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Silencing of genes linked to neurodegenerative diseases

mRNA silencing in vitro and in vivo using LNA™ GapmeRs

Dr. Anastasia Khvorova
Dr. Anastasia Khvorova works at the RNA Therapeutics Institute at the University of Massachusetts Medical School. Her lab focuses on the development of novel oligonucleotide-based therapeutics, as well ways to improve delivery and tissue distribution. They have been using LNA™ longRNA GapmeRs to silence mRNAs linked to neurodegenerative diseases.


What is the background for your current project involving LNA™ GapmeRs?

We became interested in using LNA™ longRNA GapmeRs as a positive control for the study of gene function, both in vitro and in vivo mouse and rat animal models. Currently we are working with LNA™ GapmeRs on two projects, aiming to silence two different genes which are valid therapeutic targets for neurodegenerative diseases.


For the first target, how did you perform the in vitro experiments and analyze the results?

For our first target, we were able to identify from the literature some “hotspots” within the target mRNA that are known to be more accessible to antisense oligonucleotides. We tested three different LNA™ GapmeRs targeting these hotspot regions (using lipid mediated transfection of HeLa cells, and measuring the target mRNA levels by QuantiGene branched DNA assay). By testing a range of different concentrations of the LNA™ GapmeRs we were able to measure the IC50 (the concentration of LNA™ GapmeR required for 50% knockdown), and we were very surprised that the IC50 of all three LNA™ GapmeRs was 40 pM or lower. One LNA™ Gapmer was able to achieve 90% knockdown at a concentration of just 20 pM. The IC50 values indicated that these LNA™ longRNA GapmeRs were much more potent than what we had previously seen with other LNA GapmeRs targeting other genes as well as other antisense chemistries e.g. MOE oligonucleotides where typically we observe an IC50 in low nM range.

Figure: RNAi Max mediated dose response in HeLa cells. % target mRNA normalized to untreated.



Have you tried delivering the LNA™ longRNA GapmeRs in the absence of transfection reagent?

We have tested the LNA™ longRNA GapmeRs in HeLa cells, CHO cells and in mouse primary cortical neurons by passive uptake in the absence of transfection reagent (gymnosis). We observed that the LNA™ GapmeRs were very efficiently internalized – after 24 hours the cytoplasm was fully loaded with the LNA™ GapmeR (observed using a fluorescently labeled LNA™ GapmeR). Interestingly gene silencing was efficient in CHO cells and Primary Neurons but less so in HeLa cells. This confirms the importance of cellular biology on rate of uptake. In CHOs and Primary neurons, ~ 2.5 uM concentration was necessary to induce 80% silencing with IC50 values in 600 nM range after 72 hours treatment. Some slight toxicity was observed at highest concentations used. (3 µM).

Figure: Passive uptake of LNA™ longRNA GapmeRs in HeLa cells. Blue = nucleus (DAPI), Red = 2 uM LNA™ longRNA GapmeR (fluorescently labeled). Imaged using confocal microscope. Efficient passive internalization observed in 99% of cells with preferential cytoplasmic localization.



What has been your experience so far using LNA™ longRNA GapmerR in vivo ?

For the first target, we are starting to assess the efficacy of the LNA™ GapmeRs in vivo , in a mouse model. We have tested a number of other types of chemically modified oligonucleotides designed to enable delivery in the brain, and in comparison the LNA™ GapmeRs look very promising. We have performed some initial experiments where we have injected a single dose of fluorescently labeled LNA™ GapmeR intra-cranially, to assess the biodistribution. We have used quite a high amount of LNA™ GapmeR (2 nmoles) but the results are very promising. We observed that 24 hours after the injection the whole brain was stained, indicating that the LNA™ GapmeRs are delivered highly efficiently in vivo throughout the brain after direct injection.

Figure: In vivo distribution of LNA™ longRNA GapmeR in mouse brain 24 hours following a single intra-cranial injection of 2 nmoles fluorescently labeled LNA™ longRNA GapmeR. Good distribution was observed throughout the mouse brain and into cortical and hindbrain structures.



For the second target, were you able to adopt the same strategy?

For our second target, we were not able to identify any known accessible sites on the target mRNA from the literature, so we decided to let Exiqon identify these with their design algorithm. We screened around 40 different LNA™ GapmeRs designed by Exiqon. This time we tested the LNA™ Gapmers in vitro using CHO cells and RNAi Max. Overall we obtained a very good hit rate – all but one of the LNA™ GapmeRs did knockdown the target mRNA and several of the LNA™ GapmeRs were extremely potent with IC50s of around 25 nM. Some compounds showed significant toxicity but we were able to select several nontoxic gapmers to move forward. The best candidates were tested in CHO cells and demonstrated very efficient gymnotic uptake and silencing with IC50 on average in 800nM range after 72hr incubation. Next we plan to test the best candidates from our screen in primary neurons by passive uptake.

Figure: LNA GapmeR™ efficacy in CHO cells upon gymnotic delivery (no lipid).



For the second target, did you face any particular challenges?

We did observe that the negative control LNA™ GapmeR resulted in some toxicity. As an additional control, we also measure the effect of the LNA™ GapmeRs on a housekeeping gene. For some of the LNA™ GapmeRs [at 100 nM in the presence of the lipid] against our second target, we observed knockdown of the housekeeping gene as well as the target mRNA, which may be indicative of toxicity. Since we had tested a number of different LNA™ GapmeRs we were able to select those which show potent knockdown of the target mRNA, but with no effect on the housekeeping gene or toxicity.


What are the next steps in the current project and how do you plan to perform them?

Next we plan to test different injection regimes in our in vivo mouse model for the first target, using multiple injections and different concentrations of the LNA™ GapmeR. We also plan to measure the knockdown of the target mRNA in vivo , to demonstrate that not only do we get efficient biodistribution of the LNA™ GapmeR, but that we are also able to inhibit the target gene in vivo .


How do you view the potential of LNA™ longRNA GapmeRs, in particular for in vivo applications?

LNA™ longRNA GapmeRs are superior to other chemistries in terms of in vivo biodistribution. With MOE oligonucleotides we need to use higher doses and repetitive administration to achive a biodistribution pattern similar to that observed with the LNA™ GapmeRs using a single and much lower dose.
Our preliminary results indicate that LNA™ GapmeRs in vivo might be active without the use of pumps to deliver the oligonucleotides. This experiment is on-going and if successful, then LNA™ GapmeRs will provide a major advance and enable in vivo functional genomics studies in the field of neurodegenerative disease.


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