‘Gene ther­apy 2.0’ is chang­ing medicine

Sci­en­tists are rush­ing to fig­ure out how to use the gene-edit­ing tool to stop dev­as­tat­ing dis­eases. AN­TO­NIO RE­GAL­ADO re­ports.

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DOC­TORS SAY THE DIS­EASE is ter­mi­nal, but they tell you less about liv­ing with it. About the girls who don’t see past your wheel­chair, or how the phone stops ring­ing. It’s you and Mum count­ing the birthdays and fig­ur­ing out what you can’t do this year. Dupree says he got by in high school, but in col­lege de­pres­sion gripped him. “I didn’t know how I could keep go­ing,” he says.

The prob­lem is that Dupree’s body doesn’t make dys­trophin, a pro­tein in mus­cle fi­bres that acts like a shock ab­sorber. With­out it, your bi­ceps, calf mus­cles and di­aphragm slowly turn to a fat-like sub­stance. You end up on a ven­ti­la­tor, and then your heart stops.

Dys­trophin is man­u­fac­tured by a gene that is among the largest in the hu­man genome. It con­sists of 79 com­po­nents known as ex­ons, each an in­struc­tion for one in­gre­di­ent of the pro­tein. Dupree’s prob­lem, he says, is a “pseudo” exon – as though in the mid­dle of this epic recipe some­one added an in­struc­tion that read: “Stop the cook­ing.” There are thou­sands of ways a gene this size can go wrong, and Dupree’s mu­ta­tion – a sin­gle let­ter of DNA that reads “G” in­stead of “T” – is unique, so far as sci­en­tists know.

Dupree, who ma­jored in bio­chem­istry and hopes to be­come a ge­netic coun­sel­lor, has some­times imag­ined what life would be like if that small er­ror were not there.

A year ago, in De­cem­ber 2015, he learnt how a tech­nol­ogy called CRISPR might make that pos­si­ble. A sci­en­tist named Eric Olson had re­quested some of Dupree’s blood a few months ear­lier, and Dupree had agreed. Soon he was rolling through the lab on his Tilite wheel­chair so Olson, a bi­ol­o­gist at the Univer­sity of Texas South­west­ern Med­i­cal Cen­tre, could show him the re­sults – and what some sci­en­tists now pre­dict is the like­li­est way to cure Duchenne.

Us­ing CRISPR, which makes it pos­si­ble to snip DNA open at a pre­cisely cho­sen spot, a team at the hospi­tal had mod­i­fied Dupree’s cells in a dish, cut­ting through the ex­tra exon. The edit­ing process re­quired only a sin­gle step and had taken three days. In an im­age taken with a mi­cro­scope, Dupree’s cells were clouded with green puffs of per­fect dys­trophin.

“I try to be re­al­is­tic with my ex­pec­ta­tions,” he says. “But that gave me a sense of, ‘Wow, this is here’.”

The po­ten­tial to pre­cisely and eas­ily “edit” any genome us­ing CRISPR is chang­ing the way we think about na­ture. The CRISPR tech­nique is of­ten likened to a “search and re­place” func­tion for DNA. To lab­o­ra­tory sci­en­tists, it might bet­ter be com­pared to the dis­cov­ery of fire. Ev­ery day they pub­lish an av­er­age of eight sci­en­tific ar­ti­cles de­scrib­ing new uses of the tech­nol­ogy – or merely re­flect­ing on its ex­po­nen­tially ex­pand­ing pos­si­bil­i­ties, such as de­signer ba­bies en­gi­neered with de­sir­able traits and mos­qui­toes with DNA pro­grammed to make them go ex­tinct.

Among th­ese pos­si­bil­i­ties, the chance to end the pain and suf­fer­ing of peo­ple like Dupree is CRISPR’S most com­pelling, if still dis­tant, prom­ise. In early-stage lab ex­per­i­ments, aca­demic sci­en­tists are show­ing that gene edit­ing of­fers new ways to at­tack cancer, to knock out HIV and hep­ati­tis in­fec­tions, even to re­verse blind­ness and deaf­ness. Com­pa­nies aren’t far be­hind. Three star­tups in the Bos­ton area have al­ready raised a com­bined US$1 bil­lion and part­nered with some of the world’s big­gest drug com­pa­nies such as Bayer and No­var­tis. “None of us can an­tic­i­pate where this tech­nol­ogy will end up,” says Olson. “I’m op­er­at­ing un­der the premise that it will take us far­ther than we can imag­ine.”

