Targeting aberrant epigenetics by nanomedicine

描述（由申请人提供）：急性髓性白血病（AML）是最常见的成人白血病之一，对大多数患者来说仍然是致命的疾病。地西他滨或5-阿扎胞苷抑制异常DNA甲基化可恢复细胞增殖、分化和凋亡的正常模式，然而，临床反应仅限于少数造血系统恶性肿瘤。此外，化学治疗剂的脱靶摄取和低效递送导致不期望的副作用。因此，迫切需要将新型治疗剂靶向递送至白血病细胞。我们的长期目标是开发创新的纳米载体，并阐明控制DNA甲基化的调控机制，从而作为开发可用于减轻疾病过程的治疗方案的先决条件。具体的假设是，当用作联合治疗时，DNA低甲基化剂（硼替佐米、miR 29 b和Sp1 siRNA）之间存在协同效应，并且当通过基于脂质体或脂质聚合复合物的纳米载体递送时，这种协同效应可能会增强。 我们的假设基于以下观察结果：1）miR 29 b直接或间接破坏AML中的所有DNA甲基转移酶（DNMT），从而导致DNA低甲基化。2)蛋白酶体抑制剂硼替佐米通过miR 29 b上调消耗Sp1/NF：B依赖性DNMT 1转录并消除DNMT 3a和3b表达，进而引起DNA低甲基化。3)Sp1/NF：B复合物是调控miR 29 B和DNMT转录的中心调节因子。两种调节剂的抑制剂均显示出令人鼓舞的抗DNMT活性。4)化学化合物或寡脱氧核糖核苷酸（ODN）的靶向基于脂质的纳米载体递送显示出更显著的靶向下调，从而在体外和体内具有更显著的抗肿瘤活性。基于这些观察结果，本提案的实验重点是纳米载体的设计、合成和优化以及硼替佐米与miR 29 b或Sp1 siRNA组合的协同效应。 具体目的旨在提供各种制剂中药物递送功效和特异性的综合评估，以及硼替佐米、miR 29 b和sp1 siRNA作为单一疗法或联合疗法的体外和体内药理学活性的评价。具体目标是：1.设计、合成和优化靶向脂质体（Lips）和脂质聚合复合物（LP），用于将硼替佐米和Sp1 siRNA或miR 29 b递送到AML细胞系和AML患者原代细胞中。我们将通过以下方式开发合适的靶向脂质体制剂：i）使用聚碳酸酯膜挤出合成纳米载体，然后远程加载药物或新开发的微流体（MF）方法，然后与靶向配体缀合，ii）表征靶向纳米载体的大小和大小分布、药物递送功效、细胞摄取和毒性。 2.确定Lip-bortezelatin与LPs-Sp1 siRNA或Lip-bortezelatin与LPs-miR 29 b的组合在AML细胞系和患者原代细胞中的药理学活性。我们将使用i）蛋白质印迹和定量PCR（qPCR），ii）LC/MS/MS和iii）MTS和PI/AV染色证明通过靶向纳米载体递送的DNA低甲基化剂对DNMT调控网络的同时药理学修饰协同诱导体外DNA低甲基化。 3.在鼠模型中对Lip-bortezelatin与LPs-Sp1 siRNA或Lip-bortezelatin与LPs-miR 29 b的组合的药理学活性进行临床前体内评价。我们将通过使用蛋白质印迹、qPCR、LC/MS/MS在白血病荷瘤小鼠中进行的药代动力学/药效学（PK/PD）研究和临床疗效评估，确定实现体内协同作用的组合的有效药理学剂量。 该项目将通过跨学科方法进行，由具有转化研究和实验治疗学（Liu，Marcucci和加尔宗），PK/PD（Chan），纳米工程（JLee和RLee）专业知识的研究人员进行。如果成功，这项研究将推进对纳米科学的理解，增强对蛋白酶体系统，miR和DNA甲基化在白血病发生中的作用的理解，并建立抑制异常DNMT活性的基本概念。

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) represents one of the most common adult leukemia and remains as a deadly disease for most patients. Inhibition of aberrant DNA methylation by decitabine or 5-azacitidine restores normal patterns of cell proliferation, differentiation and apoptosis, however, the clinical response is restricted to a minority of hematopoietic malignancies. Further, off-target uptake and low efficient delivery of chemotherapeutic agents leads to undesirable adverse effects. Hence, there is an immediate need for targeted delivery of novel therapeutic agents to leukemic cells. Our long-term goals are to develop innovative nanocarriers and to elucidate the regulatory mechanisms controlling DNA methylation thereby leukemogenesis as a prerequisite to the development of therapeutic protocols that can be used to attenuate the disease process. The specific hypothesis is that there exists a synergistic effect among DNA hypomethylating agents (bortezomib, miR29b and Sp1 siRNA) when used as combination therapy and this synergism may be enhanced when delivered by liposome- or lipopolyplexe-based nanocarriers. We base that hypothesis on the observations that 1) miR29b disrupts all DNA methyltransferases (DNMTs) directly or indirectly in AML thereby leading to DNA hypomethylation. 2) proteasome inhibitor bortezomib depletes Sp1/NF:B-dependent DNMT1 transcription and abolishes DNMT3a and 3b expression via miR29b upregulation, in turn causing DNA hypomethylation. 