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，它可以形成一个截尾四面体。这个结构是由我们的六聚体环构成的，四面体结构的四个边都包含六聚体环，但每个环都包含3个h形的交叉连接器，连接到其他环。这种类型的结构允许掺入多达12个功能实体，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了这些结构。纳米粒子的形状和大小与功能有关的假设似乎是正确的。至少在一定程度上，由于更好的吸收，我们发现，使用结合的Dicer底物PLK1敲除靶基因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，我们也在与Cryo-EM核心合作，进一步表征这些粒子。—以前，可编程六聚体RNA环被开发用于多达六种不同功能的控制递送。为了在不影响纳米粒子拓扑结构的情况下增加功能化的潜力，我们在RNA环的双链区域引入了间隙。采用分子动力学模拟的方法来评估设计的动态行为和灵活性的变化。然而，模拟所显示的变化不能被常规技术如非变性聚丙烯酰胺凝胶电泳和动态光散射清楚地证实。此外，在人外周血单个核细胞原代培养的体外分析中，没有发现对新组装的免疫识别有任何差异。为了解决这些不足，我们引入了一种计算机辅助的定量策略，该策略基于对云母/空气界面上研究的RNA纳米颗粒的afm分辨变形分析算法。我们通过人工图像分析验证了这种计算方法，并将其与模拟预测结果拟合。提出的纳米颗粒修饰策略和随后的基于原子力显微镜的分析为未来基于核酸的纳米技术的发展提供了一个广谱的方法。----利用rna作为靶向药物来控制患病细胞的典型方法依赖于对靶向细胞状态的预分析，然后递送药物，例如反义、抗ir或siRNA，从而分离治疗步骤和诊断步骤。我们开发了一套基于上下文敏感rna的逻辑开关，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释放的条件激活或失活作为表达rna的功能。这些开关使用RNA/DNA杂交设计，显著限制了与核酸酶降解相关的问题。为了控制基于RNA的纳米颗粒的可交付功能和稳定性，在开发由RNA/DNA杂交构建的计算设计纳米颗粒时，融合了DNA和RNA的特性。这些分子在血清中具有更高的稳定性，可以附着荧光标记物进行跟踪，并且能够分离功能元素的成分，使其失活，但在互补支点的控制下允许稍后激活，通过互补支点可以调整重新结合的动力学。可切割的底物siRNA可以分成两个组分，每个组分由RNA/DNA杂交组成。互补RNA单链支点可以代替DNA用于杂交的构建。这两个杂交种，当转染到细胞中，由于结点和计算确定的杂交种和产物之间的热力学差异，重新组合成两个产物。从热力学的角度来看，RNA支点的使用是有利的，因为它减少了解压缩杂合体和产生功能性RNA元件所需的单链末端的长度。从设计的角度来看，RNA支点可以是功能性DS RNA的一部分，或其他潜在的RNA片段，从而减小了所产生的杂交双工的大小并最大限度地减少了设计限制。含有3种Dicer底物sirna的基于rna的杂交体，可协同同时靶向HT29肿瘤中凋亡相关基因，经过细胞培养的重要测试，目前已在一项全面的小鼠研究中使用，该研究部分由发明开发计划资助。初步结果令人鼓舞，肿瘤内和尾静脉注射均能抑制肿瘤生长。目前正在使用其他递送剂进行进一步的研究。----由于我们可以用基于rna的纳米颗粒控制免疫反应，我们一直在与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（部分由UMD-NCI合作伙伴关系资助的综合癌症研究基金）。与乔斯特·奥本海姆（Joost Oppenheim）小组合作，我们发现了可能显著的体内结果，显示10只免疫能力强的小鼠中有3只“治愈”。大多数其他小鼠的肿瘤都有明显的消退。计划进行进一步的实验，以更全面地描述作用途径。在细胞培养和体内递送基于rna的纳米结构对于使用这些药物的治疗方法的发展至关重要。由于核酸酶的作用，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。在脂质方面，我们构建了由DOTAP、DOPE和DSPE-PEG2000组成的靶向癌细胞的递送剂（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）合作开发光活化聚合物，用于递送我们的rna纳米颗粒。结果在这里也非常令人鼓舞，显示最小的泄漏没有激光治疗和显著的功能，当激光治疗。HPPH是第二代氯基光敏剂，在食管癌的临床试验中显示出巨大的治疗潜力。我们与Birla技术与科学研究所的Sunil Dubey合作，开发并验证了一种生物分析方法，用于估计大鼠血浆中的HPPH（一种用于光动力治疗的化合物），该方法使用带PDA检测器的高效液相色谱法。使用HPPH LNP的Colon-26小鼠使用PDT显示出更好的疗效。我们还测试了用于将siRNA传递到肿瘤和大脑的亲水分子囊泡GLH-19和GLH-20配方。我们在两个地方都表现得很好，包括由于穿越血脑屏障的问题而难以瞄准的大脑。我们还用分子动力学分析了各种配方的稳定性，这很好地解释了我们在实验中看到的结果。—我们的两项专利的独家许可是与一家初创公司建立的，该公司是在NCI纳米挑战之外建立的。该公司的计划是将我们的rna纳米颗粒用于胶质母细胞瘤。