Sci­en­tists know the gene er­rors re­spon­si­ble for about 5,000 in­her­ited dis­or­ders and se­quenc­ing labs dis­cover some 300 more each year. Some are one-in-a-bil­lion syn­dromes. Duchenne is at the other ex­treme; it is one of the most com­mon


in­her­ited dis­eases, af­fect­ing 1 in 4,000 boys. Girls are af­fected rarely and to a lesser de­gree.

Gene edit­ing could be a way to erase such dis­eases with a one-time, per­ma­nent al­ter­ation of a per­son’s DNA. It’s a step be­yond con­ven­tional gene ther­apy – the 30-year-old idea of in­sert­ing en­tire re­place­ment genes into a per­son’s cells, usu­ally us­ing a virus. That ap­proach is im­prac­ti­cal for some dis­eases. The gene for dys­trophin, for in­stance, is too large to fit in­side a virus, as CRISPR’S DNAs­nip­ping pro­teins can. Some­times a faulty gene that’s do­ing harm needs to be si­lenced, so adding a new one won’t help. CRISPR’S abil­ity to delete and swap out ge­netic letters makes a huge new range of treat­ments pos­si­ble.

Some doc­tors are now call­ing CRISPR “gene ther­apy 2.0”. To be sure, even gene ther­apy 1.0 has yet to fully ar­rive. Af­ter 30 years of re­search, sci­en­tists are still learn­ing how to use viruses to move ge­netic in­struc­tions into a liv­ing per­son’s cells. Only two gene-re­place­ment treat­ments for in­her­ited dis­ease have ever been ap­proved, both in Europe.

But Olson says he is con­vinced CRISPR is the most plau­si­ble way to stop Duchenne. Early this year, he showed he could re­pair mu­ta­tions in mice with mus­cu­lar dys­tro­phy af­ter send­ing viruses stuffed with CRISPR in­gre­di­ents into their veins. “A mouse is not a boy, but we think we know ex­actly what needs to be done,” Olson says. If it works, he adds, “this is a cure, not a treat­ment”.

Olson says the very first hu­man test of a CRISPR ther­apy in a pa­tient with Duchenne could be­gin in two years in what would be a small, ex­ploratory clin­i­cal trial in­volv­ing just a few boys. Work­ing with Jerry Men­dell of Na­tion­wide Chil­dren’s Hospi­tal in Ohio, a cen­tre for gene-ther­apy stud­ies, they ex­pect to give the treat­ment to mon­keys dur­ing the next 12 months, a pre­lude to hu­man tests. The re­searchers will also be look­ing to see whether the CRISPR gene ther­apy has un­ex­pected ef­fects. Ac­ci­den­tal ed­its are a par­tic­u­lar con­cern.

Dupree, who is fol­low­ing events in the lab, says he’s not ex­pect­ing much for him­self. He knows the stud­ies could take years and since his mu­ta­tion is unique, he’d need a ther­apy tai­lored just for him. “I am more ex­cited about the im­pli­ca­tions sci­en­tif­i­cally than any treat­ment for me,” he says. But his mother, Deb­bie Dupree, says chat boards and Face­book pages where par­ents gather are al­ready alight with ques­tions. “There is a lot of talk. Peo­ple want to know when it will be avail­able,” she says.

Duchenne pa­tients and their fam­i­lies won’t be the only ones anx­iously ask­ing that ques­tion. Count­less oth­ers fac­ing deadly can­cers or HIV, as well as sickle-cell ane­mia and nu­mer­ous other ge­netic dis­eases, could soon be watch­ing the fate of those Crispr-al­tered cells in Olson’s lab. Are they the be­gin­ning of a new era of medicine or merely one more promis­ing re­search re­sult that will never make it out of the lab? In par­tic­u­lar, re­searchers

will need to solve the next chal­lenge: safely and ef­fec­tively edit­ing DNA in cells through­out a hu­man body, thus turn­ing CRISPR from an in­valu­able lab tool into a med­i­cal cure.

