Targeting aberrant epigenetics by nanomedicine

描述(申请人提供)：急性髓系白血病(AML)是最常见的成人白血病之一，对大多数患者来说仍是一种致命的疾病。用地西他滨或5-氮胞苷抑制异常的DNA甲基化可恢复正常的细胞增殖、分化和凋亡模式，但临床反应仅限于少数血液系统恶性肿瘤。此外，非靶点摄取和低效率的化疗药物输送会导致不良的不良反应。因此，迫切需要将新的治疗药物靶向输送到白血病细胞。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去甲基化试剂(Bortezomib、miR29b和Sp1 siRNA)在用于联合治疗时存在协同效应，并且这种协同效应可能在脂质体或脂多糖为基础的纳米载体中被增强。我们基于这样的假设：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Bortezomib通过上调miR29b的表达，抑制Sp1/NF：b依赖的DNMT1转录，取消DNMT3a和3b的表达，从而导致DNA低甲基化。3)Sp1/NF：B复合体是调节miR29b和DNMT转录的中心调控因子。这两种调节剂的抑制剂都显示出令人鼓舞的抗DNMT活性。4)靶向脂基纳米载体递送化合物或寡核苷酸(ODN)显示出更显著的靶向下调，从而在体外和体内具有更明显的抗肿瘤活性。基于这些观察，这项建议的实验重点是纳米载体的设计、合成和优化，以及Bortezomib与miR29b或Sp1 siRNA的协同效应。具体目的是全面评估各种剂型的药物传递效果和特异性，并评估Bortezomib、miR29b和SP1 siRNA作为单一疗法或联合疗法的体外和体内药理活性。1.设计、合成和优化靶向脂质体(Lips)和脂多聚体(LPs)，用于将Bortezomib和Sp1 siRNA或miR29b导入AML细胞和AML患者原代细胞。我们将通过以下方式开发合适的靶向脂质体配方：i)使用聚碳酸酯薄膜挤出合成纳米载体，然后远程装载药物或新开发的微流控(MF)方法，然后连接到靶向配体；ii)靶向纳米载体的尺寸和粒度分布、药物传递效率、细胞摄取和毒性的表征。2.测定Lip-bortezomib与Lps-Sp1 siRNA或Lip-bortezomib与Lps-miR29b在AML细胞株和患者原代细胞中的药理活性。我们将同时用靶向纳米载体递送的DNA去甲基化药物修饰DNMT调控网络，并在体外使用i)Western印迹和定量聚合酶链式反应(QPCR)、ii)LC/MS/MS和iii)MTS和PI/AV染色来协同诱导DNA去甲基化。3.在小鼠模型上对Lip-bortezomib与Lps-Sp1 siRNA或Lip-bortezomib与Lps-miR29b的药理活性进行临床前体内评价。我们将通过在携带白血病的小鼠身上进行药代动力学/药效学(PK/PD)研究，使用Western印迹、qPCR、LC/MS/MS和临床疗效评估来确定在体内实现协同作用的组合的有效药理剂量。该项目将由具有翻译研究和实验治疗学(Liu，Marcucci和Garzon)、PK/PD(Chan)、纳米工程(JLee和RLee)专长的研究人员通过跨学科方法进行。如果成功，这项研究将推进对纳米科学的理解，加强对蛋白酶体系统、miR和DNA甲基化在白血病发生中作用的理解，并为抑制异常的DNMT活性奠定基础。

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.