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的RNA纳米结构，形成截断的四面体。该结构是从我们的六聚体环构建的，四面体结构的4个侧面都包含六角形环，但每个环都包含3个H形的跨界连接器，与其他环。这种类型的构造允许将多达12个功能实体（例如DICER底物，信标和/或适体）掺入。我们发现，细胞似乎比其他一些RNA纳米构造更好地占据了这些构建体。关于功能性的纳米颗粒形状和大小重要的假设似乎是正确的。至少部分是由于更好的吸收，我们发现靶向基因诱导细胞死亡的敲低，使用掺入的DICER底物PLK1比其他一些颗粒更有效。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，以进一步表征这些粒子。为了增加功能化的潜力，对纳米颗粒拓扑的影响很小，我们将差距引入了RNA环的双链区域。分子动力学模拟用于评估设计灵活性的动态行为和变化。然而，模拟所建议的变化无法通过传统技术（例如非元素化聚丙烯酰胺凝胶电泳和动态光散射）清楚地证实。同样，在人外周血单核细胞的原发性培养物中进行的体外分析并未揭示新组合的免疫学识别的任何差异。为了解决这些缺陷，我们引入了计算机辅助的量化策略，该策略基于对云母/空气界面上研究的RNA纳米颗粒的算法AFM分辨变形分析。我们通过手动图像分析验证了这种计算方法，并将其拟合到模拟预测的结果中。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广谱方法。 ----利用RNA作为靶向剂来控制患病细胞的典型方法依赖于靶向靶向的细胞状态的预先分析，然后递送剂，例如反义，抗杀菌剂或siRNA，从而将治疗步骤和诊断性步骤分开。我们开发了一组基于上下文敏感的RNA逻辑开关，将这两个步骤结合到一个逻辑系统中。这允许单链或双链RNA的释放的条件激活或失活作为表达RNA的函数。开关是使用RNA/DNA杂种设计的，这显着限制了与核酸酶降解有关的问题。 ---为了控制基于RNA的纳米颗粒的可交付功能和稳定性，将DNA和RNA的性质合并为由RNA/DNA杂交构建的计算设计的纳米颗粒的开发。这些分子允许血清中的稳定性更高，荧光标记的附着以进行跟踪以及将功能元件灭活的功能元素的组成部分拆分，但是可以在互补的脚趾控制下以后的激活来调节重新相关的动力学。可滴定的底物siRNA可以分为两个组成部分，每个组件由RNA/DNA杂交组成。互补的RNA单链脚趾而不是DNA可以用于杂种的构建。当将两种杂种转染到细胞中时，由于脚趾和杂种和产物之间的计算确定的热力学差异，将其重组成两种产物。从热力学的角度来看，RNA脚趾的使用是有利的，因为它减小了解压缩混合动力并生成功能性RNA元素所需的单链末端的长度。从设计的角度来看，RNA Toehold可以是功能性DS RNA的一部分，也可以是其他潜在的RNA部分的一部分，可降低大小并最小化所得混合双链体的设计约束。基于RNA的杂种含有3个DICER底物siRNA，用于同时在HT29肿瘤中同时靶向与凋亡相关的基因的靶向，此后，在细胞培养物进行了大量测试后，在一项由发明开发计划中部分资助的全面小鼠研究中，在细胞培养物中进行了重大测试。最初的结果看起来令人鼓舞，表明肿瘤内肿瘤生长的迟缓在肿瘤内，并且通过尾静脉注射更多。正在使用替代递送剂进行进一步的研究。 ----由于我们可以通过基于RNA的纳米颗粒来控制免疫反应，因此我们一直与Joost Oppenheim-CCR合作，而Chris Jewell-UMD则利用这些特性来激活抗癌治疗的免疫系统（部分由UMD-NCI综合癌症研究赠款的UMD-NCI合作伙伴关系资助）。与Joost Oppenheim Group合作，我们发现体内结果可能具有重要意义，显示了3张10个免疫能力小鼠中的“治愈”。其他大多数小鼠表现出显着的肿瘤回归。计划进一步的实验以更全面地表征作用途径。 ---在细胞培养和体内基于RNA的纳米构建物的递送对于使用这些药物的治疗方法开发至关重要。非修饰的裸rNA由于核酸酶的血清而在血清中的半衰期短，并且由于其负电荷而难以越过细胞膜。因此，我们正在开发脂质和聚合物制剂。在脂质的情况下，我们已经构建了由DOTAP，DOPE和DSPE-PEG2000组成的递送剂，以靶向癌细胞（与ESTA Sterneck，CCR合作）。实验看起来很积极。此外，我们正在与Jonathan Lovell（Buffalo U）合作，开发光活化聚合物，用于传递基于RNA的纳米颗粒。在这里，结果也非常令人鼓舞，在没有激光治疗的情况下显示出最小的泄漏，当激光治疗时具有明显的功能。 HPPH是第二代基于氯素的光敏剂，在治疗食管癌的临床试验中显示出巨大的治疗潜力。我们与Sunil Dubey（Birla Technology of Science与Sunil Dubey）合作使用了一种使用PDA检测器的高性能液相色谱法估算了大鼠血浆中HPPH（用于光动力疗法中使用的化合物）的生物分析方法。 - 使用HPPH LNP使用PDT表现出优异的功效。我们还测试了Bolaamphiphile囊泡GLH-19和GLH-20制剂，用于将siRNA递送至肿瘤和大脑。我们向两个位置表现出良好的输送，包括大脑，由于与跨越血脑屏障有关的问题，这很难针对。还通过分子动力学分析了所测试的各种配方的稳定性，这很好地解释了我们在实验中看到的结果。-- - 我们两项专利的专有许可是通过一家从NCI纳米座上建立的初创公司建立的。该计划是为了将我们的基于RNA的纳米颗粒用于胶质母细胞瘤。