Novel cell therapy for anemia of CKD

描述(由申请人提供)：慢性肾脏疾病(CKD)影响大约7%的美国人口，并导致产生促红细胞生成素(EPO)的肾小管周围成纤维细胞形成疤痕和丢失。目前，CKD的EPO缺乏性贫血的治疗方法是重组EPO类似物注射，这些注射最近出现了不良副作用，如中风、心脏病发作和深静脉血栓形成的风险增加，这可能会阻止这种疗法的进一步使用。尽管这些副作用的机制尚不清楚，但很明显，每周或每月推注EPO类似物并不能概括这种重要激素的生理调节，推注可能会改变EPO信号通路。因此，迫切需要开发治疗慢性肾脏病贫血的替代疗法。在这里，我们描述了一种创新的实验设计，使用非病毒转座子介导的基因转移来开发一种治疗慢性肾脏病贫血的新策略。针对EB病毒(EBV)等持久性(潜伏)病毒的转基因T淋巴细胞，在慢性病毒抗原刺激下能在体内长期存活(8年)。此外，临床前和最近的临床研究表明，通过激活共同转移的自杀基因，T细胞很容易被诱导凋亡，提供了额外的一层安全和控制。因此，我们假设，病毒特异性T细胞经过基因改造后，可以诱导 表达的EPO和单独可诱导的自杀基因代表了持续安全治疗CKD贫血的理想候选细胞群。在特定的目的1中，我们建议修饰病毒特异性的小鼠T细胞，以诱导表达EPO和自杀基因，并将它们注入野生型和CKD小鼠中，以测试它们在体内调节红细胞压积水平的有效性。具体目标2重点是将这些基因修饰扩展到人类T细胞，并在体外测试它们通过慢性病毒抗原刺激长期繁殖的能力，以及诱导表达EPO和在需要时进行选择性诱导细胞消融的能力。在具体目标3中，我们将评估转基因人类T细胞的功能 并确定在体外转基因表达的EPO存在和不存在的情况下，EBV特异性T细胞的频率及其对EBV抗原的反应。

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) affects an estimated 7% of the US population and results in scarring and loss of peritubular fibroblasts which produce erythropoietin (EPO). EPO-deficient anemia of CKD is currently treated with recombinant EPO analog injections that have recently been associated with undesired side effects such as increased risk of stroke, heart attacks, and deep vein thrombosis which may preclude further use of this therapy. Although the mechanisms of these side effects are unclear, it is clear that bolus dosing of EPO analogs either weekly or monthly does not recapitulate the physiologic regulation of this important hormone and bolus dosing may alter EPO signaling pathways. Thus, there is a critical need to develop alternative therapies for anemia of CKD. Herein we describe an innovative experimental design using non-viral transposon-mediated gene transfer to develop a new strategy for therapy of anemia of CKD. Genetically modified T lymphocytes whose specificity is directed to persistent (latent) viruses such as Epstein-Barr virus (EBV) survive long-term (>8 years) in stable numbers in vivo due to chronic viral antigen stimulation. Moreover, preclinical and recent clinical studies have shown T cells can be readily induced to apoptose by activation of a co-transferred suicide gene, providing an additional layer of safety and control. We therefore hypothesize that virus specific T cells genetically modified to inducibly express EPO and a separately inducible suicide gene represent an ideal candidate cell population for sustained and safe treatment of anemia of CKD. In specific aim 1, we propose to modify virus specific murine T cells to inducibly express EPO and a suicide gene and we will infuse them into wild type and CKD mice to measure their effectiveness in regulating hematocrit levels in vivo. Specific aim 2 focuses on extending these genetic modifications to human T cells and testing them in vitro for their ability to be propagated long-term via chronic viral antigen stimulation, as well as inducibly express EPO and undergo selectively induced cell ablation if needed. In specific aim 3, we will evaluate the functionality of genetically modified human T cells from patients with CKD and determine the frequency of EBV-specific T cells and their response to EBV antigen in the presence and absence of transgenically expressed EPO ex vivo.