Novel cell therapy for anemia of CKD

描述（由申请人提供）：慢性肾脏疾病（CKD）影响约7%的美国人口，导致产生促红细胞生成素（EPO）的小管周围成纤维细胞的瘢痕形成和丢失。目前，重组EPO类似物注射治疗CKD的EPO缺乏性贫血，但最近出现了意想不到的副作用，如卒中、心脏病发作和深静脉血栓形成的风险增加，这可能会阻止该疗法的进一步应用。虽然这些副作用的机制尚不清楚，但很明显，每周或每月给药EPO类似物并不能概括这种重要激素的生理调节，而且大剂量给药可能会改变EPO信号通路。因此，迫切需要开发CKD贫血的替代疗法。在这里，我们描述了一个创新的实验设计，利用非病毒转座子介导的基因转移来开发一种治疗慢性肾病贫血的新策略。基因修饰的T淋巴细胞特异性针对持续性（潜伏）病毒，如eb病毒（EBV），由于慢性病毒抗原刺激，其在体内长期（80 ~ 8年）稳定存活。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细胞可以很容易地诱导凋亡，提供额外的安全和控制层。因此，我们假设，通过基因修饰诱导表达EPO和单独诱导自杀基因的病毒特异性T细胞代表了持续和安全治疗CKD贫血的理想候选细胞群。在具体目标1中，我们提出修饰病毒特异性的小鼠T细胞来诱导表达EPO和自杀基因，我们将把它们注入野生型和CKD小鼠体内，以测量它们在体内调节红细胞压积水平的有效性。特异性目标2侧重于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和在必要时进行选择性诱导细胞消融的能力。在特定目标3中，我们将通过测定EBV特异性T细胞的频率以及它们在体外存在和不存在转基因EPO的情况下对EBV抗原的反应来评估来自CKD患者的转基因人T细胞的功能。

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 by determining the frequency of EBV-specific T cells and their response to EBV antigen in the presence and absence of transgenically expressed EPO ex vivo.