一个简单的替代，以立体定向注射miRNA的脑特异性敲除
Summary
MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.
Abstract
MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.
Introduction
微小RNA已成为新的治疗目标，因为它们在基因表达和直接的证据参与疾病的调节普遍作用。 miRNA是正在积极探讨了他们的潜力作为药物靶点1,2。另外，在miRNA表达的改变与几种疾病3和仿真这些变化由miRNA表达的人工扰动的相关联的可用于研究参与疾病表现的细胞途径。 miRNA的靶向药物组织特异性递送目前是miRNA的基础的药物发展的一个重大挑战。拮抗和miRNA模仿是有希望的代理商扰动miRNA水平4-6。但是，特特点增强了它们的特异性和功效必须掺入RNA拮抗剂的设计中，可以使用之前在体内扰动miRNA表达。
微RNA是特别相关在目前无法治愈的神经变性疾病和神经发育疾病的目标。血 – 脑屏障构成限制拮抗在大脑的输送。立体定向注射广泛用于啮齿动物模型，提供分子的特定位置在大脑7。它需要技巧，仪器仪表和时间大量的投资。立体定向注射是侵入性的，包括外科手术，造成至少轻微伤害和被限制在本地投递。同时优先使用靶向神经元的使用细胞穿透肽的可以应对这些限制，因为它们可以通过跨血管途径给药，但违反血脑屏障。这种由狂犬病毒糖蛋白（RVG）衍生肽，以前是用来提供的siRNA对乙型脑炎病毒在小鼠8。我们发现，使用的肽为微小RNA拮抗剂递送中，miRNA可有效在小鼠大脑9撞倒。
内容】“>的miRNA的第二个主要挑战击倒源于miRNA的小尺寸和密切相关的序列同种型的存在。我们采取的示例的mmu-的miR-29家族，包括三个密切相关的同种型中，miR-29a的，b和c。RNA拮抗剂一般也沿着主链修饰以增加它们的稳定性和使它们耐受核酸酶的攻击。锁核酸（LNA的）提供，它们提高的热稳定性，甚至进一步的优点导致了超越目标降解空间位阻10。所有沿主干引入的修改可能是有效的，但昂贵的。前面我们已经看到，超过最优数量的修改不能进一步提高疗效。因此，RNA拮抗剂的设计涉及到RNA拮抗剂的优化改造。到复杂的微小RNA拮抗剂与亲神经肽，带电七溴到壬精氨酸扩展非共价地被使用。 D型精氨酸残基被使用的，因为它们赋予更高的稳定性，因为它们不被蛋白酶敏感的切割。七溴到壬精氨酸延伸充当有效的细胞渗透剂，虽然它们不赋予细胞类型特异性。通过共价连接的RVG肽与壬精氨酸连接体，亲神经，产生细胞穿透肽。肽的正电荷的残基带负电荷的核酸骨架相互作用，以形成复合物。这些复合物可以用于有效转染的DNA或RNA导入培养的细胞和体内进入组织。
Protocol
Representative Results
Discussion
Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.
Materials
| Vortex | |||
| Restrainer or Decapicone | |||
| Narrow runway | ~70-cm-long, ~5-cm-wide with ~5-cm-high walls. | ||
| Reagents | |||
| Fluorescently labelled oligonucleotides (siGLO) | GE Healthcare Dharmacon INC | D0016300120 | |
| 10% sterile D-glucose | |||
| Antagomir-29 | Exiqon | custom synthesis | |
| Antagomir-control | Exiqon | custom synthesis | |
| Neuropeptide RVG | G.L.Biochem (Shanghai) Ltd. | custom synthesis | >98% purity |
| Neuropeptide RVM | G.L.Biochem (Shanghai) Ltd. | custom synthesis | >98% purity |
| 其他 | |||
| Cotton | |||
| Warm water | |||
| Insulin syringes | |||
| Absorbent sheets | |||
| Ink | |||
| Brush | |||
| Antiseptic |
References
- Roshan, R., Ghosh, T., Scaria, V., Pillai, B. MicroRNAs: novel therapeutic targets in neurodegenerative diseases. Drug discovery today. 14, 1123-1129 (2009).
- Maes, O. C., Chertkow, H. M., Wang, E., Schipper, H. M. MicroRNA: Implications for Alzheimer Disease and other Human CNS Disorders. Current Genomics. 10, 154-168 (2009).
- Soifer, H. S., Rossi, J. J., Saetrom, P. MicroRNAs in Disease and Potential Therapeutic Applications. Mol Ther. 15, 2070-2079 (2007).
- Bader, A. G., Brown, D., Winkler, M. The Promise of MicroRNA Replacement Therapy. Cancer research. 70, 7027-7030 (2010).
- Stenvang, J., Petri, A., Lindow, M., Obad, S., Kauppinen, S. Inhibition of microRNA function by antimiR oligonucleotides. Silence. 3, 1-17 (2012).
- Trang, P., et al. Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice. Molecular Therapy. 19, 1116-1122 (2011).
- Barbash, S., Hanin, G., Soreq, H. Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome. J Vis Exp. (76), e50170 (2013).
- Kumar, P., et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature. 448, 39-43 (2007).
- Roshan, R., et al. Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice. RNA. 20, 1287-1297 (2014).
- Kaur, H., Wengel, J., Maiti, S. Thermodynamics of DNA−RNA Heteroduplex Formation: Effects of Locked Nucleic Acid Nucleotides Incorporated into the DNA Strand. 生物化学. 47, 1218-1227 (2008).
- Griffiths-Jones, S., Grocock, R. J., Van Dongen, S., Bateman, A., Enright, A. J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research. 34, D140-D144 (2006).
- Kaur, H., Babu, B. R., Maiti, S. Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LNA). Chemical Reviews. 107, 4672-4697 (2007).
- Guyenet, S. J., et al. A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia. J. Vis. Exp. (39), (2010).
- Bergen, J. M., Park, I. -. K., Horner, P. J., Pun, S. H. Nonviral Approaches for Neuronal Delivery of Nucleic Acids. Pharmaceutical Research. 25, 983-998 (2008).
- Zou, L. -. L., Ma, J. -. L., Wang, T., Yang, T. -. B., Liu, C. -. B. Cell-Penetrating Peptide-Mediated Therapeutic Molecule Delivery into the Central Nervous System. Current Neuropharmacology. 11, 197-208 (2013).
- Hwang, D. W., et al. A brain-targeted rabies virus glycoprotein-disulfide linked PEI nanocarrier for delivery of neurogenic microRNA. Biomaterials. 32, 4968-4975 (2011).
