En simpel alternativ til Stereotaktisk Injection til Brain Specifik Knockdown af miRNA
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
MikroRNA'er er dukket op som hidtil ukendte terapeutiske mål på grund af deres universelle rolle i reguleringen af genekspression og direkte beviser for involvering i sygdom. MiRNA bliver aktivt udforsket for deres potentiale som lægemiddel rettet mod 1,2. Endvidere er ændringer i miRNA ekspression er forbundet med adskillige sygdomme 3 og simulering af disse ændringer ved kunstig forstyrrelse af miRNA ekspression kan anvendes til at studere cellulære veje, der er involveret i sygdommen manifestation. Vævsspecifik levering af miRNA målretning narkotika er i øjeblikket en stor udfordring for miRNA baseret lægemiddeludvikling. Antagomirs og miRNA efterligner er lovende midler til forstyrrende miRNA niveauer 4-6. Særlige funktioner, der forbedrer deres specificitet og effekt har dog skal indarbejdes i udformningen af antagomirs, før de kan bruges til in vivo forstyrrelse af miRNA udtryk.
MikroRNA'er er særligt relevante som mål i øjeblikket uhelbredelige neurodegenerative og neuro-udviklingsmæssige sygdomme. Blod-hjerne-barrieren udgør en begrænsning af leveringen af antagomirs i hjernen. Stereotaktiske injektioner er meget udbredt i gnavermodeller at levere molekyler til bestemte steder i hjernen 7. Det kræver dygtighed, omfattende investeringer i instrumentering og tid. Stereotaktiske injektioner er invasive, indebærer kirurgi, forårsager mindst mindre skade, og er begrænset til lokal levering. Anvendelsen af cellepenetrerende peptider med en præference for målretning neuroner kan imødegå disse begrænsninger, da de kan leveres gennem trans-kar rute, men bryde blod-hjerne-barrieren. Et sådant peptid afledt af rabiesvirus glycoprotein (RVG), er tidligere blevet anvendt til at levere siRNA mod japansk encephalitis virus i mus 8. Vi fandt, at bruge peptid til antagomir levering, kan miRNA være effektivt slået ned i musen hjernen 9.
Indholdsproduktion "> Den anden store udfordring af miRNA knock-down skyldes den lille størrelse af miRNA og tilstedeværelsen af nært beslægtede sekvens isoformer. Vi tager eksemplet med MMU-miR-29 familie, som består af tre nært beslægtede isoformer, miR-29a , b og c. Antagomirs generelt også ændres langs rygraden til at øge deres stabilitet og gøre dem modstandsdygtige over for angreb fra nukleaser. Locked Nucleic Acids (LNA) tilbyder en yderligere fordel, at de forbedrer termisk stabilitet og endda føre til at målrette nedbrydning over og ud sterisk hindring 10. Introduktion modifikationer langs rygraden kan være effektiv, men dyrere. Vi har tidligere set, at modifikationer udover et optimalt antal ikke yderligere at forbedre effektiviteten. Udformningen af antagomir involverer derfor den optimale ændring af antagomir.Til kompleks antagomir ikke-kovalent med neurotropisk peptid, en ladet hepta- til nona-arginin forlængelse er brugt. D-argininrester anvendes, da de medfører højere stabilitet, da de ikke er modtagelige for spaltning med proteaser. Hepta- til Nona-arginin strækninger fungere som effektive cellepenetrerende midler, selv om de ikke giver celletypespecificitet. Ved kovalent binding RVG peptidet til nona-arginin linker, neurotropisk, cellepenetrerende peptid blev genereret. De positivt ladede rester af peptidet interagerer med negativt ladet nukleinsyre rygrad, til dannelse af komplekser. Disse komplekser kan anvendes til effektivt at transficere DNA eller RNA i dyrkede celler og in vivo i væv.
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…
Divulgations
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 |
| Other | |||
| Cotton | |||
| Warm water | |||
| Insulin syringes | |||
| Absorbent sheets | |||
| Ink | |||
| Brush | |||
| Antiseptic |
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