Over the course of 2020, you heard a number of announcements from us about our collaboration with Ionis Pharmaceuticals on an ASO for prion disease. We published on our clinical strategy and discussions with regulators [Vallabh 2020a], preclinical data in mice [Minikel 2020], and the natural history of biomarkers from our clinical research study at MGH [Vallabh 2020b]. We got officially listed on Ionis’s pipeline and Dr. Anne Smith, who is leading clinical development, spoke to the patient community at CJD Foundation’s virtual conference. While those are all the updates directly relevant to our prion disease program, I’m always keeping my eye on interesting developments in the science of ASOs and other RNA-targeting therapeutics more broadly. Over the course of the past year there were several news stories and/or papers that caught my eye that I never yet got around to blogging about. Here is a roundup.

an oral ASO against PCSK9

The ASO on which we’re collaborating with Ionis will be delivered intrathecally, meaning via a lumbar puncture. Currently this is the only realistic way to get ASOs into the brain. One question we often get from people in the prion disease community is “when will this be a pill?” The answer is not for a long time. Intrathecal delivery of ASOs to the CNS builds on years of work to develop comparable ASOs for spinal muscular atrophy and other diseases, and even someone invents a way to deliver ASOs to the brain orally, developing and honing that technology, demonstrating its safety, and applying it to our disease in particular, will take years. Still, it’s interesting to keep an eye on new technological developments that could eventually be relevant. Therefore, I was interested to see a conference abstract late last year that an oral ASO, albeit not for a brain indication, is now in preclinical development [Gennemark 2020]. Update: a paper is now published: [Gennemark 2021]. The actual drug in question is an ASO against PCSK9 being developed for a liver indication, hypercholesterolemia. The abstract describes preclinical development, with testing in rats, dogs, and monkeys. Bioavailability in the liver was about 7% in dogs. The compound doesn’t match rat sequence, but a proof-of-concept compound against a different target achieved 78% knockdown in rats. They mention that there was a reduction (though they don’t say how much) in plasma LDL in monkeys at doses of 28-56 mg/day. A PCSK9 ASO is currently in a clinical trial (NCT04641299) with subcutaneous delivery, but I haven’t yet heard anything about a human trial of oral delivery. This is exciting, but remember that the liver is perhaps the easiest tissue to hit with a drug, while brain is perhaps the hardest. An ASO pill for brain diseases is still likely a long ways off.

risdiplam: approval of a splice-modulating small molecule

Risdiplam, a small molecule designed to modulate splicing of SMN2 RNA in spinal muscular atrophy, obtained FDA approval. It is the third disease-modifying therapy to be approved for spinal muscular atrophy, after nusinersen (an ASO) and onasemnogene abeparvovec (an AAV gene therapy), and the first to be given as an oral tablet. But, much more excitingly, it is a novel type of therapy. Until now, the only approved small molecule drugs targeting RNA were linezolid and tedizolid, two antibiotics that bind a pocket in the bacterial 23S ribosomal RNA [Warner 2018]. Risdiplam binds the complex of the SMN2 RNA with the U1 snRNP [Campagne 2019], causing SMN2 exon 7 to be included, thus giving rise to a functional SMN protein. As a modulator of the splicing of a human RNA, it represents a whole new therapeutic mechanism and a flagship for small molecules targeting RNA. PTC Therapeutics, partnered with Roche/Genentech, spent many years developing splice modulators for SMA [Naryshkin 2014, Ratni 2016, Ratni 2018], and they even brought an earlier analogue, RG7800, to the clinic [Kletzl 2019] before pivoting to risdiplam. Both preclinical and Phase I clinical studies of risdiplam showed good safety, pharmacokinetics, and an ability to induce full-length SMN2 RNA just as hoped [Poirier 2018, Sturm 2018]. As far as I could tell, the Phase II/III data have not yet been published, so all we have to go on is Genentech’s press release and the FDA label. Those indicate that, for instance, 90% of risdiplam-treated infants survived after a year of treatment, a timepoint where, based on natural history data, only ~25% of untreated infants would have been expected to survive.

