Computational and Experimental RNA Nanobiology

计算和实验 RNA 纳米生物学

• 批准号: 10014517
• 负责人: Bruce Shapiro
• 金额: $ 131.28万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014517
• 关键词: Address; Affect; Air; Algorithms; Apoptosis; Binding Proteins; Biocompatible Materials; Biologic Development; Blood - brain barrier anatomy; Brain; Buffaloes; CCR; Cell Culture Techniques; Cell Death; Cell membrane; Cells; Charge; Clinical Trials; Collaborations; Colon; Complementary RNA; Complex; Computer Assisted; Computing Methodologies; Cryoelectron Microscopy; DNA; Detection; Development; Dimensions; Disease; Double-Stranded RNA; Elements; Environment; Extravasation; Formulation; Funding; Future; Generations; Genes; Glioblastoma; HPPH; High Pressure Liquid Chromatography; Human; Hybrids; Image Analysis; Immune; Immune response; Immune system; Immunologics; In Vitro; Injections; Institutes; Kinetics; Lasers; Legal patent; Length; Licensing; Lipids; Location; Logic; Malignant Neoplasms; Malignant neoplasm of esophagus; Manuals; Methodology; Methods; Mica; Modification; Molecular; Molecular Analysis; Molecular Conformation; Mus; Nanostructures; Nanotechnology; Nucleic Acids; PLK1 gene; PUVA Photochemotherapy; Pathway interactions; Peripheral Blood Mononuclear Cell; Photosensitization; Plasma; Polyacrylamide Gel Electrophoresis; Polymers; Program Development; Property; RNA; RNA Stability; RNA analysis; Rattus; Receptor Cell; Research Project Grants; Scanning Probe Microscopes; Science; Serum; Shapes; Side; Small Interfering RNA; Structure; System; Tail; Techniques; Technology; Testing; Therapeutic; Thermodynamics; Untranslated RNA; Veins; Vesicle; anticancer research; anticancer treatment; aptamer; base; behavior change; cancer cell; catalyst; chlorin; design; detector; disorder control; experimental study; flexibility; genetic information; in vivo; invention; knock-down; light scattering; molecular dynamics; nanobiology; nanodevice; nanoparticle; nuclease; off-patent; particle; programs; simulation; synthetic biology; targeted agent; therapeutic development; tumor; tumor growth; uptake; yeast two hybrid system

Recently we developed a new type of RNA nanostructure that forms a truncated tetrahedron. The structure was built from our hexameric ring where 4 sides of the tetrahedral structure each contain the hexmeric ring, but each ring contains 3 H-shaped crossover connectors to the other rings. This type of construct allows for the incorporation of up to 12 functional entities such as Dicer substrates, beacons and/or aptamers. We found that cells seem to take up these constructs better than some of the other RNA nanoconstructs. The hypothesis that nanoparticle shape and size matter regarding functionality seems to be true. Due, at least in part, to the better uptake we found that knockdown of targeted genes to induce cell death, using incorporated Dicer substrate PLK1 is more efficacious than some of our other particles. Several different methods were used to verify the assembly of this particle including the newly acquired atomic force microscope (AFM). Currently we are also collaborating with the Cryo-EM core to further characterize these particles.---Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduced gaps into the double-stranded regions of the RNA rings. Molecular dynamics simulations were used to assess the dynamic behavior and the changes in the flexibility of the designs. The changes suggested by simulations, however, cannot be clearly confirmed by conventional techniques such as nondenaturing polyacrylamide gel electrophoresis and dynamic light scattering. Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of the new assemblies. To address these deficiencies, we introduced a computer-assisted quantification strategy, which is based on an algorithmic AFM-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validated this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis provided a broad-spectrum approach for the future development of nucleic acid-based nanotechnology. ----Typical methodologies that utilize RNAs as targeting agents to control diseased cells rely on pre-analysis of the state of the cells to be targeted followed by delivery of an agent e.g. antisense, antimiR or siRNA, thus separating the therapeutic step and the diagnonostic step. We developed a set of context-sensitive RNA-based logic switches that combines both steps into one logic system. This permits the the conditional activation or deactivation of the the release of single-stranded or double-stranded RNAs as a function of expressed RNAs. THe switches are designed using RNA/DNA hybrid significantly limiting issues related to nuclease degredation. ---To achieve control over deliverable functionality and stability of RNA-based nanoparticles, the properties of DNA and RNA were merged in the development of computationally designed nanoparticles that were constructed from RNA/DNA hybrids. These molecules allow higher stability in blood serum, attachment of fluorescent markers for tracking, and the ability to split the components of functional elements inactivating them, but allowing later activation under the control of complementary toeholds by which the kinetics of re-association can be tuned. Diceable substrate siRNA could be split into two components, each consisting of an RNA/DNA hybrid. Complementary RNA single-stranded toeholds rather than DNA can be used in the construction of the hybrids. The two hybrids, when transfected into cells recombine into two products due to the toeholds and the computationally determined thermodynamic difference between the hybrids and the products. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it reduces the length of the single stranded ends required to unzip the hybrids and generate the functional RNA element. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and minimizing the design constraints of the resulting hybrid duplexes. RNA-based hybrids containing 3 Dicer substrate siRNAs for synergistic simultaneous targeting of apoptosis-related genes in HT29 tumors are now being used, after significant testing in cell cultures, in a comprehensive mouse study funded, in part, by the Invention Development Program. Initial results look encouraging showing retardation of tumor growth both intratumorally and more so by tail vein injection. Further studies are being performed using alternative delivery agents. ----Since we can control immune response with RNA-based nanoparticles, we have been collaborating with Joost Oppenheim- CCR, and Chris Jewell-UMD to take advantage of these properties to activate the immune system for anti-cancer treatment (funded in part by an UMD-NCI Partnership for Integrative Cancer Research grant). Working with Joost Oppenheim group we found possibly significant in vivo results showing a "cure" in 3 out 10 immune competent mice. Most of the other mice showed signficant regression ot their tumors. Further experiments are planned to characterize more fully the pathways of action. ---The delivery of RNA-based nanoconstructs in cell culture and in vivo is essential for the development of therapeutic methodologies using these agents. Non-modified naked RNAs have short half-lives in blood serum due to nucleases and have difficulty crossing cell membranes due to their negative charge. Thus, we are developing lipid and polymer formulations. In the case of the lipids we have constructed delivery agents consisting of DOTAP, DOPE and DSPE-PEG2000 to target cancer cells (in collaboration with Esta Sterneck, CCR). Experiments look quite positive. In