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.

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
<ol>
	<li>Retrieve the mature miRNA sequence from miRBase11 (<a href="http://www.mirbase.org/">http://www.mirbase.org/</a>).</li>
	<li>Retr...

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The authors have nothing to disclose.

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...

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 targets1,2. 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 miRNA levels4&#8211;6. However, special features that enhance their specificity and efficacy have to be incorporated into the design of antagomirs before they can be used for in vivo perturbation of miRNA expression.
MicroRNAs are especially relevant as targets in currently incurable neurodegenerative and neuro-developmental diseases. The blood-brain barrier poses a restriction to the delivery of antagomirs in the brain. Stereotactic injections are widely used in rodent models to deliver molecules to specific locations in the brain7. It requires skill, extensive investment in instrumentation and time. Stereotactic injections are invasive, involve surgery, cause at least minor injury and are restricted to local delivery. The use of cell penetrating peptides with a preference for targeting neurons can counter these limitations since they can be delivered through the trans-vascular route but breach the blood brain barrier. Such a peptide derived from the Rabies Virus Glycoprotein (RVG), was previously used to deliver siRNA against Japanese Encephalitis Virus in mice8. We found that using the peptide for antagomir delivery, miRNAs can be effectively knocked down in the mouse brain9.
The second major challenge of miRNA knock-down arises from the small size of miRNAs and the presence of closely related sequence isoforms. We take the example of mmu-miR-29 family which consists of three closely related isoforms, miR-29a, b and c. Antagomirs are also generally modified along the backbone to increase their stability and render them resistant to attack by nucleases. Locked Nucleic Acids (LNAs) offer a further advantage that they enhance thermal stability and even lead to target degradation over and beyond steric hindrance10. Introducing modifications all along the backbone can be effective but expensive. We have earlier seen that modifications beyond an optimal number may not further enhance the efficacy. The design of the antagomir therefore involves the optimal modification of the antagomir.
To complex the antagomir non-covalently with the neurotropic peptide, a charged hepta- to nona-arginine extension is used. D-Arginine residues are used since they confer higher stability as they are not susceptible to cleavage by proteases. Hepta- to nona-arginine stretches act as efficient cell penetrating agents, although they do not confer cell type specificity. By covalently linking the RVG peptide to the nona-arginine linker, a neurotropic, cell penetrating peptide was generated. The positively charged residues of the peptide interact with the negatively charged nucleic acid backbone, to form complexes. These complexes can be used to effectively transfect DNA or RNA into cultured cells and in vivo into tissues.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

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.

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...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

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.

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

a-simple-alternative-to-stereotactic-injection-for-brain-specific

Research

JoVE Journal

Neuroscience

12.6K Views.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. 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.