3) Sp1/NF:B complex is a central regulator governing both miR29b and DNMT transcription. Inhibitors for both regulators display encouraging anti-DNMT activity. 4) targeted lipid-based nanocarrier delivery of chemo-compounds or oligo deoxyribonucleotides (ODNs) display more significant target downregulation thereby more pronounced anti-tumor activity in vitro and in vivo. Based on these observations, the experimental focus of this proposal is on the nanocarrier design, synthesis and optimization as well as the synergistic effect of bortezomib combined with miR29b or Sp1 siRNA. The specific aims are designed to provide a comprehensive assessment of drug delivery efficacy and specificity in a variety of formulations and the evaluation of in vitro and in vivo pharmacological activity of bortezomib, miR29b and sp1 siRNA as monotherapy or combination therapy. The specific aims are to: 1. Design, synthesize and optimize targeted liposomes (Lips) and lipopolyplexes (LPs) for delivering bortezomib and Sp1 siRNA or miR29b into AML cell lines and AML patient primary cells. We will develop suitable targeted liposomal formulations by i) synthesis of nanocarriers using polycarbonate membrane extrusion followed by remote-loading of the drug or newly developed microfluidic (MF) methods and then conjugated to targeting ligands, ii) characterization of targeted nanocarriers for the size and size distribution, drug delivery efficacy, cellular uptake and toxicity. 2. Determine the pharmacological activity of the combination of Lip-bortezomib with LPs-Sp1 siRNA or Lip-bortezomib with LPs-miR29b in AML cell lines and patient primary cells. We will demonstrate that simultaneously pharmacological modification of DNMT regulatory network by targeted-nanocarrier delivered DNA hypomethylating agents synergistically induces DNA hypomethylation in vitro using i) Western blot and quantitative PCR (qPCR), ii) LC/MS/MS and iii) MTS and PI/AV staining. 3. Perform preclinical in vivo evaluation of the pharmacological activity of the combination of Lip- bortezomib with LPs-Sp1 siRNA or Lip-bortezomib with LPs-miR29b in murine models. We will define the effective pharmacological dose of the combinations achieving synergisms in vivo by pharmacokinetic/pharmacodynamic (PK/PD) studies in leukemia-bearing mice using Western blot, qPCR, LC/MS/MS and the assessment of clinical efficacy. This project will be carried out through an interdisciplinary approach by investigators with expertise in translational research and experimental therapeutics (Liu, Marcucci and Garzon), PK/PD (Chan), nanoengineering (JLee and RLee). If successful, this investigation will advance the understanding of nanosciences, enhance the understanding of the roles of proteasome system, miR and DNA methylation in leukemogenesis and establish a fundamental concept for the inhibition of aberrant DNMT activities.