CRISPR EVOLVED IN­SIDE BAC­TE­RIA, over bil­lionyear time scales, as a form of im­mu­nity against viruses. Bac­te­ria col­lect and store short snip­pets of DNA from viruses that have in­vaded them, spac­ing the seg­ments out through their own genome in a pat­tern called “clus­tered reg­u­larly in­ter­spaced short palin­dromic re­peats” – the term that gives CRISPR its acro­nym. When re­in­fected with one of th­ese viruses, bac­te­ria can cre­ate copies of th­ese ge­netic snip­pets, which zip up let­ter for let­ter with the new virus’s DNA – sig­nalling to a spe­cialised cut­ting en­zyme that it should at­tach it­self and close, pin­cer-like, onto the vi­ral genome and sever it.

By 2013, teams of sci­en­tists in Bos­ton, Berke­ley and Seoul separately showed this nat­u­rally oc­cur­ring bac­te­rial im­mune process could be sim­pli­fied and re­pur­posed to cut DNA in hu­man cells. Though sci­en­tists had pre­vi­ously cre­ated gene-edit­ing pro­teins, th­ese were dif­fi­cult to de­sign and build com­pared with the so­lu­tion bac­te­ria had de­vised. “In­stead of ver­sion 2 or ver­sion 3, it was ver­sion 3 tril­lion,” says Tom Barnes, chief sci­en­tist of the CRISPR startup In­tel­lia Ther­a­peu­tics in Cam­bridge, Mas­sachusetts. “It went from no labs work­ing on it to ev­ery­one work­ing on it.”

In­tel­lia is one of a trio of star­tups that have set up shop around Bos­ton and raised about US$300 mil­lion each to cre­ate CRISPR treat­ments; the oth­ers are Edi­tas Medicine and CRISPR Ther­a­peu­tics. Barnes says CRISPR vastly sim­pli­fies gene edit­ing be­cause of the way the cut­ting works. Just as bac­te­ria spot and slice the vi­ral ge­netic ma­te­rial, CRISPR can zero in on spe­cific stretches of hu­man DNA. The only in­gre­di­ents needed are an edit­ing en­zyme – one named Cas9 is used most of­ten – and a short “guide”, or length of ge­netic letters, to tell it where to cut.

It seems sim­ple, but us­ing it to cre­ate hu­man treat­ments is any­thing but. And there’s one hitch that’s of­ten over­looked: “edit­ing” is a bit of a mis­nomer. Sci­en­tists have mastered cut­ting into DNA, which gives them some­thing akin to a “delete” key for genes, in ad­di­tion to the “add” func­tion of­fered by tra­di­tional gene ther­apy. But they can’t as eas­ily re­write genes let­ter for let­ter, an as­pect of the tech­nol­ogy still be­ing de­vel­oped.

For now, that mostly lim­its them to sit­u­a­tions where delet­ing genes, or parts of them, is use­ful. Duchenne is one of those. An­other is sickle-cell dis­ease, a con­di­tion that in the United States af­fects mostly African-amer­i­cans. Med­i­cal re­searchers have given it rel­a­tively lit­tle at­ten­tion in the past, but there’s an ob­vi­ous DNA cut that might solve it, mean­ing a po­ten­tially el­e­gant cure. Now Mitchell Weiss, a haema­tol­o­gist who treats peo­ple with sickle-cell at St. Jude Chil­dren’s Re­search Hospi­tal in Mem­phis, says ev­ery gene-edit­ing com­pany is call­ing him. “The in­ter­est right now is in­cred­i­ble,” he says. “Be­fore, no one was in­ter­ested. No one cared. But they need a proof of prin­ci­ple, and this is a good one.”

In ad­di­tion to find­ing the kind of ge­netic prob­lem to which CRISPR of­fers a so­lu­tion, com­pa­nies need a way to get the CRISPR in­struc­tions into the body. Most are count­ing on viruses for that job, but In­tel­lia’s strat­egy is to pack­age CRISPR into fatty blobs that liver cells suck up, just as if they were choles­terol. In Au­gust 2016, at the an­nual CRISPR meet­ing in Cold Spring Har­bor, New York, re­searchers from the com­pany showed that a sin­gle dose could al­ter the genomes of at least half the cells in a mouse’s liver. If In­tel­lia can suc­cess­fully edit liver cells in a per­son, that may let the com­pany treat a slew of pre­vi­ously unas­sail­able meta­bolic con­di­tions like a form of hered­i­tary amy­loi­do­sis, in which painful plaques build up in the body.

