Vincent Dion simply wanted to understand how unstable, repetitive regions of the genome contract and in some cases expand, inciting disease. He started by cutting across both strands of a set of repeats, then tried just nicking one strand. “We ended up with this very surprising result,” said Dion, of the University of Lausanne in Switzerland. From nicking, “you end up with almost only contractions.” Suddenly, he realized, he had a potential therapeutic on his hands (Cinesi et al., 2016).An intron in the C9orf72 gene, linked to ALS, can grow and grow, topping 4,000 repeats of the GGGGCC sequence in some carriers (Cruts et al., 2015). But Dion and others are working on a variety of ways to reduce or eliminate expansions linked to repeat disorders using one of modern biology’s hottest tools: CRISPR-Cas gene editing. Some researchers are using the endonuclease Cas9, targeted to the disease-linked repeat with a guide RNA, simply to snip out these expanded repeat sequences out of the genome. But others, including Dion, have found many creative ways to modify the CRISPR system to contract or silence these expanded repeats, either at the DNA or RNA level.
Some, like Dion, tackle the repeat-riddled genome in the hopes of developing a one-shot treatment that eliminate these sequences for good. Others are taking aim at transcription and translation of these repeat sequences, with techniques that sidestep much of the risk of off-target effects on other, important genes in the genome (see Oct 2017 news). All of these approaches are all still in early, research stages. And, all face similar challenges en route to the clinic, including safety, tolerability and the delivery of the therapeutic to a broad swathe of cells in the nervous system.
“I think the potential is very exciting…but those are the hurdles,” said Tom Cooper of Baylor College of Medicine in Houston. He’s careful not to be too optimistic about treatments coming out of this kind of research anytime soon, but did note that these editing technologies are improving quickly. For example, scientists are working on high-fidelity versions of the Cas9 enzyme that minimize the danger from off-target effects (Kleinstiver et al., 2016).
The new CRISPR-based techniques join several others under development targeting C9orf72 ALS, including antisense oligonucleotides and antibodies (see Oct 2017 news).
RNA Roadblocks and ShreddersRepeat tracts are dangerous, but they’re also a vulnerable spot, said Eric Wang of the University of Florida. During transcription, RNA polymerase II often struggles to synthesize these sequences (Kramer et al., 2016; Siboni et al., 2015). It’s not certain why, said Wang, but it likely has to do with secondary structures formed by the repetitive DNA strands or the DNA-RNA hybrids that can be generated during this process. “Transcribing repeats is like pushing [RNA polymerase II] up a mountain,” he said.
Wang’s team wondered if they could further reduce the synthesis of expanded repeat RNAs, including those associated with C9orf72, by placing roadblocks in front of the enzyme’s path. The longer the repeat, the more room to place these obstacles, they reasoned. Wang’s team lays out their approach in the November 2 Molecular Cell (Pinto et al., 2017).
They used a deactivated version of Cas9 (dCas9) as the physical barrier, with the help of a guide RNA to direct the “dead” endonuclease to the repeat-riddled gene. Their first target: myotonic dystrophy. In this disease, triplet CTG repeats form RNA foci that sequester the splicing factor muscleblind, altering the transcriptome for the worse.
In muscle cells from people with myotonic dystrophy type 1, the approach significantly reduced the number of cells containing RNA foci and restored RNA splicing. The researchers also checked for off-target effects on other CTG or CAG repeat tracts in the genome, and found no changes to gene expression in the associated genes.To further evaluate this approach, Wang’s team asked whether this potential treatment could reduce key symptoms of myotonic dystrophy in a well-established mouse model of the disease. They injected an AAV6 virus encoding dCas9 and the CTG guide RNA into the tail veins of HSALR mice harboring 250 CTG repeats in the 3’UTR of a human skeletal muscle actin gene, at two days old. The roadblocks reduced, somewhat, the tell-tale signs of the disease at five weeks, including myotonia. However, the treatment did not stop the synthesis of expanded repeat RNAs or repair splicing in all muscle cells. “We got a little bit of rescue, but it wasn’t great,” said Wang. Changing the delivery vehicle or dosage might help, he suggested.
