Intrathecal siRNA injection for spinal neuron gene knockdown - Your experiences and tips?

Hi all,

I want to knock down some genes in superficial dorsal horn neurons. I’m avoiding using viral vectors right now and siRNA injection intrathecally looks like an attractive option.

I know that this has been done before, with varying success based on the gene of interest and the transfection method:

What are you experiences and thoughts? I’m going to use siRNA from Dharmacon in mice.

Some questions:

  • How many injections (how many days?)
  • How many days until I should expect knockdown (3 days, 7 days?)
  • What delivery method (PEI, Lipofectamine, naked siRNA?)
  • What dose?

I know there aren’t definitive answers to these questions, and I’m going to have to just try stuff and determine empirically, but if anyone has some standard parameters that have worked for them, I’m all ears.

While I have never done intrathecal injections, I do a lot of nerve injections and we usually allow around 2 days for retrograde transport, after that we perform our injury and it works most of the time. The key factor is how efficient your siRNA is. In some cases we have done several injections every other day and this has achieved the knockdown.

About the delivery method we modify the siRNA with a thiol group on the 5’ end so we can link it with a transfection agent. We use a lipophilic molecule called penetratin and this gives us good results. No idea about the dose when doing it intrathecally.

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Thanks @lfqueme. This is interesting. Do you have more information on the penetratin method? Sciatic injections of siRNA sounds pretty great. @tberta used another effective and crafty method. He used an RVG peptide (which targets the ACh receptor) to facilitate the uptake of siRNA by sciatic nerve axons. See this paper;

I’ll let you know how the intrathecal goes.

Here is the paper from Davidson et al. that describes the technique to link the siRNAs to penetrating.

Also this is the paper that show the technique describing how we choose which of the siRNA in the smarpool we would modify with the thiol group. (as modifying every siRNA in the pool proves way too expensive and it’s not really necessary)

You can ask for the thiol modification to Dharmacon when you order them.

Marc-Andre Dansereau does siRNA injections intrathecally. I will send him a link to this discussion as he might have more insights on the technique.

Hey guys -

We mostly used Dicer-substrate siRNA for in vivo studies. More efficient, less immune activation. The group of M. Behlke has a lot of papers on that.

  • We injected 5 µg dayly, for two days. In rats,
  • We saw knockdown 24h and 5 days after the last injection
  • We didn’t try naked. We were told it wouldn’t work. PEI and Lipofectamine are pronociceptive by themselves. Induce interferons, I think. Might not be an issue for your work. We used a protein called transductin for a while, but they don’t make it anymore. There might be something similar out there. We now use lipidic nanoparticles from Precision Nanosystems, and it works great in our hands.

However, with intrathecal injection, we don’t have any proof so far that we are getting into dorsal horn neurons. We get the DRGs alright, but we never saw any siRNA reaching the L4-L6 dorsal horn.

Hope this help!

@MADansereau and @lfqueme

Thanks so much for the insights. This is very useful.

@MADansereau Interesting that you only get DRG and not spinal neurons. I’ll try with PEI and see what happens. I’ll report back here with my results.

In the past we have used PEI to knockdown gene in both DRG and SC.

Briefly, siRNA was dissolved in RNase-free water at the concentration of 1 μg/μl as stock solution, and mixed with polyethyleneimine (PEI, Fermentas Inc., Glen Burnie, MD), 10 min before injection, to increase cell membrane penetration. PEI was dissolved in 5% glucose, and 1 μg of siRNA was mixed with 0.18 μl of PEI. siRNA (2 μg) was intrathecally injected twice 2 and 1 day before the experiment.

To note, we were mostly targeting DRG in my experiments, but same protocol has been used in the lab to target SC.

For the siRNA, I suggest the Trilencer-27 siRNA kit from Origene. It contains three different siRNAs targeting different regions of the gene of interest and minimal interferon response (

Hi @MADansereau

I was looking for options for transfecting neurons in vivo, and I came across this video. And i was like, “I know him!”

Looks like those LNPs are pretty effective. I want to try to inject them directly into the spinal cord to see if I can target spinal neurons.

Would you recommend the LNPs? Looks like it’s a pretty involved system (read: expensive $$)