What’s ob­vi­ous is that it will be eas­ier to get CRISPR to work in some parts of the body than oth­ers. The eas­i­est task is prob­a­bly delet­ing genes in blood cells, since th­ese cells can be re­moved from a pa­tient and then put back. Al­ready, a Chi­nese drug com­pany has opened a study to cre­ate su­per­charged im­mune cells to bat­tle cancer, and sci­en­tists at the Univer­sity of Penn­syl­va­nia have an­nounced sim­i­lar plans with the fi­nan­cial back­ing of the bil­lion­aire in­ter­net en­tre­pre­neur Sean Parker.

If you’re look­ing for gene edit­ing’s Ever­est, it’s prob­a­bly rewrit­ing DNA in the hu­man brain – say, to treat Hunt­ing­ton’s dis­ease. Edit­ing mus­cle cells lies some­where in the mid­dle of the dif­fi­culty scale. Ge­net­i­cally, it’s a good can­di­date. Even with just a delete key, Olson says, up to 80% of mus­cu­lar dys­tro­phy cases could be treated. Ini­tially, the edit­ing treat­ment he’s work­ing on will tar­get a hot spot in the dys­trophin gene – exon 51, in which Edi­tas has also sig­nalled an in­ter­est. Delet­ing that exon could treat about 13% of Duchenne cases.

The most sig­nif­i­cant un­known is whether it will be pos­si­ble to edit enough mus­cle cells and make enough dys­trophin in a hu­man body. “I think this rep­re­sents the most promis­ing strat­egy,” Olson says. “But the thing that has to go right is that it has to be ef­fi­cient.” Mus­cles, in­clud­ing the heart, glutes, and bi­ceps, make up 40% of a per­son’s body mass – bil­lions and bil­lions of cells. So far, in his mice, Olson has suc­ceeded in pro­duc­ing dys­trophin in 5-25% of mus­cle fi­bres. It’s half cal­cu­la­tion and half spec­u­la­tion, but he thinks that edit­ing 15% of the mus­cle cells in a boy will be enough to slow, if not halt, mus­cu­lar dys­tro­phy.

When I last spoke to Olson, he was rush­ing to a phone meet­ing to drum up com­mer­cial sup­port for his idea of start­ing a hu­man test for a Duchenne treat­ment. He has been talk­ing with sev­eral com­pa­nies in­clud­ing Edi­tas, prob­a­bly the best-known of Bos­ton’s trio of CRISPR star­tups. It has Bill Gates and Google as in­vestors.

The com­pany, founded by sev­eral of the in­ven­tors of CRISPR tech­nol­ogy, also de­clared an early in­ter­est in Duchenne, li­cens­ing work done at Duke Univer­sity. But its chief op­er­at­ing of­fi­cer, San­dra Glucks­mann, says it isn’t pro­vid­ing up­dates on the Duchenne pro­gram.

In fact, Edi­tas has been ly­ing low. CRISPR could po­ten­tially treat so many dif­fer­ent dis­eases that the com­pany has been re­luc­tant to an­nounce what its do-or-die project will be, and prov­ing any CRISPR drug is ef­fec­tive could eas­ily take a decade.

That puts Glucks­mann in a tough po­si­tion. On week­ends she an­swers emails from des­per­ate par­ents: “Could CRISPR cure my child?”. In the­ory the an­swer may be yes, but about a quar­ter of the time Glucks­mann has never even heard of the ill­ness be­fore. The an­swer Edi­tas has been giv­ing to the par­ents of boys with mus­cu­lar dys­tro­phy has been par­tic­u­larly dis­ap­point­ing: “I am very sorry to hear about your son. Un­for­tu­nately, we are still in the very ear­li­est stages of re­search.”

ONE THING THAT’S AL­READY AP­PAR­ENT is that many in­her­ited ge­netic dis­eases will re­quire tai­lor­ing a CRISPR treat­ment to very spe­cific mu­ta­tions – those af­fect­ing small sub­sets of pa­tients or even in­di­vid­ual peo­ple. Take Dupree, who lives less than a mile from Olson in a Dal­las sub­urb. His mu­ta­tion is unique, and it’s not near exon 51, so he wouldn’t be helped by the first CRISPR treat­ment Olson is de­vel­op­ing.

