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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

This will require tools to measure oligonucleotides in wastewater and more studies to see how they are broken down. NB: tomorrow and Friday there won’t be #apaperaday as I’ll be traveling back home. Monday is for now an unknown but Tuesday-Friday will feature an @erdera.bsky.social GAM special

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Regardless I agree with authors that monitoring for environmental risks is important, especially now that oligonucleotides are also used in agriculture

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Alnylam has studied whether modified nucleotides used in siRNAs were integrated into the DNA and this did not happen if I recall correctly (was presented at #OTS2018 )

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Especially now siRNAs are also developed as pesticides looking for off target effects becomes more pertinent Authors discuss the risk for mutagenesis and integration of the oligonucleotides into the DNA. However this is a low risk as the oligonucleotides are designed to target RNA rather than DNA

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

They can be directly toxic due to the modifications. However authors argue the modifications were optimized to not be toxic in humans. Authors also worry for off target effects: blast analysis is usually done for human transcripts but not for all environmental organisms

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

They propose that nucleases may help to break them down (however the chemical modifications used have rendered them resistant to nucleases…). Authors consider different ways oligonucleotides can cause toxic effects.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Authors propose concentration steps and improving mass spectrometry to assess oligonucleotide levels. Authors argue that oligonucleotides can be hydrolysed and perhaps degraded by UV in wastewater.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Mass spectrometry for now is not sensitive enough Authors propose splint PCR as an option. However I can say from personal experience that this does not work well for bulky modifications, at least not equally well for all ASO sequences.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Back to the paper: authors discuss methods to detect the oligonucleotides. A PCR won’t work because the oligonucleotides are too short. Also I'm not sure if polymerase would work for all chemical modifications used

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

PMOs used to treat Duchenne patients in the USA are given every week at 30-80 mg/kg doses and excretion via urine is >90%. This amounts to 100-200 gram dosed per year for a 50 kg patient…

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

The delivery route plays a role here too: more excretion is expected for systemic treatment than for intrathecal delivery. Something authors do not consider: regimen and dose. While inclisiran is used by many, it is dosed as a low dose once every 6 months.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

From ADME (absorption distribution metabolism and excretion) studies it can be estimated how much oligonucleotides are excreted. Authors argue that amounts excreted by rare disease patients are negligible while inclisiran may result in kgs excreted amounts in the EU.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Authors introduce different oligonucleotide modalities: aptamers, single stranded antisense oligonucleotides (ASO) and siRNA. 60 different chemical modifications have been published, however only 8 chemical modifications are present in approved oligonucleotide treatments

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

The oligonucleotides will end up in the wastewater after they are excreted via urine and feces of treated patients Authors argue that it is unclear whether this poses an environmental risk or not. They discuss this in this perspective paper

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Authors introduce there are now 25 approved modified oligonucleotide therapies, mostly for rare disease but inclisiran is more generally used to treat cardiovascular disease. These oligonucleotides are chemically modified and contain 20-60 nucleotides

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 21h

Final #OTS2026 #apaperaday is a different type of paper than normal: a perspective on environmental risks of modified oligonucleotide therapies by Hofstetter et al in environmental science and technology Doi 10.1021/acs.est.6c05885

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

The annual Oligonucleotide Therapeutics Society cookie exchange has taken place at last. 🇨🇦🇳🇱#OTS2026 Thanks Masad for the Canadian cream cookies. Enjoy the stroopwafels!

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

Also: something not working is also a result and I appreciate authors sharing their results because they showcase that even though you expect a protein to be functional, does not mean that it will be.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

Thus they assumed the protein might be functional. However now we know the skipped protein is not cleaved. Authors stress that even though the approach of exon 19 skipping will not be therapeutic the study does give insight in protein processing

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

Authors discuss that when skipping inframe exons assessing if the resulting protein is functional is crucial. Agreed! Authors outline they had performed in silico analysis revealing exon 19 encodes a small part of a stealth domain. But the protein has 4 such domains & this is the least conserved

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

The wild type and exon 19 deleted proteins were expressed. However the beta subunit was not cleaved for the exon 19 deleted protein, which explains why it is not functional: without cleavage the protein cannot be assembled and you get no functioning enzyme.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

This resulted in a minor, not significant increase in hydrolase activity. It did not increase protein levels or reduce LAMP2, a biomarker increased in patients. To assess if exon 19 deleted protein was functional authors made overexpression constructs for wildtype, mutated & exon 19 deleted protein

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

A frame shifting deletion in exon 19 is the most common variant. As exon 19 is in frame authors wondered whether skipping it could bypass the variant while still giving rise to a functional protein. Authors designed and tested ASO to induce exon 19 skipping in patient fibroblasts.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

Without these tags hydrolases do not go to their proper location and are degraded. GlcNAc-PT is a large protein that consists of an alpha beta and gamma subunit. The alpha and beta unit are encoded by the same gene and the protein is then cleaved to form the units that assemble into an enzyme

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

MLII is a lysosomal storage disease characterized by developmental delays and bone malformations amongst others. Patients generally die in the first decade of life MLII is caused by lack of the GlcNAc-PT enzyme which is involved in tagging hydrolases with M6P groups.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 13/10/2026

#apaperaday #OTS2026 special continues with a pick by Goncalves et al from orphanet journal of rare diseases on testing ASOs for exon skipping for mucolipidosis type II (ML II). Doi 10.1186/s13023-026-04430-2

