For day one, see here. For day two, see here.

Summary of day two

Highlights of yesterday were the longer talks - James Shorter’s talk on ‘reversing phase transitions’ (i.e. about prion-like proteins and methods of turning them back to their native conformation), and the talks by Christine Holt and Jik Nijssen’s about translation in axons. There were some very cool real-time videos of transcriptional and translational activity in axons. Matthew Wood’s talk about improving design & delivery of therapies was of interest - there’s more detail on that the talks later today.

It seems clear that misfolded protein aggregation is a major feature of ALS. However, this seems to occur to a bunch of different proteins - TDP-43, C9orf72, SOD1, FUS, all of which contain ‘prion-like’ domains (domains with specific amino acid ), and all of which have been observed in aggregated form in neurons of individuals suffering from ALS. These aggregates seem to self-catalyse and spread. Extrapolating a little, maybe they spread along the brain connectome during disease progression (c.f. Jill Meier’s talk from Day one). And, excitingly, maybe aggregation can be reversed (c.f. James Shorter’s talk). Similar effects are known for other neurodenerative diseases, e.g. alpha-synuclein in Alzheimer’s. What was less clear to me is how all these different proteins relate to ALS. Can each of them cause ALS on their own? Do they catalyse misfolding of each other? There’s also still a bit of a question as to whether this is a cause or a symptom. However, there is evidence that reversing the aggregation reverses degradation. Is ‘neurodegenerative disease’ really a spectrum of damage due to aggregation of a variety of different misfolded proteins?

(There’s also a category of talk I have real trouble with. They go like this: we know disease or treatment X is involved in disease in humans. So we’ll make organism Y (= mice, or rats, or zebrafish, or less problematically stem-cell derived neurons) and we’ll do thing Z to them (where Z = knock out or knock down the gene, or overexpress it, or insert a humanised genetic variant, or inject some prion-like protein, etc.). Then we’ll observe what happens. Look, things happen! This often seems problematic to me as a) it’s often purely observational and b) it’s often pretty unclear how results can or could ever be interpreted in terms of human ALS. Not all this type of work suffers from these problems, but from my fairly naive vantage point I often find it difficult to tell and wish speakers would spend more time on motivation.)

Today’s session which is largely about novel approaches to therapy and clinical trials, see the bottom of this post for a summary.

Sesson 7

Russell McLaughlin - why GWAS is (still) useful in ALS Russell is the winner of the ENCALS young investigator award. Begins with the genetic architecture of ALS. Genes affect the phenotype. Back in 90’s we might have thought about individual genes (like SOD1). As time went on more genes were discovered. A picture of genetic heterogeneity emerged. But now talking about polygenic risk. Individuals will carry different amounts of this depending on alleles carried. Brief cartoon of a GWAS. Manhattan plot for ALS, 8 loci appear above the ‘genome-wide significant’ line. van Rheenen et al Nature Genetics 2016. But there’s extra signal in the rest of the genome. E.g. polygenic risk scores for prediction. Plot showing small amounts of prediction that increase as you include more SNPs (but remains low overall). Conclusion: a lot more ALS genes remain to be discovered. What will we discover in future? Shows LDScore and GTEx expression, signal is in central nervous system. Comparison with schizophrenia. Cross-trait prediction: does genetic schizophrenia risk predict ALS? Answer is yes - schizophrenia genes predict ALS status (quite a bit better than ALS genes do). This seems quite specific to schizophrenia. Does that mean ALS / SCZ should co-occur? Says no (cartoon of a joint distribution of liability, with a vertical threshhold for SCZ and horizontal one for ALS, sets don’t overlap much). SNP chips don’t just predict disease. Population genetics of Ireland and Britain, showing a PCA-like analysis of Britain and Ireland. Iceberg analogy (what’s left to discover is below the surface).

Bart Swinnen (Leuven) - Pur-alpha provides a potential link between RNA toxicity and loss-of-function in C9orf72 ALS. Is there evidence for RNA toxicity? And what is its mechanism? Uses a zebrafish model. I didn’t listen to this, c.f. comments above.

Ziqiang Lin (KCL) MRI imaging reveals frontal cortial and cerebellar deficits in TDP-43(Q331K) knock-in mice. Still not very happy about this kind of talk. Anyway, tensor-based morphometry demonstrated brain volume changes in mutatnts. Primarily in frontal and cerebellular cortex. E.g. orbital cortex, volume decreased by 13%, P=0.027 (I didn’t catch the counts but they were something like 15 of each mouse).

