Long non-coding RNAs and Parkinson’s DiseaselncRNA silencing using LNA™ GapmeRs
Assoc. Prof. Antony Cooper
Associate Professor Antony Cooper
is the divisional head of Neuroscience in the Garvan Institute of Medical Research (Sydney, Australia). His lab’s research focuses on the area of neurodegenerative disease, and specifically understanding the basis of Parkinson’s Disease.
How did you come to be interested in long non-coding RNAs?
We are working on Parkinson’s Disease and trying to understand the causes of this disease. We are interested in Neurogenomics - identifying the genomic and transcriptomic contribution to neurodegenerative diseases.
We realized that if you are only looking at the mRNA transcripts associated with the ~20,000 protein-coding genes, then you are probably overlooking the 50-100,000 long non-coding RNAs in the human genome, so you are only getting part of the story.
We looked at genome-wide association studies that have identified sections of the genome that are associated with Parkinson’s Disease. Interestingly, many SNPs associated with Parkinson’s Disease and many other diseases, are not in protein-coding exons – in fact they are 50, 100 or 200 kb away from the nearest protein-coding gene.
We found by looking at RNA-seq data that there were some RNA transcripts located near disease-associated SNP positions outside protein-coding exons. So we investigated these long non-coding RNAs (lncRNAs). We have gone on and characterized a number of interesting lncRNAs, whose regulation changes early in the development of Parkinson’s Disease.
What is the background for your current project involving LNA™ GapmeRs?
We identified a particular lncRNA encoded a significant distance from the nearest protein-coding gene. This specific lncRNA is very interesting because it’s expression level goes down early in susceptible brain regions of Parkinson’s Disease patients - before pathology is observed in that part of the brain.
Our in silico bioinformatics analysis indicated that this lncRNA had the potential to regulate a number of protein-coding genes, raising the possibility that the expression of these target genes may be regulated by the lncRNA. Some of these target genes have very direct ties with Parkinson’s Disease. So that is very exciting.
We wanted to achieve an efficient knockdown of this lncRNA and then test the expression levels of the potential target genes by qRT-PCR.
What, if any, was your previous experience with long non-coding RNA inhibition?
We designed our own antisense oligonucleotides to try to knock down the lncRNA. The ones we designed did not have the same LNA™ modifications as Exiqon’s LNA™ GapmeRs. Also, the problem with lncRNA is they can have a lot secondary structure within the molecule. That may mean that some lncRNAs are more difficult to target with antisense oligos than others. This differs from mRNA which are somewhat more linear so any one mRNA may be as accessible as another. So we did not have great success with the antisense oligonucleotides we designed, in terms of percentage of lncRNA knockdown.
When researchers try to use siRNA or shRNA they might get 70% knockdown, and many people are quite content with that – perhaps choosing to overlook the 30% that still remains. But we did not want to ignore the 30% so that is why we approached Exiqon to try the LNA™ GapmeRs.“We were not content with 70% knockdown using siRNA and other methods. We wanted to push it as far as we could.”
How did you perform the experiment and analyze the results?
Exiqon designed LNA™ GapmeRs for the lncRNA sequence we submitted, then synthesized and delivered them. The LNA™ GapmeRs have been very successful for us. The fact that we are buying a repeat order speaks of our satisfaction with the product.“We have been very pleased with the LNA™ GapmeRs designed by Exiqon.”
We started off using them in HeLa cells as we wanted to use the easiest and simplest model to ask the question “can this lncRNA regulate these putative target genes?” In the HeLa cells we used the Lipofectamine+ transfection reagent and got >95% knockdown of the lncRNA, which is quite impressive - pretty close to a knock-out.
We’ve also used the LNA™ GapmeRs in the neuroblastoma cell line SH-SY-5Y with Lipofectamine+ transfection reagent. With these cells, we typically got 70 – 80% knockdown. As these cells differentiate they become more neuronal in character that also affects how well they transfect. “In HeLa cells we got >95% knockdown – pretty close to a knock-out. We also used neuroblastoma cells.”
In HeLa cells, we looked at a few of the target genes by qRT-PCR and we found that when the lncRNA is knocked down by the LNA™ GapmeR, the target genes are also down-regulated.
How do feel about the results so far?
To be honest, I am always someone who – if I find a good product, which I believe Exiqon’s is - I tell other people about it. An indication of how good Exiqon’s product is, is that I have suggested to a number of my colleagues that, if they are having trouble of getting efficient knockdown of whatever transcript they are interested in, they should consider contacting Exiqon. The combination of Exiqon’s designs and the LNA™ GapmeRs provided can likely improve their knockdown efficiency.“I have suggested to colleagues that they should use GapmeRs”
What do you know about the location of your lncRNA? Do you know if it’s located mainly in nucleus or cytoplasm?
Data from studies done by other people suggests that the lncRNA may be enriched in the nucleus. We cannot exclude that it may also be present in the cytoplasm, but a nuclear location would make sense as it would be consistent with our current thinking that this lncRNA may affect the expression of its target genes.
What do you find to be the main benefits of the Exiqon LNA™ GapmeRs from Exiqon? I think there are three factors which really contribute to the success of the LNA™ GapmeRs: Exiqon’s design algorithm which identifies the regions of the lncRNA most accessible to antisense oligonucleotides, the LNA™ modifications, and the RNase H mechanism of action, which gives a catalytic advantage because the LNA™ GapmeR is available for subsequent rounds of binding and cleavage. All that comes together to give you a much more efficient knockdown and a better way of assessing what are the consequences to the cell when the transcript is at a much lower level.
The end result is what is important, and the LNA™ GapmeRs clearly gave us better results than the other approaches that we tried, and that is all that matters.“LNA™ GapmeRs clearly gave us better results than the other approaches that we tried, and that is all that matters.”
What are the next steps in the project?
Our work now has expanded to look at every part of the genome, to look for genes that are associated with Parkinson’s Disease and ask what transcripts might come from these other regions of the human genome associated with Parkinson’s Disease.
What is fascinating about the lncRNAs is that ~30% of them are primate/human specific. That is exciting because we are looking at factors contributing to human diseases, but the challenge is that most animal models do not produce many of these lncRNAs.
To address this we are collaborating to produce human neuron cells from Induced Pluripotent Stem (IPS) cells so that we can test what are the consequences to the neuron if we knockdown the lncRNA. Differentiation is a slow process and it is a challenge to achieve and maintain a good level of knockdown over a longer period of time.