CpG-siRNA Conjugates to Target Acute Myeloid Leukemia

描述（由申请人提供）：siRNA有效递送到体内特定细胞群仍然是成功治疗应用的主要挑战。包括急性髓系白血病（AML）细胞在内的造血细胞，由于转染效率低，需要使用慢病毒载体，对siRNA的传递构成了特殊的问题。我们最近证明了细胞内受体的配体，如TLR9，可以用作细胞特异性siRNA递送的靶向部分。新型双功能CpG-siRNA偶联物通过将siRNA与CpG寡核苷酸（ODN）合成而成，可特异性靶向和沉默小鼠TLR9阳性免疫细胞（包括dc、巨噬细胞和B细胞）中的基因。与裸siRNA相比，配备CpG片段的siRNA分子在没有任何转染或包装试剂的情况下迅速被靶细胞内化并定位到核内体中。我们的初步研究表明，利用针对人类细胞刺激优化的CpG序列产生的新的CpG- sirna版本允许基因特异性靶向人类tlr9阳性肿瘤细胞，如AML。我们证明了局部和全身给药cpg - sirna靶向致癌和/或促生存基因诱导肿瘤细胞死亡和抑制异种移植的人类AML肿瘤的生长。为了优化CpG- siRNA策略在抗癌治疗中的应用，我们建议明确tlr9介导的基因沉默的分子机制和细胞内事件。我们还需要延长CpG-siRNA试剂的稳定性和循环半衰期，以最大限度地提高其全身给药的功效。通过化学修饰和cpg - sirna的多聚，将产生耐核酸酶和长寿命的缀合物。接下来，我们计划使用CpG-siRNA策略靶向STAT5，这是一种介导绝大多数AML肿瘤存活的转录因子。我们将评估优化的CpG-STAT5 siRNA对弥散性AML肿瘤模型和白血病患者原发性白血病母细胞的影响。此外，我们将测试CpG-STAT5 siRNA对正常人类免疫细胞活力和免疫激活的影响。拟议研究的结果有可能克服限制siRNA治疗应用的主要障碍，允许沉默目前不可药物的靶基因，如STAT5，从而阻断AML的增殖和生存。我们预计，随着对tlr9介导的沉默效应机制的更好理解，以及针对弥散性肿瘤优化的CpG-siRNA偶联物，拟议的研究将产生一个适用于AML和其他血液系统恶性肿瘤的广泛临床应用的技术平台。从长远来看，这项工作有可能产生新的、更有效和更安全的治疗方法，为各种形式的血癌患者提供更多的治疗选择。

DESCRIPTION (provided by applicant): Efficient delivery of siRNA to specific cell populations in vivo remains a major challenge to successful therapeutic applications. Hematopoietic cells, including acute myeloid leukemia (AML) cells, pose special problem for siRNA delivery due to low transfection efficiency, which requires the use of lentiviral vectors. We recently demonstrated that ligands for intracellular receptors, such as TLR9, can be used as targeting moieties for cell-specific siRNA delivery. Novel dual-function CpG-siRNA conjugates, generated by synthetically linking siRNA to a CpG oligonucleotide (ODN), target and silence genes specifically in TLR9- positive immune cells including DCs, macrophages and B cells in mice. In contrast to the naked siRNA, the siRNA molecules equipped with a CpG moiety, are rapidly internalized by target cells and localized into endosomes in the absence of any transfection or packaging reagents. Our preliminary studies show that a new CpG-siRNA version, generated by utilizing the CpG sequence optimized for human cell stimulation, allows for gene targeting specifically in human TLR9-positive tumor cells, such as AML. We demonstrated that local as well as systemic administration of CpG-siRNAs targeting oncogenic and/or pro-survival genes induced tumor cell death and inhibited growth of xenotransplanted human AML tumors. To optimize CpG- siRNA strategy for use in anticancer therapy, we propose to define the molecular mechanisms and intracellular events involved in TLR9-mediated gene silencing. We also need to prolong stability and circulation half-life of CpG-siRNA reagents to maximize their efficacy for systemic administration. The nuclease-resistant and long-lived conjugates will be generated through chemical modifications and multimerization of CpG-siRNAs. Next, we plan to use the CpG-siRNA strategy to target STAT5, a transcription factor mediating survival of the vast majority of AML tumors. We will assess the effect of optimized CpG-STAT5 siRNA against disseminated AML tumor models and primary leukemic blasts from leukemia patients. Additionally, we will test the effect of CpG-STAT5 siRNA on viability and immune activation of normal human immune cells. Results from the proposed studies have potential to overcome major hurdles limiting the application of siRNA therapeutics, allowing for silencing of currently non- druggable target genes, like STAT5, thereby blocking AML proliferation and survival. We anticipate that with better understanding of the mechanism(s) underlying TLR9-mediated silencing effect and with CpG-siRNA conjugates optimized for use against disseminated tumors, the proposed studies will produce a technology platform applicable to broad clinical application against AML and other hematologic malignancies. In a long- term perspective, this work has potential to generate novel, more effective and safer therapeutics, expanding treatment options for the benefit of patients with various forms of blood cancers.