As for C9orf72 ALS, a treatment like Wang’s could, theoretically, be of benefit too. In HeLa cells harboring 120 C9orf72 repeats, this approach did minimize production of poly-glycine-proline typically produced by repeat-associated translation of the repeat region, suggesting that it may help reduce the expanded repeat’s potential toxicity. Wang intends to test this strategy out in a C9orf72 BAC mouse model developed by Laura Ranum, also at the University of Florida (Liu et al., 2016).
Meanwhile at the University of California in San Diego, Gene Yeo and colleagues are also aiming to eliminate these disease-linked repeat-heavy RNAs, but are targeting them after they’re transcribed. As they reported in the August 24 Cell, they hooked Cas9 to an RNA endonuclease, and designed guide RNAs to recruit the hybrid enzyme to the repeat region of these expanded messenger RNAs (see Oct 2017 News, Batra et al., 2017). Once the enzyme snips these RNAs at least once, Yeo said, cellular endonucleases can take over and destroy them completely.In muscle cells from people with myotonic dystrophy, CUG RNA-targeted Cas9 eliminated RNA foci and restored proper RNA splicing patterns. Yeo said he also has reversed these splicing defects in mice.
Yeo’s group also designed a similar strategy to tackle C9orf72 ALS. Whether this approach, however, is likely to be of benefit remains an open question, in part because it is unclear how to evaluate its potential efficacy. Scientists are not yet certain if the repeat RNAs, the dipeptides, or other factors are the true root of C9orf72 ALS. So they don’t know what they need to correct for a therapeutic and, therefore, how to design an assay to develop one. But Yeo’s RNase approach did eliminate RNA foci from a COS cell model of the disease.
For C9orf72-based ALS, both Yeo’s and Wang’s approaches seem reasonable, said Cooper, who commented on the papers in a Molecular Cell preview (Rao and Cooper, 2017).
A key question, according to Cooper, is whether these approaches have any effect on the other copy of the C9orf72 gene, which usually only has a couple of repeats in the intronic region. That’s an important point because reduced levels of C9orf72 protein might contribute to motor neuron vulnerability in the disease.
Wang wasn’t sure what would happen to the normal C9orf72 allele. Based on his analysis, shorter stretches of CAG repeats were unaffected, presumably because they don’t have space for many roadblocks to latch on. But he noted that a GC-rich sequence such as the repeats linked to C9orf72 ALS might behave differently, so this would have to be evaluated experimentally for those sequences.
In Yeo’s case, he thinks his Cas9 approach will have a higher affinity for the lengthier repeats and therefore, likely destroy only expanded C9orf72 expanded repeat RNAs.
What about other, non-repeat-based forms of inherited ALS? Wang’s approach relies on long stretches of repeats in the genome, so it would not be able to treat diseases caused by point mutations. Yeo’s RNA scissors theoretically could, but he doesn’t think this particular approach will be necessary for those forms of genetic disease. He figured there are other strategies in the works, such as antisense and RNA interference, that could reduce levels of the toxic proteins involved in ALS due to mutations.
Dabbling in DNA
Dion, meanwhile, is pursuing a different approach to tackle repeat expansions, leveraging a modified form of the Cas9 enzyme to contract them (Cinesi et al., 2016). He, too, is initially developing this approach as potential treatment for triplet repeat disorders, including myotonic dystrophy. Like C9orf72’s GGGGCC tracts, these repeat sequences become unwieldy once they lengthen, expanding and contracting. This seems to be due to damage that occurs during DNA replication or repair, which is subsequently repaired incorrectly due to the repetitive nature of the sequence.
