CpG-siRNA Conjugates to Target Acute Myeloid Leukemia

CpG-siRNA 缀合物靶向急性髓系白血病

• 批准号: 8894451
• 负责人: Marcin Kortylewski
• 金额: $ 34.86万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8894451
• 关键词: Acute Myelocytic Leukemia; Agonist; B-Cell Lymphomas; B-Lymphocytes; Biochemical; Biodistribution; Blast Cell; Blood Circulation; Cell Culture Techniques; Cell Death; Cell membrane; Cells; Chemicals; Clinical; Complex; Confocal Microscopy; Cytoplasm; Disease; Drug Kinetics; Endosomes; Event; Gene Combinations; Gene Silencing; Gene Targeting; Genes; Growth; Half-Life; Hematologic Neoplasms; Hematopoietic; Hematopoietic Neoplasms; Human; Immune; In Vitro; Intracellular Transport; Lentivirus Vector; Life; Ligands; Link; Measures; Mediating; Modeling; Modification; Molecular; Multiple Myeloma; Mus; Oligonucleotides; Oncogenic; Pathway interactions; Patients; Population; Primary Neoplasm; RNA Interference; RNA Processing; Reagent; Reporting; Resistance; Serum; Signal Transduction; Small Interfering RNA; Specificity; Stat5 protein; TLR9 gene; Technology; Testing; Therapeutic; Therapeutic Effect; Toxic effect; Transfection; Translations; Transport Process; Work; base; cancer therapy; clinical application; conventional therapy; cytotoxic; design; immune activation; immunotoxicity; improved; in vitro Assay; in vivo; innovation; interest; leukemia; macrophage; neoplastic cell; novel; nuclease; receptor; research study; targeted delivery; trafficking; transcription factor; tumor; uptake

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.

描述（由申请人提供）：siRNA在体内有效递送至特定细胞群仍然是成功治疗应用的主要挑战。造血细胞，包括急性髓性白血病（AML）细胞，由于低转染效率而对siRNA递送造成特殊问题，这需要使用慢病毒载体。我们最近证明，细胞内受体的配体，如TLR 9，可以用作细胞特异性siRNA递送的靶向部分。通过合成连接siRNA与CpG寡核苷酸（ODN）而产生的新型双功能CpG-siRNA缀合物特异性地靶向并沉默小鼠中TLR 9阳性免疫细胞（包括DC、巨噬细胞和B细胞）中的基因。与裸siRNA相反，配备有CpG部分的siRNA分子在不存在任何转染或包装试剂的情况下被靶细胞快速内化并定位于内体中。我们的初步研究表明，通过利用针对人类细胞刺激优化的CpG序列产生的新的CpG-siRNA版本允许在人类TLR 9阳性肿瘤细胞（例如AML）中特异性地进行基因靶向。我们证明了局部以及全身施用靶向致癌基因和/或促存活基因的CpG-siRNA诱导肿瘤细胞死亡并抑制异种移植的人AML肿瘤的生长。为了优化CpG-siRNA策略用于抗癌治疗，我们建议定义TLR 9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。将通过CpG-siRNA的化学修饰和多聚化产生核酸酶抗性和长寿命的缀合物。接下来，我们计划使用CpG-siRNA策略来靶向STAT 5，STAT 5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-STAT 5 siRNA对播散性AML肿瘤模型和来自白血病患者的原发性白血病原始细胞的作用。此外，我们将测试CpG-STAT 5 siRNA对正常人免疫细胞的活力和免疫活化的影响。来自所提出的研究的结果有可能克服限制siRNA治疗剂应用的主要障碍，允许沉默目前不可药物化的靶基因，如STAT 5，从而阻断AML增殖和存活。我们预计，随着对TLR 9介导的沉默效应的机制的更好理解，以及针对播散性肿瘤优化的CpG-siRNA缀合物，拟议的研究将产生适用于AML和其他血液恶性肿瘤的广泛临床应用的技术平台。从长远的角度来看，这项工作有潜力产生新的，更有效和更安全的治疗方法，扩大治疗选择，使各种形式的血癌患者受益。

项目成果

Intracellular processing of immunostimulatory CpG-siRNA: Toll-like receptor 9 facilitates siRNA dicing and endosomal escape.

• DOI: 10.1016/j.jconrel.2013.06.007
• 发表时间: 2013-09-28
• 期刊: Journal of controlled release : official journal of the Controlled Release Society
• 作者: Nechaev S;Gao C;Moreira D;Swiderski P;Jozwiak A;Kowolik CM;Zhou J;Armstrong B;Raubitschek A;Rossi JJ;Kortylewski M
• 通讯作者: Kortylewski M

TLR9 signaling through NF-κB/RELA and STAT3 promotes tumor-propagating potential of prostate cancer cells.

• DOI: 10.18632/oncotarget.4029
• 发表时间: 2015-07-10
• 期刊: Oncotarget
• 作者: Moreira D;Zhang Q;Hossain DM;Nechaev S;Li H;Kowolik CM;D'Apuzzo M;Forman S;Jones J;Pal SK;Kortylewski M
• 通讯作者: Kortylewski M

Push and release: TLR9 activation plus STAT3 blockade for systemic antitumor immunity.

• DOI: 10.4161/onci.27441
• 发表时间: 2014-01-01
• 期刊: Oncoimmunology
• 影响因子: 7.2
• 作者: Kortylewski M;Kuo YH
• 通讯作者: Kuo YH

TLR9-Targeted STAT3 Silencing Abrogates Immunosuppressive Activity of Myeloid-Derived Suppressor Cells from Prostate Cancer Patients.

• DOI: 10.1158/1078-0432.ccr-14-3145
• 发表时间: 2015-08-15
• 期刊: Clinical cancer research : an official journal of the American Association for Cancer Research
• 作者: Hossain DM;Pal SK;Moreira D;Duttagupta P;Zhang Q;Won H;Jones J;D'Apuzzo M;Forman S;Kortylewski M
• 通讯作者: Kortylewski M

TLR9 signaling in the tumor microenvironment initiates cancer recurrence after radiotherapy.

• DOI: 10.1158/0008-5472.can-13-1314
• 发表时间: 2013-12-15
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Gao C;Kozlowska A;Nechaev S;Li H;Zhang Q;Hossain DM;Kowolik CM;Chu P;Swiderski P;Diamond DJ;Pal SK;Raubitschek A;Kortylewski M
• 通讯作者: Kortylewski M