I blogged about splice-modulating small molecules five years ago, and noted that one concern was whether they could modulate the target with sufficient specificity. ASOs may target, say, a 20 nucleotide RNA sequence, which often is enough to have no perfect matches anywhere else in the transcriptome. In contrast, risdiplam only appears to make direct contact with just 3 nucleotides in SMN2 RNA, and the RNA sequence motif for which the compound is active is no longer than 11 nucleotides [Campagne 2019]. PTC’s first SMN2 splice modulator resulted in differential expression of 12 genes [Naryshkin 2014], and Novartis’s competing compound, branaplam (then called NVS-SM1) affected expression of 175 genes [Palacino 2015]. I had wondered whether the efficacy on the one target of interest could really outweigh the safety implications of affecting tens or hundreds of other genes. The new clinical data appear to finally prove that risdiplam’s specificity is, in fact, sufficient to confer a rather favorable safety/efficacy balance. Some of the drug’s specificity may be conferred directly by the sequence that the drug binds, but rather by the fact that the drug turns a weak 5’ splice site into a strong one [Campagne 2019]. Thus, in order to be affected by the drug, a splice site must not only have the right sequence, but also be poised at just the right level of baseline U1 snRNP binding, not too strong and not too weak, such that the drug makes a difference. Meanwhile, the fact that some splice-modulating small molecules are not quite perfectly specific has pointed to intriguing new applications. Novartis’s branaplam, originally developed for SMA, turns out to also downregulate expression of HTT, and they’ve now pivoted to developing it as a therapy for Huntington’s disease. Whether it will prove safe and effective in that indication remains to be seen, but at least, this appears to provide a precedent for the notion that RNA-targeting small molecules could potentially be used to knock down a gain-of-function disease gene — their application may not be limited to correcting splice defects.

a dose-limiting toxicity in Angelman’s syndrome

A Phase I/II trial (NCT04259281) of GTX-102, an intrathecal ASO developed by Ultragenyx for Angelman’s syndrome, was paused this past fall after a significant safety event. An Ultragenyx press release stated that the dose levels in the trial were 3.3, 10, 20, and 36 mg. As reported by FierceBiotech, one patient in the 20 mg group and four in the 36 mg group had a lower limb weakness progressing to inability to walk about 1-4 weeks after dosing. This weakness gradually resolved, and the press release stated that some clinical improvement in disease symptoms was observed and even outlasted the lower limb weakness. Thus, there is no indication that development of GTX-102 will be halted, though it looks like dosing will be adjusted. The press release states that nothing like this was observed in the monkey toxicology studies that led up to clinical trials.

This development caught my notice because it might represent the first indication of a dose-limiting toxicity for an intrathecal ASO in humans. (None of Ionis’s intrathecal ASOs that have completed at least one trial in humans — nusinersen, tominersen, and tofersen — had any significant safety issues observed.) Still, it’s not yet clear how relevant the GTX-102 observations will be to ASOs more broadly. First, this adverse event may be specific to this one compound; there is not yet reason to suspect it represents any sort of broader class effect. Second, if it is a class effect, we don’t yet know what the “class” is — as far as I can tell, the exact chemical composition and sequence of GTX-102 have not been disclosed, though it is presumably one of the many ASOs enumerated in patent US20200370046A1. ASOs come in a lot of different chemistries, and some of them have toxic liabilities that others don’t — for instance there is literature on the specific safety issues with locked nucleic acid (LNA) ASOs [Burel 2016], which do not seem to occur with 2’MOE ASOs. Overall, the GTX-102 news certainly doesn’t dampen our enthusiasm for developing an ASO for prion disease, but it is something we’ll be keeping our eye on in case it affects how dosing levels are selected or what safety issues drug companies or regulators want to monitor for in future clinical trials.