addition, we are working with Jonathan Lovell (U of Buffalo) on the development of photoactivatable polymers for the delivery of our RNA-based nanoparticles. Results are very encouraging here too, showing minimal leakage without laser treatment and significant functionality when laser treated. A second-generation chlorin-based photosensitizer, HPPH shows tremendous therapeutic potential in clinical trials in treatment of esophageal cancer. We, in collaboration with Sunil Dubey (Birla Institute of Technology & Science) have developed and validated a bioanalytical method for estimation of HPPH (a compound used in photodynamic therapy) in rat plasma using High Performance Liquid Chromatography with PDA detector. --Colon-26 mice using an HPPH LNP showed superior efficacy using PDT. We have also tested bolaamphiphile vesicles GLH-19 and GLH-20 formulations for delivery of siRNA to tumors and to the brain. We showed good delivery to both locations, including the brain which is difficult to target due to issues related to crossing the blood-brain barrier. The stability of the various formulations tested were also analyzed by molecular dynamics, which explained quite well the results we were seeing experimentally.---An exclusive license of two of our patents was established with a startup company that was established out of the NCI Nanochallenge. The plan is for the company is to use our RNA-based nanoparticles for glioblastoma.

最近，我们开发了一种新型 RNA 纳米结构，可形成截顶四面体。该结构是由我们的六边形环构建的，其中四面体结构的 4 个侧面均包含六边形环，但每个环包含 3 个与其他环相连的 H 形交叉连接器。这种类型的构建体允许掺入多达 12 个功能实体，例如 Dicer 底物、信标和/或适体。我们发现细胞似乎比其他一些 RNA 纳米结构更好地吸收这些结构。纳米粒子的形状和尺寸与功能有关的假设似乎是正确的。至少部分由于更好的吸收，我们发现使用掺入的 Dicer 底物 PLK1 敲低靶基因来诱导细胞死亡比我们的一些其他颗粒更有效。使用了几种不同的方法来验证该粒子的组装，包括新获得的原子力显微镜（AFM）。目前，我们还与 Cryo-EM 核心合作，进一步表征这些颗粒。---此前，可编程六聚体 RNA 环被开发用于控制输送多达六种不同的功能。为了增加功能化的潜力而不影响纳米颗粒拓扑结构，我们在 RNA 环的双链区域引入了间隙。分子动力学模拟用于评估动态行为和设计灵活性的变化。然而，通过非变性聚丙烯酰胺凝胶电泳和动态光散射等传统技术无法清楚地证实模拟所暗示的变化。此外，对人外周血单核细胞原代培养物的体外分析并未显示新组装体的免疫识别存在任何差异。为了解决这些缺陷，我们引入了一种计算机辅助定量策略，该策略基于对云母/空气界面上研究的 RNA 纳米颗粒进行 AFM 解析变形分析的算法。我们通过手动图像分析并将其拟合到模拟预测结果来验证这种计算方法。所提出的纳米颗粒修饰策略和随后的基于 AFM 的分析为基于核酸的纳米技术的未来发展提供了一种广谱的方法。 ----利用RNA作为靶向剂来控制患病细胞的典型方法依赖于对要靶向的细胞的状态进行预分析，然后递送试剂，例如，反义、antimiR或siRNA，从而将治疗步骤和诊断步骤分开。我们开发了一套基于 RNA 的上下文敏感逻辑开关，将这两个步骤组合到一个逻辑系统中。这允许根据表达的RNA有条件地激活或失活单链或双链RNA的释放。这些开关采用 RNA/DNA 混合体设计，显着限制了与核酸酶降解相关的问题。 ---为了实现对基于 RNA 的纳米粒子的可传递功能和稳定性的控制，DNA 和 RNA 的特性被合并到由 RNA/DNA 杂合体构建的计算设计纳米粒子的开发中。这些分子在血清中具有更高的稳定性，可以附着用于追踪的荧光标记，并且能够分裂使它们失活的功能元件的成分，但允许随后在互补立足点的控制下激活，从而可以调整重新结合的动力学。可切割的底物 siRNA 可以分成两个部分，每个部分都由 RNA/DNA 杂合体组成。互补RNA单链立足点而非DNA可用于构建杂交体。当转染到细胞中时，这两种杂交体由于立足点以及计算确定的杂交体和产物之间的热力学差异而重组成两种产物。从热力学的角度来看，RNA立足点的使用是有利的，因为它减少了解压杂交体并生成功能性RNA元件所需的单链末端的长度。从设计角度来看，RNA 立足点可以是功能性 DS RNA 或其他潜在 RNA 部分的一部分，从而减小尺寸并最大限度地减少所得杂合双链体的设计限制。经过在细胞培养物中进行大量测试后，含有 3 个 Dicer 底物 siRNA 的基于 RNA 的杂交体用于协同同时靶向 HT29 肿瘤中的凋亡相关基因，目前正在一项由发明开发计划部分资助的综合小鼠研究中使用。初步结果看起来令人鼓舞，显示肿瘤内的肿瘤生长受到抑制，尾静脉注射的效果更明显。正在使用替代递送剂进行进一步研究。 ----由于我们可以用基于RNA的纳米颗粒控制免疫反应，我们一直与Joost Oppenheim-CCR和Chris Jewell-UMD合作，利用这些特性来激活免疫系统进行抗癌治疗（资助部分由 UMD-NCI 综合癌症研究合作伙伴资助）。与 Joost Oppenheim 小组合作，我们发现可能显着的体内结果显示，10 只具有免疫能力的小鼠中，有 3 只被“治愈”。大多数其他小鼠的肿瘤显示出显着的消退。计划进行进一步的实验以更全面地描述作用途径。 ---在细胞培养物和体内传递基于 RNA 的纳米结构对于开发使用这些药物的治疗方法至关重要。由于核酸酶的原因，未修饰的裸 RNA 在血清中的半衰期较短，并且由于其负电荷而难以穿过细胞膜。因此，我们正在开发脂质和聚合物配方。就脂质而言，我们构建了由 DOTAP、DOPE 和 DSPE-PEG2000 组成的递送剂来靶向癌细胞（与 Esta Sterneck、CCR 合作）。实验看起来相当积极。此外，我们正在与 Jonathan Lovell（布法罗大学）合作开发可光活化聚合物，用于递送基于 RNA 的纳米颗粒。这里的结果也非常令人鼓舞，显示无需激光治疗的泄漏最小，并且激光治疗后具有显着的功能。 HPPH 是第二代二氢卟酚基光敏剂，在食管癌治疗的临床试验中显示出巨大的治疗潜力。我们与 Sunil Dubey（比尔拉技术与科学研究所）合作开发并验证了一种生物分析方法，使用带有 PDA 检测器的高效液相色谱法估算大鼠血浆中的 HPPH（一种用于光动力疗法的化合物）。 --使用 HPPH LNP 的 Colon-26 小鼠在 PDT 中表现出卓越的功效。我们还测试了用于将 siRNA 递送至肿瘤和大脑的双亲性囊泡 GLH-19 和 GLH-20 制剂。我们显示出对两个位置的良好传递，包括由于与穿过血脑屏障相关的问题而难以瞄准的大脑。所测试的各种配方的稳定性也通过分子动力学进行了分析，这很好地解释了我们在实验中看到的结果。---我们的两项专利的独家许可是与一家在 NCI 纳米挑战中成立的初创公司建立的。该公司的计划是使用我们的基于 RNA 的纳米颗粒治疗胶质母细胞瘤。