Computational and Experimental RNA Nanobiology

最近，我们与加州大学圣巴巴拉分校的Luc Jaeger合作开发了一种新型的RNA纳米结构，它可以形成一个截断的四面体。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。我们发现细胞比其他一些RNA纳米结构更能吸收这些结构。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，由于更好的吸收和我们认为更好的处理能力，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惊人地验证该粒子的组装，包括原子力显微镜（AFM）和cro - em，分别与CCR的光学显微镜和图像分析实验室和电子显微镜实验室合作。----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。我们开发了一组基于AND和NOT功能的上下文敏感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。此外，由于杂交的DNA部分缩短，所需的DNA更少。由于较短的双链DNA螺旋产物，这具有减少先天细胞免疫反应的额外优势。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，从而减小了大小并简化了所产生的杂交双工的设计。在一项由发明开发计划资助的综合性小鼠研究中，含有3种Dicer底物sirna的基于rna的杂交体在HT29肿瘤中协同同时靶向凋亡相关基因，在细胞培养中进行了重要测试后，已经在体内进行了测试。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CCR的Joost Oppenheim合作，利用这些特性来激活免疫系统进行抗癌治疗。与乔斯特·奥本海姆的团队合作，我们在体内发现了显著的结果，10只免疫能力强的老鼠中有3只“治愈”了。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。—除了在另一个项目标题下描述的使用小分子靶向各种潜在药物的RNA基序之外，我们一直在探索，通过使用3D建模，分子动力学和分子对接，使用小分子配体停靠到特定的RNA基序，从而改变由这些基序构建的纳米结构的动力学，形状和功能。我们已经证明这种影响是可能的，比如。靶标生物物理特性的变化。进一步探索这种类型的纳米设计正在研究中。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。我们与布法罗大学的Jonathan Lovell合作开发了磺化光活化聚合物，用于递送rna纳米颗粒。这个概念是用聚焦的近红外光照射肿瘤，这些可光激活的聚合物会特异性地将药物释放到肿瘤中，而在正常组织中几乎没有任何释放，而正常组织没有受到光的影响。我们已经显示了最小的泄漏没有激光治疗和显著的功能时，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的结肠-26小鼠使用PDT显示出更好的疗效。我们还与本古里安大学的Eli Heldman合作，测试了用于将siRNA递送到肿瘤和大脑的亲bolaaamphi亲性囊泡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授权是与一家初创公司Sixfold建立的，该公司是由NCI纳米挑战成立的。该公司正在使用并进一步开发基于rna的纳米颗粒来靶向各种肿瘤类型。路易斯维尔大学的Michael Nantz和康涅狄格大学的Xiulang Lu正在合作开发一种基于肟醚的相对较新的脂质。初步结果显示，它们很有希望作为靶向肺癌的递送剂。-与哈佛医学院的Xandra Breakefield合作，也正在寻求开发将RNA装载外泌体的技术，以用作潜在的递送剂。该技术利用带高度正电荷的蛋白质来实现这种装载。初步结果看起来很有希望。

Recently we developed a new type of RNA nanostructure that forms a truncated tetrahedron in collaboration with Luc Jaeger, UC Santa Barbara. 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 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 and we believe better processivity, 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 stunningly verify the assembly of this particle including an atomic force microscope (AFM) and Cryo-EM in collaborations with the Optical Microscopy and Image Analysis Lab and with the Electron Microscopy Lab respectively, of CCR. ----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 based on AND and NOT functionalities 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 hybrids, significantly limiting issues related to nuclease degradation. ---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. In addition, less DNA is needed since the DNA portion of the hybrid is shortened. This has the added advantage of reducing innate cellular immune responses as a result of shorter double stranded DNA helical products. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and simplifying the design of the resulting hybrid duplexes. RNA-based hybrids containing 3 Dicer substrate siRNAs for synergistic simultaneous targeting of apoptosis-related genes in HT29 tumors have been tested in vivo after significant testing in cell cultures, in a comprehensive mouse study funded, in part, by the Invention Development Program. 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, to take advantage of these properties to activate the immune system for anti-cancer treatment. Working with Joost Oppenheim's group we found 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.---Besides using small molecules to target various RNA motifs for potential drugs as described under another project heading, we have been exploring, by the use of 3D modeling, molecular dynamics and molecular docking, the use of small molecule ligands that dock to specific RNA motifs that in turn alters the dynamics, shape and functionality of the nano constructs built from these motifs. We have shown that such affects are possible, i,e. changing of the biophysical characteristics of the targets. Further exploration of this type of nano design is being investigated.---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. We have worked with Jonathan Lovell, U of Buffalo, on the development of sulfonated photoactivatable polymers for the delivery of our RNA-based nanoparticles. The concept being that the use of focused near infrared light on tumors that have been administered these photoactivatable polymers will specifically release drugs into the tumor with little of any release in normal tissues that have not be effected by the light. We have shown 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 by 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 in collaboration with Eli Heldman, Ben Gurion University. 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, Sixfold that was established out of the NCI Nanochallenge. The company is using and further developing our RNA-based nanoparticles for targeting various tumor types.--- A relatively new lipid based on the use of oxime-ethers is being explored in collaboration with Michael Nantz, University of Louisville and Xiulang Lu, University of Connecticut. Preliminary results look quite promising for their use as a delivery agent in target lung cancer.--- A collaboration with Xandra Breakefield, Harvard Medical school is also being pursued to develop techniques to load exosomes with RNA for to be used as potential delivery agents. The technique utilizes highly positively charged proteins to enable this loading. Preliminary results look promising.