Angela Genge (Montreal) - on clinical trials in ALS. Where are we? Riluzole approved 23 years ago (though not everywhere). Edaravone approved in Japan, South Korea and USA, being evaluated at Health Canada and the EMA. It took many years to progress to the pivotal trial, done in Japan. Final study was carefully crafted based on the results on the previous study to pick patients for the study. Many other drugs failed. What has been learned? Placebo arms can be a problem - because to prove efficacy the placebo arm has to progress. Need a way of stratifying or randomising to esnure placebos progress. Tolerability is a problem - side effects have important consequences on the ability to power a study; are we prepared for drugs that have a benefit but a side effect? Including patients too far into disease can be a problem - does this jeopardise results? Maybe should look for early / pre-disease individuals. Rapid and very slow progressors can also be a problem. And how to handle genetics? Classic way is: don’t do a genetic test unless there is a family history. But this can give false negatives. In speaker’s clinic, decided to screen every probably ALS patient for genes for which a drug is in development: SOD1 and C9orf72, and for FUS in under-30s. (Have never seen ‘a walking talking TDP-43 mutant’). Surprised to find half of SOD1 patients that went into trial, had no family history. Without screening would have missed 50% of patients. Says you have to ask family history twice. Because after 1st ask, they will go home and talk to family & physician and discover there was history of neurodegernative diseases, and fatal diseases that went un-diagnosed. Now new design principles. Try to force homogeneity in population & speed of progression; make effective use of biomarkers to monitor progress; set long enough duration of trial (difficult because it costs ~1 million per month of study during recruitment period); and look carefully at time from onset for enrollment. Now will talk about North American side. In Jan this year was a draft guidance document issued by FDA (maybe this). One piece of guidance is, if looking at something like intrathecal delivery (i.e. not something easy like a pill), also develop and test the delivery device at the same time. Also efficacy needs to be clinically meaningful on symptoms, function, or mortality (with a stress on the latter). Safety consideration: need adequate number of patients and duration - even for a re-purposed drug. Study design must be randomised, placebo controlled, double-blind, add-on, dose response, time-to-event. Efficacy endpoint: survival or function, but also could be new endpoint if appropriate. Functional endpoints should be early and frequent with safety assessments. Now the ‘Orphan Drug Act’. She notes we have lots of healthy mice running around, but not so many patients. Quotes one prominent member of this conference “I don’t believe anything in mice”. ‘Orphan drug’ is one intended for use in a rare disease. This means < 200K people in U.S. population. This means get strong financial incentives if the drug is ‘designated’ as orphan, and 7 year marketing exclusivity. Moreover can apply before phase I or throughout development programme. Designated based on disease/condition, prevalence, and scientific rationale (which can be based on clinical, lab, or in vitro data). E.g. in 2012, 30% of orphan drugs were designated exclusively based on animal model or in-vitro based evidence. Notes EMA and Swiss processes is different. So where are we? We have outcome measures that are validated and reliable? Yes but can improve upon them. What about biomarkers that correlate with clinical changes? Maybe. Are we identifying patients early enough? (Says we should specifically focus on possible and lab-supported probably, i.e. very early, patients.) Do we have good diagnostic criteria? Finally, how much change in an outcome measure is necessary to say it’s a significant improvement (in terms of difference in quality of life)? Does this depend on the expense and difficulty of treatment? We need a view on this. Last slides are her opinion: let’s make tighter selections on patients going in. Says we should all be on a mission to capture patients in the first 6 months after diagnosis. Reduce delays. Put minimum scores (e.g. min ALSFRS). Collect blood on every patient in order that can assess genetic makeup. Determine rate of progression (for example, based on ALSFRS monthly for 3 months before screening). Promote the idea for algorithm (c.f. Prize4Life, ENCALs prediction model). Avoid run-in designs (which is effectively wasting 3 months at start before treatment starts).

Questioner asks about getting people early diagnosis, says it is very very difficult to get people prior to one year after diagnosis. Speaker agrees, advocacy groups need to get awareness to a place where we incentivise that. Also refers to clinician friend who, when patient is young, tends to take longer to make diagnosis. So get GPs to send patients earlier.

Ruben van Eijk (Utrecht) Evidence-based trial design. Endpoint of today’s talk is ‘time to fall asleep’. For ALS event is onset, disease stage, or survival. Could be measured in ms (for cultures), days or years in patients. Gold standard in ALS is to have little doubt about efficacy and unaffected by ‘deblinding’. Systematic review of ALS trials 2000-2018. Eexpramipexole (2013) 942 patients, Olesoxime 513 patients (2014), Pentoxifylline (2006) 400 patients - all showed similar (I think hypothesised) efficacy. Which sample size is right for this? Definition of ‘event’. How to incorporate unexpected event (like tracheostomy)? 4 of those trials had death as ‘event’. Observed survival rates lower than hypothecated survival rates (I think he’s saying this is true across trials in his table). Movie about TRICALS website, a tool for evidence-based trial design for ALS. (Looks pretty cool but I can’t find a link I think but this seems to be down.) Also contains documentation (and code) for statistical analysis being performed. That is very nice (but offline). Aim 1: standardization (and centralisation) of ALS trial design 2: provide guidance and reduce erroneous assumptions 3: implmentation platform for advanced trial methodology.

