A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of 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

MicroRNAs have emerged as novel therapeutic targets due to their universal role in the regulation of gene expression and direct evidence for involvement in disease. MiRNAs are being actively explored for their potential as drug targets^1,2. Further, alterations in miRNA expression are associated with several diseases³ and simulation of these changes by artificial perturbation of miRNA expression can be used to study the cellular pathways involved in disease manifestation. Tissue specific delivery of miRNA targeting drugs is currently a major challenge for miRNA based drug development. Antagomirs and miRNA mimics are promising agents for perturbing....

Protocol

Note: All the procedure including animal subjects have been approved by Institutional Animals Ethics Committee (IAEC) at the Institute of Genomics and Integrative Biology, New Delhi (IGIB/AEC/10/2013). This protocol is specifically adjusted for targeted delivery of Antagomir-29 in the brain and knockdown of miR-29.

1. Antagomir Design Strategy

1. Retrieve the mature miRNA sequence from miRBase¹¹ (http://www.mirbase.org/).
2. Retrieve the sequences of related miRNA family members using the “Gene Family” link in the miRBase record
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Results

Using the procedure presented here, complexes of 50microgram fluorescently labeled oligonucleotide (FLO) and ~850microgram RVG peptide of 1:15 molar charge ratio (FLO : peptide) were prepared and injected only once through tail vein. Complex of non-neurotropic Rabies Virus Matrix (RVM) peptide and FLO was used as a delivery control. Next day, mice brain and liver were isolated and single cell suspensions were prepared. Cells were observed under microscope for green fluorescence. FLO-RVG complex was successfully delivered.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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