Dion’s team stumbled upon this potential treatment approach while pursuing a basic research question: How do different types of DNA damage affect repeat repair? The team hoped to understand how expansions and contractions occur by inducing them in the genome in cell culture.The researchers first used zinc finger nucleases or CRISPR-Cas9 endonucleases to create double-stranded breaks in cultured human cells harboring 89 CAG repeats, characteristic of triplet expansion disorders. This resulted in nearly equivalent amounts of contractions and expansions. Then they tried a modified form of the Cas9 endonuclease, Cas9 D10A, which only nicks one strand. This time, they saw at least twice as many contractions as expansions. The results suggested that a modified form of the CRISPR-Cas9 system may preferentially shrink expansions.
Dion has not proven how this strategy creates contractions. One theory is that the nickase snips one strand of the repeats multiple times, generating a wide gap. The strand left intact forms a hairpin (see figure). When the cell’s internal DNA repair system comes along to repair the gap, it simply splices the hairpin out, leading to a contraction.
Regarding off-target effects, the researchers sequenced seven sites of other CAG repeats in the genomes of nine different clones, and found no evidence of contraction or expansion. That doesn’t mean there is no off-target nicking, Dion noted; all he can say for now is that the probability of off-target editing seems to be less than one percent.
The normal copy of the gene with short repeats would likely be safe from the nickase, because it would not have many sites in which Cas9 would nick to create a large gap. This is important in diseases such as C9orf72 ALS, because a further drop in C9orf72 protein levels may leave neurons more vulnerable to the disease.
Dion’s team also found no evidence of contraction for repeat tracts with 42 or less repeats – at least CAGs. “So long as you’re in the normal range, nothing would happen” he said. The hope, according to Dion, is that this approach could shrink repeat expansions down to a length not associated with disease, or, at least, to a less-toxic size that would result in later onset.
Wang thought this potential treatment approach was “really neat.” However, it will be important to try out its ability to reduce repeat length in non-mitotic cells, like neurons, he added, because the repair mechanisms likely differ in other cell types.
“The effect in other systems such as patient lines will be important to assess,” commented Adrian Isaacs of University College London, “particularly as some expansions were also observed. The overall efficacy is not clear.”
Indeed, Dion’s group is now evaluating their technique in several iPS and mouse models of repeat expansion disorders, though they have not yet attempted it with C9orf72 ALS yet. It’s hard to predict what might happen, Dion said, given that repair and instability mechanisms, and therefore their ability to contract, may depend on the specific sequence of the repeat expansion. Not much is known about how these G4C2 repeat sequences in the C9orf72 gene exapand and contract in vivo.
Meanwhile, several groups aim to tackle repeat disorders, including C9orf72 ALS, by simply removing these repetitive sequences from the genome once and for all. The approach uses Cas9 to cleave either side of the repeat region, so that the repeats drop out. The flanking regions are then glued back together by the intracellular DNA repair machinery.
That is the approach some researchers are taking in myotonic dystrophy, too. In recent proof-of-principle studies, two teams used this strategy to remove repetitive sequences from the DMPK gene in muscle cells derived from people with the disease. The approach prevented the formation of RNA foci and the alteration of RNA splicing. Importantly, the strategy did not affect the expression of other genes nearby. (van Agtmaal et al., 2017; Provenzano et al., 2017).
The same approach could potentially be used to tackle C9orf72 ALS. The expanded repeats sit conveniently in an intron, giving scientists some room to safely cleave on either side.Two groups in Los Angeles are pursuing this cut-and-run strategy. At the University of California, Los Angeles, Giovanni Coppola and colleagues are developing their approach in patient-derived iPS cells. They selected cut sites four base pairs from the repeats’ start at one end, and 49 nucleotides down on the other. Repeat excision prevented the rise of toxic RNA foci, according to their analysis published as a preprint on BioRχiv (Pribadi et al., 2016).
This approach in principle could leave small insertions and deletions (indels) behind in the genome. But since these repeat expansions occur in an intron of the C9orf72 gene, these genetic changes may not necessarily be a cause for concern, Coppola’s team wrote (Pribadi et al., 2016).
