Novel Cellular Lifespan Extension for Tissue Engineering

描述（申请人提供）：血管组织工程是一个快速发展的领域。最近，一些研究小组已经证明了利用患有血管疾病的老年人的细胞培养血管的可行性。这些血管有一天可能会被植入动脉旁路。然而，培养血管的成功与否很大程度上取决于供体的年龄。由于寿命有限，来自老年供体（即60岁以上）的细胞表现出非常差的生长特性，通常不能形成融合的血管组织。我们最近的研究表明，延长寿命对于从老年供体的血管细胞中成功培养动脉至关重要。逆转录病毒介导的人类端粒酶逆转录酶（hTERT）的过表达延长了细胞寿命，并使70岁以上患者的动脉培养成为可能。为了成功，传统的hTERT需要在很长一段时间内表达，以延长端粒和延长寿命。然而，考虑到最近逆转录病毒治疗的严重联合免疫缺陷患者的癌症并发症，对于使用逆转录病毒进行hTERT递送存在重大关切。此外，组成型过表达hTERT本身可使细胞易发生恶性转化。因此，虽然延长分化细胞的寿命可能对许多类型的组织的工程设计至关重要，但持续的逆转录病毒递送hTERT是有问题的。最近，一种hTERT突变体与poll DNA结合蛋白融合。这种突变体与细胞DNA结合并迅速延长端粒，从而在短时间内延长细胞寿命。由于它的快速作用，pot1-hTERT可以通过更短暂的非逆转录病毒途径——腺病毒或直接核感染来传递。我们预计瞬时传递pot1-hTERT将影响细胞寿命延长，而不会伴随逆转录病毒和持续hTERT表达相关的风险。在这项应用中，我们将研究老年血管细胞中瞬时po1 - htert表达的生物学，作为老年人细胞寿命延长和组织工程的范例。我们将确定瞬时hTERT-pot1对寿命、表型、细胞周期调节因子和工程血管形成的影响。我们预计，作为一种强大的工具，pot1-hTERT可能具有普遍的适用性，可以为老年人设计多种类型的组织。

DESCRIPTION (provided by applicant): Vascular tissue engineering is a rapidly accelerating area. Recently, several groups have shown the feasibility of culturing vessels using cells derived from elderly humans with vascular disease. These vessels may one day be implantable for arterial bypass. However, success in culturing vessels has depended strongly on the age of the donor. Because of limited lifespan, cells from older donors (i.e. over 60 years of age) display very poor growth properties and often fail to form confluent vascular tissues. We have recently shown that lifespan extension critical for success in culturing arteries from vascular cells from older donors. Retrovirally-mediated overexpression of the human telomerase enzyme reverse transcriptase (hTERT) extends cell lifespan and enables the culture of arteries for patients older than 70 years. To be successful, conventional hTERT needs to be expressed over a long time period to lengthen telomeres and extend lifespan. However, significant concerns exist regarding use of retroviruses for hTERT delivery, in light of recent cancerous complications in retrovirally-treated patients with severe combined immunodeficiency. In addition, constitutive over-expression of hTERT could itself pre-dispose cells to malignant transformation. Hence, while lifespan extension of differentiated cells will likely continue to be critical for engineering many types of tissues, sustained retroviral delivery of hTERT is problematic. Recently, a mutant of hTERT fused with the poll DNA binding protein has been described. This mutant binds to cellular DNA and extends telomeres very rapidly, thereby providing lifespan extension of cells in a short time period. Because of its rapid action, pot1-hTERT may be delivered via more transient, nonretroviral approaches - either adenoviral, or by direct nucleofection. We anticipate that transient delivery of pot1-hTERT will affect cellular lifespan extension, without concomitant risks associated with retroviruses and sustained hTERT expression. In this application, we will study the biology of transient pot1-hTERT expression in elderly vascular cells, as a paradigm for cellular lifespan extension and tissue engineering in the elderly. We will determine effects of transient hTERT-pot1 on lifespan, phenotype, cell-cycle regulators, and on engineered vessel formation. We anticipate that pot1-hTERT may have general applicability as a powerful tool to enable the engineering of multiple types of tissues for older humans.