But there’s no ques­tion in Olson’s mind that Dupree’s mu­ta­tion is cor­rectable too, given the tech­nique can po­ten­tially tar­get any spot on the genome. Dupree now sees at least a glim­mer of a chance that some­one could make a CRISPR treat­ment just for him. “It’s only given once, and maybe it’s not that ex­pen­sive,” he says. “It made me think about how it could be done, be­cause I see things mov­ing closer.”

Pae­di­a­tri­cian-in-chief Ron­ald Cohn at Toronto’s Hospi­tal for Sick Chil­dren, who is also a mus­cu­lar dys­tro­phy doc­tor, is cer­tain that with CRISPR one-of-a-kind treat­ments are pos­si­ble and even likely. Last De­cem­ber, he pub­lished a paper show­ing cor­rec­tions of sev­eral rare mu­ta­tions – again in cells in a lab dish, in­clud­ing some taken from a child with dwarfism and oth­ers from an­other boy with Duchenne. That boy, 14-year-old Gavriel Rosen­feld, is the son of close friends of Cohn’s in Lon­don. They run a char­i­ta­ble foun­da­tion that Cohn ad­vises.

Cohn is a new­comer to CRISPR. A few years ago, he was study­ing hi­ber­nat­ing squir­rels. They don’t move for months, yet their mus­cles aren’t any worse for it. That is the sort of “we might just find some­thing” ap­proach favoured in ba­sic-re­search labs. Now, with gene edit­ing, he sees a di­rect path to cur­ing some­one he knows. Since cor­rect­ing Gavriel’s cells, Cohn’s lab has also cre­ated a mouse model that shares his mu­ta­tion. Like Dupree’s, the mu­ta­tion is one of a kind, and within a few weeks Cohn’s lab will start treat­ing the mice.

But then what? Cohn says he doesn’t know. How would you even test a drug de­signed for one per­son? Who would pay for it? He says he vis­ited Health Canada, the coun­try’s reg­u­la­tor, and was told to come back if he cured the mice. “This is go­ing to re­quire a sig­nif­i­cant re­think­ing,” he says. “The fact that you and I are hav­ing this con­ver­sa­tion is the be­gin­ning of the par­a­digm shift.”

Cohn’s ap­proach of cor­rect­ing in­di­vid­ual mu­ta­tions has stirred hope among par­ents of boys with Duchenne. “This is a CURE!!!” one wrote on the web. His lab has used CRISPR to fix mu­ta­tions in cells taken from sev­eral boys he knows, and a wait­ing list he keeps in a spread­sheet cur­rently lists 53 chil­dren with mus­cu­lar dys­tro­phy. The par­ents of all of them want to know if their child could be helped by gene edit­ing.

If a gene-ther­apy study like the one Olson plans is suc­cess­ful, and if CRISPR reaches enough mus­cle cells, there might be a strong ar­gu­ment that a one­off treat­ment would work. Af­ter all, to aim at a new mu­ta­tion all you would do is tweak the com­po­nent of CRISPR that ze­roes in on a spe­cific DNA se­quence. The price of man­u­fac­tur­ing a sin­gle


dose also might not be an ob­sta­cle. Two ex­ist­ing gene ther­a­pies ap­proved in Europe cost US$1 mil­lion and US$665,000. Even if it cost twice that, a one-time gene fix with CRISPR would be cheaper than a life­time of costly drugs, wheelchairs and de­pen­dency.

In hold­ing out the hope of in­di­vid­ual cures, Cohn ad­mits he has cre­ated some new prob­lems. He has in­vited par­ents to the lab, and lit­tle boys have tot­tered among the lab stools. But he and his stu­dents have de­cided to stop re­fer­ring to “Gavriel’s cells” or “Jake’s cells” and use nu­mer­i­cal code names in­stead. They still know who is who, but this gives them space to be im­par­tial. “I know in the back of my head, but you want to stay un­bi­ased,” says a grad­u­ate stu­dent in the lab, Ta­tianna Wong. “I can’t work on this case just be­cause I feel bad for him. I have sci­en­tific ques­tions to an­swer.”