One worry, however, is that those indels, which fall in a regulatory region, might drop levels of the C9orf72 protein even further, potentially exacerbating the disease. But Coppola’s team found this was not the case. The researchers also checked for off-target effects by sequencing the 1,000 most likely cleavage sites for each guide RNA sequence, and found no convincing evidence of off-target cuts.
Kim Staats from Justin Ichida’s group at the University of Southern California (USC) reported their progress using a similar strategy at the 2017 meeting of the Society of Neuroscience in Washington, D.C. They also use guide RNAs that bring Cas9 close to the repeats, without altering the gene’s exonic sequences. Lab members used this approach to cut out G4C2 from patient-derived iPS cells, according to Staats. Next, she is hoping to use this same strategy to remove these sequences in model mice, developed by Robert Baloh of Cedars-Sinai in Los Angeles, carrying human C9orf72 with more than 100 repeats – a key step to develop this approach as a treatment strategy for the disease (see O’Rourke et al., 2015).
Designing guide RNAs to remove the repeat region is differnet from some other CRISPR-based approaches, because the RNAs can’t simply be targeted to the repeat sequence itself, Staats pointed out. Instead, the researchers must find sequences that the Cas9 enzyme can bind between the repeats and neighboring exons. Compounding the difficulty, the instability of the repeats has led to various polymorphisms in those neighboring regions. “We should be wary that it might not be one-guide-system-fits-all,” she said.
This sequence variability makes the development of these approaches challenging, according to Staats. One such polymorphism, in the model mice, restricted the area that the guide RNA could be targeted to just a couple of dozen base pairs. “We had a very small region to work with,” said Staats.
Erase and Replace
While C9orf72 repeat expansions are the most common genetic cause of ALS, there are plenty of other disease-linked mutations, in genes such as SOD1, TARDBP, and FUS. CRISPR can offer a solution there, too. In the October 25 Science, Feng Zhang and colleagues from the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, reported a novel technique which may enable at least some of these mutations to be corrected at the RNA level (Oct 2017 News, Cox et al., 2017). Instead of Cas9, they used the endonuclease Cas13, which naturally targets RNA. They deactivated the enzyme’s cleavage ability, and hooked it to a RNA editor, ADAR2. The result was an enzyme that, when directed by a guide RNA, swaps an adenosine to an inosine, which acts as a guanine in this context.
The researchers used this technique to correct 36 different disease-linked mutations in cultured human HEK293 cells, including a G943A mutation in the gene encoding the RNA-binding protein TDP-43 that causes ALS. However, the efficiency varied from 35% down to wholly ineffective, and the enzyme made thousands of off-target edits, too. An improved version made no off-target edits in experiments with two genes; however, efficiency remained low, around 20%.
Meanwhile, scientists at Harvard University in Cambridge, Massachusetts are adapting the CRISPR-Cas9 system to correct disease-linked point mutations at the DNA level. The method, which uses a cytosine deamidase targeted by Cas9 to the disease-linked mutation, converts cytosine to thymine within DNA, without requiring a double-stranded break (Komor et al., 2016; May 2017 news, Kim et al., 2017). This minimizes the risk of indels that may form, a key downside of conventional CRISPR-Cas9 editing systems. The approach, according to the Harvard team’s estimates, may enable the correction of hundreds of disease-linked genes – including those associated with ALS.
Pros, Cons, and Unanswered Questions
With the number of editing approaches growing, a key question is which of them may be the best choice for ALS, and for which form of the disease. Correcting disease-linked mutations at the DNA or at the RNA level each have their advantages.
“For DNA, once you’ve corrected it, you’re done,” Dion pointed out. RNA-targeted treatments, in contrast, would require long-term expression of Cas9 or Cas13 endonucleases, enzymes that originate from bacteria (Chew et al., 2016). “This might come with other problems that, at the moment, we don’t know,” Dion said. Yeo, for his part, is hoping to reduce unwanted side effects by ‘humanizing’ his Cas9 enzyme.
