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

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

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! 🥳

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.

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

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

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

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

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!

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.

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).

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.

So likely this will underestimate off target options. However, BLAST will give homology to all transcripts, also those not expressed in brain or e.g. only in utero. RNAsequencing can identify direct and indirect off target effects, but this needs to be done in a relevant (human neuronal) cell line

ASOs can also cause off target effects, either through hybridization to similar sequences, or by eliciting immune responses via specific motifs. For off-target binding BLAST analysis can be performed, though this will not take into account that G-U can also bind.

Tests for cell viability or apoptosis can also be used, e.g. caspase-3 cleavage (apoptosis marker), LDH excretion (cell membrane marker) or alamarblue analysis (assessment of metabolic activity).

The toxicity is in part due to chelation of Ca++ and Mg++ ions and this is a dose related effect. Supplementing Ca++ and Mg++ in the formulation can reduce the side effects. MEA (multielectrode assay) analysis can also be used to detect an inhibition of neurotransmission.

Different techniques exist to detect the ASOs, e.g. in situ hybridyzation, miRNA scope, using fluorescent or radioactive tags (though these can change the properties of the ASOs) and antibodies that recognize the backbone or chemical modifications.