SOME VET­ER­ANS OF GENE THER­APY roll their eyes when they hear what new­com­ers think CRISPR will do. I vis­ited the vec­tor de­vel­op­ment cen­tre at St. Jude, tour­ing a cramped L-shaped lab with By­oung Ryu, an ex­pert in mak­ing viruses, who chops the air above his head and says, “peo­ple’s ex­pec­ta­tions are up here.” Ryu warns that ba­sic, un­re­solved bi­o­log­i­cal prob­lems re­main. One is whether edit­ing will work of­ten enough in cells such as those in the bone mar­row, the type that need to be changed to cor­rect sickle-cell dis­ease. If too few cells end up edited, the treat­ments won’t be ef­fec­tive. “It’s a num­bers game,” he says.

Ryu was the first em­ployee at a Bos­ton-area gene-ther­apy com­pany, Blue­bird Bio, whose stock price stag­gered down the chart af­ter its first few pa­tients didn’t all re­spond the same way. “I’m not neg­a­tive on CRISPR, but there is a re­al­ity check,” Ryu says. “It’s not com­ing to peo­ple next year. It works in the petri dish ev­ery sin­gle time, but my per­spec­tive is that genome edit­ing may hap­pen in the fu­ture but not in the near term.”

CRISPR’S fu­ture as a treat­ment de­pends heav­ily on the skills of gene ther­a­pists like Ryu. They’ve been mak­ing progress, yet so far only two gene ther­a­pies – the kind that add an en­tire gene – have reached the mar­ket to ad­dress an in­her­ited dis­or­der. One, called Strimvelis, pro­vides an out­right cure for a fa­tal im­mune de­fi­ciency and was ap­proved in 2016 in Europe. It took 15 years to test it on 18 chil­dren, and sim­i­lar tri­als had failed. “What I learned about gene ther­apy is that the rab­bit does not win the race,” says Weiss, who leads the St. Jude ef­fort to ap­ply gene edit­ing to sickle-cell dis­ease. “The tor­toise wins the race.”

Side ef­fects could also be an ob­sta­cle. CRISPR has the po­ten­tial to cause ac­ci­den­tal, un­wanted ed­its that could not be erased if they ended up writ­ten into a per­son’s genome. Cur­rently re­searchers rely on aca­demic com­puter pro­grams to pre­dict such ef­fects. But a pro­gram can’t pre­dict ev­ery­thing. Two early tests of gene ther­apy in the 2000s ac­ci­den­tally caused leukemia in sev­eral chil­dren. No one had an­tic­i­pated that con­se­quence of chang­ing the genome. Al­though Olson says he has not seen ill-ef­fects in his mice, he al­lows that CRISPR can cause “in­ad­ver­tent changes in DNA that are im­por­tant for life”. Edit­ing bil­lions of in­di­vid­ual cells in a per­son’s body, sci­en­tists ac­knowl­edge, will be the surest way to dis­cover how CRISPR can go wrong.

It may take a lot longer than we think, but sooner or later, gene edit­ing will change what medicine looks like. The biotech­nol­ogy in­dus­try be­gan in the 1970s when some­one grafted in­sulin into E. coli, show­ing that a hu­man pro­tein could be man­u­fac­tured out­side the body. Now there’s a way to change DNA where it lies, in­side your genes.

When he looked through a mi­cro­scope at his own cells in Olson’s lab, Dupree tried to take the ra­tio­nal view: here was a so­lu­tion for the next gen­er­a­tion of boys. His mother, how­ever, al­lowed her­self to hope. “I was ec­static. I re­mem­ber think­ing: this could be some­thing that works,” Deb­bie says. Duchenne is a tick­ing clock. Par­ents can’t help mak­ing the cal­cu­la­tions: this long for an­i­mal stud­ies, this many years for the first hu­man trial, that much more time un­til they know if it re­ally works. Luck­ily, Dupree’s dis­ease is the slow-mov­ing kind. The doc­tors said he’d be gone by 19, but he’s still here. Maybe he’ll still be here in 10 years, says his mother, “so they can try it on him”.

Wheel­chair bound Ben Dupree is cau­tiously hop­ing that the re­sults be­ing cooked up in the lab will one day help him.

A vir­tu­oso gene ed­i­tor: CRISPR- Cas9 (3d model) at work on a strand of DNA.

If CRISPR can reach enough of th­ese wasted mus­cle cells a one- off treat­ment could work.

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