Helene Tran (U Massechusetts) - Optimisation of preclinical nucleic acid-based therapeutics. Intro about C9orf72 repeat expansion, found in ~40% familial and ~7% sporadic ALS patients. Expansion transcribed into repeat RNA that translate into repeat peptides that form aggregates inside cells - nucleus and cytoplasm. Disease models (mice, iPSC-derived cells, etc) agree this peptide is toxic. Hypothesise can silence the repeat RNA to fix this. Use antisense oligonucleotdies that entre the cytoplasm or nucleus, hybridise to target RNA, and result in RNase H-mediated degredation. Why use ASO (single-stranded nucleic acis)? Other therapies using ASOs are now being approved. Now about optimisation. 1: identify ASOs sequences that specifically target C9orf72 repeat-containing variants Shows C9orf72 transcript variants, V1, V2, V3, differing in their use of exons. V2 is the most widely transcribed but does not express the repeat. So can target V1 and 2 without completely removing C9orf72 translation totally. Designed ASOs, found indeed it knocked down V1 V3 but not V2. Also staining indicates reduced # of repeat peptides per cell. Now mice. ASOs do not cross the blood/brain barrier, so intracerebro-ventricular injection. Tested 3 sequences, 2 were not well tolerated (sequence=GCCCCTAGCGCGCGACTC). But one was and it spread around brain. Now talking about ASO stability: inherently unstable. Modified backbone by adding phosphorothiate (PS) linkage, increasing stability. Also added sugar modification into RNA-like conformation; this increases binding affinity. Tests (more mice) indicated the modified and unmodified ASOs (ASO5) have similar effects, but modified ASO is better tolerated (lower body weight loss…that’s a bit worrying). Next tried to improve tolerability by studying dose. Found 200microgram admissible without adverse effects, which is pretty low. Added modified versions (ASO5-1 and ASO5-2) that mix PS linkage and phosphodiester (PO) linkage. Found more tolerable version. Now worked (on more mice) to study effective dosage. Further work on stability and longevity of ASO. Seems to work up to 12 weeks after injection. To conclude: first generation ASO not that bad, if you optimise can make something that is both safe and active and lasts for a while. It does not exacerbate C9orf72 haploinsufficiency (see this). Concludes, this is Robert Brown’s lab.

Questioner asks if the oligos might target any other genes, answer is it they did work that suggests it may target some intergenic regions but not other genes.

Jean-Cosme Dodart (Massachusetts) Wave lifesciences, ‘WVE-3972-01’ (This is what Helene Tran was talking about). I think it is in clinical trial (more news on this and another C9orf72 therapy) starting in 2018. Lots of details in this talk but I’m already convinced by the previous talk. One thing is visualisation of WVE-3972-01 results in monkey CNS tissue. (Poor monkeys). Toxicology studies are ongoing, clinical trials in Q4 2018.

Rubika Balendra (UCL) Another approach to targetting C9orf72 repeat expansions. C9orf72 repeat RNA formas a G-quadrupplex secondary structure (diagram of this which lookslike a sort of Escher version of a cube). Look for small molecules that stabilise C9orf72 RNA by screening small molecules. Picked 3 particular molecules. Use iPS-derived motor neurons, takes 30 days and get 90% pure motor neurons, these do exhibit C9orf72 repeat expansion expression. G-quadruplex-binding small molecules have low toxicity in these cells. Study in derived cortical and motor neurons, two molecules (called DB1246 and DB1247) were most effective in reducing quadruplexes. Lots more evidence it is working, in Drosophila. Video of live drosophila larvae dissection. Lovely. Conclusion: potential for therapy.

Maria Grazia Biferi (IM, Paris) - about a new gene therapy for SOD1-linked ALS. Prize4Life award. Replicating increase in survival in SOD1 mice. Trying to apply similar method to C9orf72 mutants. Using antisense approaches. Exploring using a gene therapy vector (AAV10). It allows longer antisense RNA to be used. Unfortunately I have drifted off to think about other things in this talk.