tofersen in SOD1 ALS

Excitingly, we learned the results from the Phase I/II trial of tofersen, Ionis Pharmaceuticals’ ASO against SOD1 for ALS [Miller 2020]. Nusinersen, the splice-modulating ASO targeting SMN2 for spinal muscular atrophy, has been FDA-approved since 2016 and we now have lots of data on that drug. But, following tominersen against HTT for Huntington’s disease [Tabrizi 2019], tofersen is now only the second intrathecal ASO designed for knockdown, as opposed to splice modulation, of a target gene, to have clinical trial results read out in humans. The trial involved five treatment arms: patients received injections of either placebo or 20, 40, 60, or 100 mg of tofersen. Each patient got five injecitons spaced over 3 months, and was followed for another 3 months afterwards. Tofersen achieved clear target engagement: at 3 months after the first dose, the concentration of SOD1 protein in patient CSF at the highest dose was reduced by 33% to 36% (depending on whether you compare only to the patients’ own baselines, or also normalize to the placebo group). That’s comparable to tominersen, which acheived 40% knockdown of mutant huntingtin in CSF. But potentially more exciting findings are from the trial’s secondary endpoints. They measured three outcomes related to ALS disease progression: ALSFRS-R score (measuring overall ability to function), slow vital capacity (ability to breathe), and handheld dynamometry megascore (muscle strength). Overall, patients at the highest dose group experienced less disease progression on all three measures than patients in the placebo group. That may provide some whiff of signal that the ASO is not only doing its job of knocking down the disease-causing protein, but also affecting the disease process. Of course, it’s important to remember that this is super preliminary. This small trial was not powered to detect change in these outcomes, so the data are noisy and there is not a clean dose-response relationship across the different tofersen dose levels. For example, ALSFRS-R in the 20 mg group declined by just 0.76 points, while in the placebo group it declined by 5.63 points, even though the 20 mg dose did not acheive appreciable target engagement (-1% to +2% change in CSF SOD1, depending on how you normalize). That example shows how any signal in these exploratory outcomes is also wrapped up with a lot of noise. A Phase III trial (NCT02623699) is now underway to definitively answer the question of whether tofersen slows progression of SOD1 ALS.

intrathecal AAV in adult humans

Finally, one study that received a fair amount of press recently reported on a new potential modality: intrathecally delivered AAV-vectored microRNA [Mueller 2020]. In other words, a small RNA designed to knock down SOD1 RNA was placed in a viral vector and injected into the CSF of adults with SOD1 ALS. This is interesting because it addresses an area where there were previously no data: intrathecal AAV in adult humans. As background, an intravenous AAV drug is approved for infants — onasemnogene abeparvovec, a virus which delivers an intact copy of SMN1 for spinal muscular atrophy [Mendell 2017]. But rodent data have for years suggested that AAV9 uptake into brain neurons is good in neonates but very weak in adult animals [Foust 2009]. That has left open the question of whether a drug like onasemnogene could ever work for an adult disorder. Early primate studies didn’t provide much basis for optimism: when 1.8×10^12 viral genomes (vg) were delivered intrathecally in macaques, the distribution was broad across the brain but very low yield [Gray 2013]. They measured both GFP positivity and the ratio of viral genomes to host diploid genomes (vg/dg), and both were around 2%. Most scientists I’ve spoken with assume that we need to engineer better AAV vectors in order to make adult brain gene therapy a reality, as explained here. Nonetheless, some scientists have hypothesized that it might be possible to achieve meaningful levels of neuronal uptake with existing AAVs, such as AAV9, if the drug is delivered intrathecally at super high doses. To date there has been a shortage of data on this topic. A recent conference abstract hinted at meaningful target engagement in monkeys with an AAV gene therapy [Thomsen 2019], but was short on detail and I didn’t manage to see the actual talk at AAN2019.

The preclinical development of the SOD1 miRNA therapy in question here had previously reported promising results in primates. They intrathecally injected macaques with a whopping 3.5×10^13 viral genomes of AAVrh10 and the vg/dg ratio was nearly 100 in bulk spinal cord, or about 5.3 when they microdissected out motor neurons [Borel & Gernoux 2018]. The treatment resulted in ~50% apparent knockdown of SOD1. Scaling from monkeys to humans, they selected a dose level of 5×10^14 vg, again delivered intrathecally, for human studies [Mueller 2020]. The trial included just 2 patients, though, so ultimately it is hard to tell what the effect was. SOD1 protein in CSF was not lowered in either patient. In the one patient who died and underwent autopsy, the vg/dg ratio in spinal cord was right around 10, substantially lower than in the monkeys but still high enough to be potentially meaningful, and SOD1 protein level was nominally lower than in a few untreated patients, but it is tough to tell whether that is treatment-related or a fluke. The injection resulted in a considerable immune response but appeared to be managed effectively with immunosupression (sirolimus and prednisone). Overall, it’s an interesting development, but we’ll need to see studies in many more patients to know whether intrathecal AAV is a potential therapeutic modality for adult CNS diseases.