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

Here we demonstrate the use of a cell penetrating peptide with a modified antisense oligonucleotide to specifically knock down micro RNA in the brain of mice. This was achieved by designing an antigo against May 29. In this study, we have used antigo males to target microRNAs lock nucleic acid modifications.

That is LNA modifications were incorporated at five different positions. In this, an incorporation of LNA modifications enhances its binding affinity to the micro RNA. Also, it gives nucleus resistance and therefore enhances the microRNA targeting efficiency.

Complexes of the ANTIGO MAR with the peptide was prepared in vitro and injected into the mice through the tail vein. The knockdown of the micro RNA resulted in behavioral changes that suggests the involvement of this microRNA in ataxia three consecutive injections were sufficient to induce noticeable change in gait after treatment, brain tissue was collected and the knockdown of the microRNA was confirmed. CPPs or self penetrating peptides have recently emerged as attractive carriers for nucleic acid delivery.

Here we have used one such arginine rich neurotropic peptide that can undergo charge based complexation with the LNA, which upon administration into the systemic circulation, will reach its designated target site that is neuronal cells in this case and ensure proper release of the LNA into these cells. The sequences of the ME 29 family members were retrieved from mease and the 16 MER antisense were synthesized with lock nucleic acid modifications at five evenly spaced positions to prepare complexes. The peptide and antigo solutions of appropriate concentrations were prepared separately in sterile 10%glucose.

Under aseptic conditions. The Antigo solution was slowly added dropwise to the peptide solution with gentle stirring. Slow addition, and gentle stirring are critical for formation of mono dispersed complexes whose size is in the tric scale.

The antigo was added in three equal parts each time, allowing a minute for mixing, followed by a minute of incubation without mixing. Rapid addition can lead to aggregation of the complexes and precipitation. Antigo complex is now ready for injection.

The complex was administered through a tail vein injection. The mice were returned to the same cage. Behavioral assays for ataxia were carried out daily, three hours post-injection.

The mouse was held by its tail, not allowing it to touch any surrounding objects. Notice the way hind limbs are splayed out for 10 seconds. In the ledge test, the mice were required to walk along the thin ledge of the cage.

The Mice walk through a thin channel leaving footprints with inked feet on a strip of thick absorbent paper ink only the hind feet. To avoid confusing data From four limbs, let us see The effect of the antigo treatment. We isolated the brain and other tissues from the treated and controlled mice.

We found that the R VG peptide delivered a fluorescently labeled siRNA specifically to the brain, but not to other tissues like liver. A non neurotropic control peptide RVM delivered the siRNA to the liver. Mice treated with ANTIER 29 showed defects in hind limb.

Clasping Controlled Mice treated with a scrambled anme could negotiate the ledge, but after treatment with anme against MARE 29, the mice showed mild defects in this assay. In the footprint Assay, there was a striking shortening of step length in the treated mice. Additionally, We confirmed through QR tpcr that levels of mid 29 A and mid 29 B were downregulated For development of micro NNA based therapeutics.

Brain specific delivery of anti microRNAs is a challenge due to blood brain barrier. Using the technique presented here, we can avoid the conventional delivery method of anti microRNAs to the brain. This method makes knockdown of microRNAs technically feasible and increases tissue specificity.