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

I appreciate the efforts made to elucidate the fundamental workings but agree with the authors that single ASO treatment will not be enough and that developing many ASOs will be difficult.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

Hematopoietic differentiation was partially improved. Authors discuss that this is because they now address missplicing of only one gene. The suggest a combination approach targeting multiple genes could be developed.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

Authors designed ASOs and the one targeting motif 1 worked best to reduce poison exon inclusion and increase EZH2 protein expression. This also increased specific histone methylation. The ASO had no effect on the missplicing of other transcripts

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

They showed that mutated SRSF binds to this motif with a pulldown experiment and that NMD proteins and splicing factors can bind to it when the mutant SRSF2 is expressed but not when the wild type SRSF2 is expressed

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

Authors generated a doxycycline inducible minigene to study things further. They showed 3 target motifs for mutated SRSF in the poison exons, of which the first was most important for exon inclusion

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

Authors validated their findings in an independent sample set. They also showed poison exon inclusion and reduced protein levels when they mutated wild type cells and less inclusion and more protein when they corrected mutated cells

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

Authors focused further analysis on EZH2, a transcript coding for a protein involved in histone methylation and thus impacting gene expression levels. The SRSF mutation caused inclusion of a poison exons, NMD and reduced levels of EZH2 protein. This caused also reduced hematopoietic differentiation

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

Authors compared splicing of mutated and wild type cells, seeing 843 cases of differential splicing, 16% of which were inducing nonsense mediated mRNA decay (NMD). They noticed that the mutation in SRSF2 caused the target motif in exons to change from GGWG to CCWG.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

In many blood cancers splicing factor mutations are found, causing misregulation of splicing to increase oncogene and/or reduce tumor suppressor gene expression. Which genes are misregulated depends on which splicing factor is mutated and how. Authors focused on SRSF mutations in AML patients

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 12/10/2026

#apaperaday #OTS2026 special with a paper by Islam et al from genes and development on using antisense oligonucleotides (ASO) to modulate oncogenic EZH2 missplicing. Contains a lot of fundamental work as well! Doi10.1101/gad.353628.126.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

Authors discuss that future work should include human IPSC lines as the mouse model is rather artificial and also has a very quick onset and loss of motor neurons so seeing treatment effects might be challenging Third #OTS2026 special coming on Monday

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

No improvement on rotarod performance was seen compared to the control ASO. Also no reduction of neurofilament light could be observed

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

Then authors injected the doxycycline stop to induce pathology mouse model with the ASO and a control ASO via intracerebroventricular injection. Also in the mouse this reduced ataxin-2 protein and TDP-43 aggregates but not total TDP-43 levels

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

In a cell line for induced TDP-43 aggregation the ASO could reduce aggregation and stress granules. TDP-43 total levels were not affected. Control ASOs were used! 🥳

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

Authors wanted to increase this alternative splicing with antisense oligonucleotides (ASO) and did an ASO walk. ASO 8-2 was most efficent in cultured cells to increase the alternative splicing and reduce normal splicing and ataxin-2 protein production.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

This was accompanied by an increase in ataxin-2. Authors noticed ataxin-2 has an alternatively spliced transcript where exon 8 is elongated by 47 nucleotides This causes a frame shift so these transcripts cannot produce ataxin-2

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

This increase enhances TDP-43 related toxicity . Authors thus speculated if Ataxin-2 down regulation could be therapeutic for ALS First they studied the relation between ataxin-2 and TDP-43 in a mouse model where stopping doxycycline treatment will trigger TDP-43 accumulation and motor neurons loss

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

Amyotrophic lateral sclerosis (ALS) is a severe progressive lethal disease caused by loss of motor neurons. TDP-43 aggregation & phosphorylation & stress granules are hallmarks of the disease. Ataxin 2 is a protein present in stress granules and levels are increased in spinal cord of ALS patients

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 09/10/2026

#apaperaday From Denver where today we have N of 1 collaborative ancillary meetings today and tomorrow we have our annual meeting prior to #OTS2026 Today’s pick is by Heinsinger et al in Neurology of Disease on splicing modulation of ataxin 2 as a strategy to treat ALS Doi 10.1016/j.nbd.2026.107582

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 08/10/2026

I appreciate the overview and the recommendations! Hope to see many of you in Denver!

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 08/10/2026

Then move to more relevant models, e.g. co-cultures or more differentiated neurons. Then finally one can use more complex cell models like brain organoids to test the effects of chronic exposure. Always include positive and negative controls!

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 08/10/2026

Start with general toxicity assays (viability), then do cell type specific assays (calcium oscillations in neuron, immune toxicity in astrocytes and glia cells). Do dose responses and validated with biological replicates and multiple cell lines.

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 08/10/2026

Immune stimulation happens via TLR9 activation. In CNS only microglia and astrocytes appear to express TLR9, but at varying level. Authors end with a recommendation for safety/toxicity in vitro studies: set up a platform with known and validated cell lines (e.g. responsive to toxic ASOs).

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Annemieke Aartsma-Rus @oligogirl.bsky.social · 08/10/2026

Also here variability is an issue as there will be differential expression also between biological replicates. So interpretation is not always easy. For immune stimulatory effects, cytokine release can be analysed in PBMCs or BJAB cells.

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