with collaborators we have explored the use of the methyguanine methyl transferase (MGMT) which protects against alykating agents such as BCNU in a non-human primate model achieving marking rates of up to 20%. Thus, we need to understand better how to culture and subject to selection human stem cells. As noted, a major goal of this project is to examine in CD34 positive hematopoietic adult human stem cells the role of the CXCR4 chemokine receptor (ligand is SDF-1) on engraftment in marrow. We showed that overexpression of CXCR4 in human CD34 hematopoietic stem cells enhanced engraftment of these cells in the NOD/SCID mouse xenotransplant model. The immunodeficiency, WHIM (warts, hypogammaglobulinemia, infections, myelokathexis apoptosis of neutrophils), is caused by truncations in the C-terminus of CXCR4. We created gene transfer vectors to over express the WHIM type mutant CXCR4 in CD34 stem cells and showed that this resulted in increased migration, adhesion and intracellular calcium flux in response to SDF-1. We showed that this was caused by a failure to downregulate or to internalize the mutant receptor providing a biochemical basis for the dominant hyperfunction abnormality of CXCR4 activity associated with WHIM. We also find that the mutant CXCR4 enhances engraftment of cells expressing this mutant receptor and it may be a useful tool to enhance engraftment. CD26 is a protease expressed on bone marrow stroma and also on some CD34 positive hematopoietic adult human stem cells. CD26 is a type IV dipeptide proteinase that can cleave and inactive SDF-1. We show that treatment of NOD/SCID mice with Diprotin A, an inhibitor of CD26 on marrow stroma, markedly enhances engraftment of CD34 positive hematopoietic adult human stem cells in this xenograft model. We believe that this could be an important therapeutic method to enhance engraftment.

该项目将外周血造血祖细胞（PBHP）研究为基因治疗的靶标或用于同种异体移植时，用于治疗影响免疫系统细胞的遗传疾病。该项目还研究了来自遗传性免疫缺陷患者的CD34阳性造血成人人类干细胞，其最终目标是为这些疾病开发造血干细胞疗法。 我们开发了新的方法和材料，这些方法和材料提高了我们将新基因进入人血干细胞的能力。 我们还正在探索Busulfan作为移植的更为干细胞特异性和免疫系统保留条件方案的潜力，因此我们需要更好地了解此类药物对人CD34阳性造血成人人类干细胞的影响。人类和动物研究的基因疗法研究的证据表明，为遗传校正的血细胞提供体内生长或生存优势可以通过增加体内校正细胞的百分比来改善基因治疗的结果。一种方法是与允许选择性富集的基因共表达治疗基因（例如X链接CGD的矫正基因）。在与合作者的研究中，我们探讨了甲基甲基转移酶（MGMT）的使用，该酶在非人类灵长类动物模型中可预防诸如BCNU之类的Alykating剂，可实现高达20％的标记率。 因此，我们需要更好地了解如何培养并受到选择人类干细胞的选择。 如前所述，该项目的主要目标是在CD34阳性造血成年人类干细胞中检查CXCR4趋化因子受体（配体是SDF-1）在骨髓植入中的作用。我们表明，在人类CD34造血干细胞中，CXCR4的过表达增强了这些细胞在点点/SCID小鼠异种移植模型中的植入。免疫缺陷（Warts，低磁性血症，感染，骨髓触及中性粒细胞的骨凋亡）是由CXCR4的C末端截断引起的。我们创建了基因转移载体，以过度表达CD34干细胞中的异想天开的突变体CXCR4，并表明这会导致迁移，粘附和细胞内钙通量增加，以响应SDF-1。我们表明，这是由于未能下调或内部化突变受体提供的，从而为与异想天开有关的CXCR4活性的显性高功能异常提供了生化基础。我们还发现，突变的CXCR4增强了表达这种突变受体的细胞的植入，它可能是增强植入的有用工具。 CD26是一种在骨髓基质和一些CD34阳性造血成人人类干细胞上表达的蛋白酶。 CD26是可以裂解和非活性SDF-1的IV型二肽蛋白酶。 我们表明，在这种异种移植模型中，用二吡啶A（一种CD26的抑制剂）Anod/SCID小鼠（一种CD26的抑制剂）显着增强了CD34阳性造血成人人类干细胞的植入。 我们认为，这可能是增强植入的重要治疗方法。