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 miRNA levels^4–6. 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.

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
   (http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009 ).
3. Reverse complement the sequence: http://www.bioinformatics.org/sms/rev_comp.html provides a convenient tool to reverse complement sequences.
4. Mark the positions to be modified by LNA using the following guidelines:
   1. Choose Thymine residues placed 3-4 bases apart for the LNA modification, since LNA pyrimidines are more stable than purines¹².
   2. Choose Cytosine residues separated by 3-4 residues if previous step produces less than five modifications.
   3. Prioritize the five modifications to the 5’ end of the antagomir, since this avoids the seed sequence.

2. Antagomir-neuropeptide Complex Preparation

Note: This is the most critical step in the protocol and it needs standardization. To standardize the protocol any fluorescently labeled oligonucleotide (FLO) can be used in place of the antagomir⁹. The peptides should be of high purity (HPLC grade, 98% purity).Sequences and charge of peptide and antagomir are given in Table 1.

1. Decide the number of moles of antagomir to be injected based on molar charge on antagomir, its toxicity and weight of the mouse. Calculate the number of moles of peptide to be injected based on molar charge ratio of antagomir : peptide.
   NOTE: Standardize molar charge ratio of antagomir : peptide for toxicity and effective delivery in the mouse brain. Use different molar charge ratios of fluorescently labeled oligonucleotide: peptide for standardization. We found that Antagomir-29 and control were both toxic at 4microgram/gram body weight of mouse.  Antagomir-29 and control were used at 2microgram/gram body weight, but this effective concentration is likely to vary for each microRNA.
2. Dilute the peptide and antagomir in separate microfuge tubes from stock solutions with sterile 10% D-glucose to the desired final concentration as calculated in step 1.
3. Keep the microfuge tube with the peptide solution on a vortex at moderate vortexing speed.
4. Add 1/3^rd of the antagomir solution to the peptide solution tube, slowly, drop wise, while it is mixed thoroughly on a vortex mixer.
5. Continue mixing of solutions on the vortex for next 1 min and then allow the mixture to stand for 1 min.
6. Repeat the step 4 and 5 two more times to mix the remaining antagomir solution with the peptide solution in the same microfuge tube. The slow addition is critical for the formation of mono-disperse complexes that are effective in transfection. Rapid addition can lead to aggregation of the complexes and their precipitation.
7. Incubate the complex for 30 min at RT without vortexing. During this time of    incubation, acclimatize the mice to the lab where injections will be performed.

3. Tail Vein Injection of Antagomir-neuropeptide Complex

1. To restrain the animal, place the mouse in a restrainer or decapicone of proper size.
2. Clean the surface of the tail using cotton swab pre-soaked in warm water (about 40°C). This will promote vasodilation and increase the visibility of the vein.
3. Approach the tail with a small volume (0.5 – 1.0 cc) insulin syringe at a 15-20° angle. Be careful not to introduce any air into the syringe. Start at the distal portion of the tail. Inject the complex. Remove the needle and apply an antiseptic swab directly to the injection site (approximately 5-10 sec) to stop any bleeding.
4. Replace the mouse in an individually ventilated cage in the group of 3-5, with the corncob and tissue paper as enrichment. Do not keep uninjected and injected mice in same cage.

4. Behavioural Assays

Note: Keep the time interval of 3-4 hr in between injection and behavioural assays to allow recovery after injection. Bring the mouse to a quiet room.  Do not disturb the animals during this period of acclimatization.

1. Acclimatize the mouse to the new room for 30 min and then start the following behavioural assays. Keep a gap of 10 min in between each behavioural assay.
2. Hindlimb clasping:
   NOTE: This test indicates motor dysfunction and neurological impairment.
   1. Lift the mouse gently by holding near base of the tail in the clear background.
   2. Check the hindlimb position for 10-15 sec. Wild type mouse splays hindlimbs away from abdomen, while an ataxic mouse tends to retract one or both hindlimbs towards the abdomen more than 50% of time suspended¹³.
   3. Return the mouse to the cage
3. Ledge test:
   1. Lift the mouse gently by holding the tail and keep it on the ledge of a cage.
   2. Observe the mouse specifically at the corner of the cage. Wild type mouse can pass the corners easily without fear and losing the balance. Ataxic mouse lose its balance, freeze or shakes while walking along the cage ledge and at the corners.
4. Mouse foot print assay:
   NOTE: For this assay keep the absorbent sheet ready, on the floor of a narrow runway (~70-cm-long, ~5-cm-wide with ~5-cm-high walls.)
   1. Hold the mouse gently by one hand and apply ink on the hind limbs by a brush.
   2. Place the absorbent sheet on the floor of a narrow runway.
   3. Allow mouse to walk or run over an absorbent sheet in a straight line in a runway from one end to other.
   4. Repeat the process two more times with each mouse using fresh absorbent sheet.
   5. Measure the distance between two consecutive steps in the forward movement. Do not include first and last few footprints where the animal is just initiating and finishing its run, respectively.

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