Another unanswered question is how often an approach that targets repeat RNAs would need to be given to treat ALS. Some studies indicate these editing systems, delivered by gene therapy, might persist for a few years (reviewed in Naldini, 2015). But suppose a person finds out they carry a C9orf72 expansion at age 30 or 40? They would presumably require treatment for decades.
“It’s still really uncharted territory,” said Wang. It’s possible booster shots might be required over time. Wang also wonders, would future treatments result in an immune reaction to the virus used to deliver the therapy the first time around? “That’s sort of unknown, because we haven’t gotten there as a community,” he said. (For a review, see Mingozzi and High, 2017.)
The advantage of editing C9orf72 repeat RNAs, or using cleavage-inactivated Cas9 to block their transcription as Wang did, lies in safety because the enzymes won’t cut off-target sites, Isaacs pointed out. If either of these approaches occasionally edit or cut up a few off-target transcripts, the diseased cells still have the gene encoding them to make more.
But targeting the genome is riskier, said Cooper. Within the genome lie many sites similar to disease-linked mutations, and microsatellites similar to disease-linked repeats. “Putting something in that’s going to mess up the genome; that’s what I would be worried about,” said Cooper. “We don’t want to be causing cancer or hitting the wrong gene.”
Those off target effects are a common problem for all CRISPR-Cas editing approaches, Isaacs said, as is efficient delivery of both the enzyme and the guide RNA into the brain, a problem common to all AAV-based therapeutics. “Delivery is likely to keep improving,” he added.
It’s not even clear how many cells in the muscle or nervous system in people with ALS need to be reached, Wang pointed out: “What proportion of nuclei do you need to correct? Is it 10%, is it 50%? I don’t think we know that.”Yeo doubts genome-targeted techniques have much of a shot, given these delivery challenges, unless they’re provided at the embryo stage (August 2017 news, Ma et al., 2017). He thinks it would be too difficult to remove the disease-linked repeats in a majority of cells—particularly in the nervous system, where non-proliferative cells neurons struggle to repair the double-stranded breaks typically formed during the CRISPR process—without dangerous off-target effects. Targeting the RNA or its synthesis will work better, Yeo suspects, because it doesn’t require that DNA repair process, and can tolerate some level of off-target editing or cleavage.
Meanwhile, Jennifer Doudna’s team at the University of California in Berkeley is working on a different approach. They are developing a CRISPR-Cas9 system that can be delivered locally, avoiding these obstacles (Staahl et al., 2017).
Another challenge is how to cram any of these potential therapies for ALS into a compact viral vector: AAV9 holds about 4.7 kilobases of DNA. “It’s really hard to fit the traditional [Streptococcus pyrogenes] Cas9 into an AAV vector,” said Staats. “It’s pretty much impossible to fit the whole thing in with the guides and reporters.”
There are plenty of solutions: In Ichida’s group, Staats and colleagues solved the issue by splitting the gene encoding the Cas9 enzyme into halves, each delivered by a separate virus. Yeo’s group used truncated versions of the Cas9 gene. And, Wang’s team turned to Cas9 from a different bacterial source, S. aureus, which is 1.5 kb smaller (Ran et al., 2015).
For now, researchers are exploring a variety of options to tackle diseases, including ALS, with CRISPR-Cas editing systems. It’s entirely possible that some technical hurdles might be solved within a couple of years, Cooper speculated. For example, a recently discovered Cas9, found in bacteria from hot springs and deep sea vents, remains active longer in circulation, opening the door to more efficient drug delivery (Harrington et al., 2017). Another emerging CRISPR-Cas9 approach enables genes to be edited in neurons at up to 30% efficiency in the brain, at least in the mouse (Nishiyama et al., 2017).
CRISPR has already jumped from the lab into clinical trials, under Phase 1 and 2 study for several cancers. And, the approach may be tested in the clinic as a treatment for the blood disorder β-thalassemia as early as next year.
“I don’t know what’s going to work better, ultimately,” said Wang. “I think a lot of these [techniques] will work if you can get the material into cells.”
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