Single-guide-RNA-directed Synergistic Activation Mediator, a novel strategy to sensitize Non-Hodgkin Lymphomas to therapeutic drugs.

项目总结 目的：非霍奇金淋巴瘤(NHL)是美国第六大常见癌症，其中 90%是B型细胞，其中60%是好斗的，40%是懒惰的。尽管所有类型的非霍奇金淋巴瘤对 最初的治疗，惰性非霍奇金淋巴瘤是无法治愈的：患者完全治愈后，疾病会复发 对治疗的反应(CR)。此外，尽管努力开发有效的治疗方法，许多侵袭性淋巴瘤仍会复发。 最终，其中30%变得无法治愈，这突显了开发新疗法或改善的必要性 现有的。 在这项建议中，我们介绍了一种新的药物增敏策略，用于目前可用的抗非霍奇金淋巴瘤药物。 该方法利用sgRNA-SAM技术，这是CRISPR工具的变体，以实现有效、具体 以及内源性Ehd3基因的持续重新激活。建议的方法是基于这样一个事实： Ehd3在从NHL标本中分离的CD34+细胞亚群中优先下调 这表明药物敏感性降低。 因此，我们的工作假设是CD34+细胞中Ehd3表达的缺失是 非霍奇金淋巴瘤细胞的药物不敏感性。因此，恢复其表达将增强其细胞毒作用 目前使用的药物。 具体目标：AIM1，将存在于一项评估患者来源的Ehd3表达的前瞻性研究中 非霍奇金淋巴瘤标本。在这里，我们的目标是确定特定的NHL亚型，其中Ehd3表达水平是 显著降低，这可能更适合于所提出的基因再激活方法。在……里面 AIM2，我们试图进行建议的sgRNA-SAM指导的Ehd3的体外开发和测试 在非霍奇金淋巴瘤建立的细胞系和患者来源的(PD)细胞中，转录重新激活。我们还将评估 该方法增强细胞对2CDA-R和CHOP-R细胞毒作用的敏感性 药物在体外。在Aim3中，我们将进行sgRNA-SAM方法作为药物增敏的体内原理验证 策略，在CHOP-R和2CDA-R方案下，使用NHL细胞的动物异种移植。

PROJECT SUMMARY Objectives: Non-Hodgkin’s lymphomas (NHLs) are the 6th most common cancers in the US, of which 90% are B cells-type, and include 60% aggressive and 40% indolent. Although all types of NHL respond well to initial therapy, indolent NHLs are incurable: the disease relapses after the patient has achieved complete response (CR) to therapy. Also, despite efforts to develop effective therapies, many aggressive lymphomas recur and eventually 30% of these become incurable, highlighting the need to develop new therapies or to improve existing ones. In this proposal, we introduce a novel drug sensitization strategy for currently available anti-NHL drugs. The approach makes use of the sgRNA-SAM technology, a variant of the CRISPR tools, for effective, specific and sustained reactivation of the endogenous Ehd3 gene. The proposed approach is based on the fact that Ehd3 is preferentially down-regulated in a subpopulation of CD34+ cells isolated from NHL specimens and which show a reduced drug sensitivity. Therefore, our working hypothesis is that loss of Ehd3 expression in CD34+ cells is a driver event in the drug insensitivity of NHL cells. Consequently, restoring its expression will potentiate the cytotoxic effects of currently used drugs. Specific Aims: Aim1, will consist in a prospective study to assess Ehd3 expression in patient-derived NHL specimens. Here, we aim to identify specific subtypes of NHLs in which Ehd3 expression levels are predominantly decreased, and which could be more suitable for the proposed gene reactivation approach. In Aim2, we seek to perform in vitro development and test of the proposed sgRNA-SAM-directed Ehd3 transcriptional reactivation, in NHL established cell lines and patient-derived (PD) cells. We will also assess the power of the approach in potentiating the cells’ sensitivity to the cytotoxic effects of 2CdA-R and CHOP-R drugs in vitro. In Aim3, we will perform in vivo proof-of-principle of sgRNA-SAM approach as a drug sensitization strategy, under the CHOP-R and 2CdA-R regimens, using animal xenografts of NHL cells.