Julian Gold (Sydney) An open-label trial of Triumeq in patients with ALS. Human endogenous retroviruses: a link to MND/ALS: The Lighthouse Project. Phase IIa study of antiretroviral therapy (which is used to treat almost everyone with HIV). Investigating safety and tolerability and efficacy parameters. 40 patients at 4 sites in Sydney and Melbourne. MND confirmed < 24 months ago. All patients HLA B*5701 negative (this is to avoid allergy to one of the drugs). Ten-wekk lead in phase, then treated with ‘open-label’ fashion with Triumec. It is a combination of three drugs (Abacivir, lamivudine,dolegurine or something), it is one tablet once a day. Screened 44, 3 dropped out for social reasons and were replaced, during treatment 5 dropped out and 35 finished study. Of 5 dropouts, 2 did so due to high liver function tests, possibly due to alcohol, 3 because of reasons unrelated to Triumec. AEs (= side effects), generally unrelated to drug. Concluded it was safe and well tolerated, not interacting with Riluzole, no vital sign indicators => primary outcome met. Secondary outcome: ALSFRS-R, forced vital capacity (FVC), neurophysiological index (NPI), biomarkers (including P75), survival. ALSFRS-R more or less flat per patient. ALSFRD-R dropped during both lead-in by about 1 point per month - relatively quick progression and during treatment by about half that. Used matched ‘historical controls’ from the GSK ‘NoGo’ study. Some evidence trajectory improved for lighthouse patients. Similarly for FVC and NPI (but there are pretty massive confidence intervals here). Kind of similar for P75 biomarker, some stratification here (that looks a bit tenuous to me because, again confidence intervals are wide). Then survival, compared with ENCALs survival curve. (Can these be compared? They are different studies). But says there appears to be something going on. What does it all mean? Parameters look like influencing patients in the right direction. Ok, why might it work? Infections limited to somatic cells only allow horizontal transmission. The most common one that we know is HIV. It lands on the cell and releases its core of RNA into the cell, and then it produces an amazing enzyme called reverse transcriptase. It converts RNA to DNA, and this is integrated into our genome. It becomes part of our genome! Species have been infected with these retroviruses over the last 40 years - leading to natural selection. Diagram of these across species. ‘HERV-K’ is the last one to have been integrated into our genome - 8% of our genome is HERVs (retrotransposons). But of these only HERV-K is still able to produce an active virus. A while back some groups looked to see if there was an association with ALS, and found increased reverse transcriptase in ALS compared to controls and other diseases. HERV-K is present at 35 different places across the genome, and can be upregulated when it produces viral particles which bud off and transfer to other cells. It’s been found to damage (only) motor neurons. Is this cause or effect? Well HERV-K introduced into transgenic mice induces MND (Wenxue Li et al 2015). So can antiretrovirals make any difference? Looked at IC90 and found components of Triuvec were more effective against HERV-K than against HIV. Is this a clue? It may certainly be worth looking at further.

Jonathan Katz (San Francisco) NP001 Phase 2 Results. Starts by saying this trial was negative. NP001 is a ph-adjusted IV formulation of sodium chlorite. Had been thought to do something. Earlier clinical trial: this kind of worked, in 136 patients. Particularly, effects in some strata analysed post-hoc. Phase 2b had 138 patients. Well balanced study. Drug was well tolerated. Primary endpoint: both groups fell by the same amount. (Although his plots show the treated patients doing better than the placebos - not much better, not significantly better, but better.) Serum IL-18 did not change so it may be that the drug didn’t work. Take-hom messages 1: beware post-hoc cohort analyses in the name of heterogeneity - statistical risk! (I guess this is garden of forking paths / researcher degrees of freedom etc.) Does ranging (including different dosages) itself doubles chance of a ‘winner’ (due to statistical noise). Was there a sufficient outside sounding board? (They mainly had investors and infrastructure members that benefitted from the trial.)

Summary

Here are a few points from today’s talks that stood out to me

1: Angela Genge’s comment that half of all ALS patients with SOD1 mutations entering clinical trials, had no family history. I.e. family history may not be a reliable marker of genetic causes.

2: Greatest progress seems to be being made on therapies for individuals carrying the C9orf72 repeat expansion. There were a couple of talks about this, but the ones on WVE-3972-01 seemed particularly compelling, with detailed work on optimising the therapy, and this is entering clinical trials.

3: I’d expected more to be said about Masitinib, which as far as I can tell is hoped to be generally protective in a mild way. It sounded like there is some difficulty getting approval for its clinical trial.

4: Very valid points from Jonathan Katz, which was really a cautionary tale about drawing unwarranted hope from post-hoc analyses of clinical trial data. (i.e. look hard enough and you can always find an effect, c.f. ‘researcher degrees of freedom’, but this does not mean it is real).

5: Julian Gold’s talk on Triumvec was very thought-provoking. I did not really believe in the effects found in clinical trials. But the hypothesis, described in the second half of the talk - about activation of latent retroviruses encoded in our DNA - was really interesting.

6: Finally, I also noted there were no talks about looking for de novo mutations (i.e mutations that occur in the patient, not inherited from parents) in sporadic ALS patients. I was a little surprised about this because I’ve seen work on finding putatively causal de novo mutations in a previous talk by Justin Ichida, and thought that might be an area of active research here.