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一种致命的疾病。地西他滨或5-阿扎胞苷对异常DNA甲基化的抑制可恢复细胞增殖、分化和凋亡的正常模式，然而，临床反应仅限于少数造血恶性肿瘤。此外，化疗药物的脱靶摄取和低效率递送导致不良反应。因此，迫切需要将新的治疗药物靶向递送到白血病细胞。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，从而将白血病的发生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，DNA低甲基化剂（硼替佐米、miR29b和Sp1 siRNA）在联合使用时存在协同作用，并且当脂质体或脂多聚树脂基纳米载体递送时，这种协同作用可能会增强。我们的假设基于以下观察结果：1)miR29b直接或间接破坏AML中所有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减少Sp1/NF: b依赖性DNMT1转录，并通过miR29b上调消除DNMT3a和3b的表达，从而导致DNA低甲基化。3) Sp1/NF:B复合体是miR29b和DNMT转录的中央调控因子。这两种调节因子的抑制剂显示出令人鼓舞的抗dnmt活性。4)针对lipid-based nanocarrier交付chemo-compounds或低聚糖脱氧核苷酸(ODNs)从而更加明显差别显示更重要的目标对这些基因的体外和体内抗肿瘤活性。基于以上观察结果，本课题的实验重点是硼替佐米纳米载体的设计、合成和优化，以及硼替佐米与miR29b或Sp1 siRNA的协同效应。具体目的是全面评估各种剂型的给药效果和特异性，以及评价硼替佐米、miR29b和sp1 siRNA作为单药或联合治疗的体内外药理学活性。具体目标是：1。设计、合成和优化靶向脂质体（Lips）和脂多复合物（LPs），用于将硼替佐米和Sp1 siRNA或miR29b递送至AML细胞系和AML患者原代细胞。我们将通过以下方法开发合适的靶向脂质体配方：i)利用聚碳酸酯膜挤压合成纳米载体，然后远程装载药物或新开发的微流体（MF）方法，然后偶联到靶向配体；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. 测定lip -硼替佐米联合LPs-Sp1 siRNA或lip -硼替佐米联合LPs-miR29b在AML细胞系和患者原代细胞中的药理活性。我们将通过Western blot和定量PCR (qPCR)， 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低甲基化药物同时对DNMT调控网络进行药理学修饰，协同诱导DNA低甲基化。3. 在小鼠模型中对Lip-硼替佐米与LPs-Sp1 siRNA或Lip-硼替佐米与LPs-miR29b联合使用的药理活性进行临床前体内评价。我们将采用Western blot、qPCR、LC/MS/MS等方法对白血病小鼠进行药代动力学/药效学（PK/PD）研究，并对临床疗效进行评估，确定联合用药在体内实现协同作用的有效药理学剂量。该项目将通过跨学科的研究方法进行，研究人员具有转化研究和实验治疗学（Liu， Marcucci和Garzon）， PK/PD (Chan)，纳米工程（JLee和RLee）的专业知识。如果成功，这项研究将促进纳米科学的理解，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，并为抑制异常DNMT活性建立一个基本概念。

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.