PEGGY PETERSON PHOTOGRAPHY C complexity of treatments the means that a sim launched to help most those at cost risk, the and Although antibody-based therapies have been in low-occurred and middle-income countries. and at least three-quarters of deaths those fromdied cardiovascular that disease year, death worldwide. About 17.7 million people and strokedisease are leading the causes of statistics published in2015,ischaemic heart attack.heart and dramatically reduces person’s the risk of a The simple procedure cholesterol trims levels involved inregulating cholesterol blood. inthe targets liver, inthe cells agene tweaking that is against cardiovascular The injection disease. BY ANTHONYKING the riskofcardiovascular disease. Advances ingene editing raise the prospect ofaone-off injection that could reduce vaccine heart-disease The Kiran Musunuru(centre) andhisteamare usinggenomeeditinginthemouselivertomodify enzymes thatregulate levelsof‘bad’cholesterol. benefit tobenefit many more people around world. the pler, one-off such fix as avaccine would of be GENE EDITING The good news is news thatThe good acombination of gene According to World Health Organization a health centrea health to get avaccination a middle-aged person into can walk onsider this scenario: it’s 2037,and ©

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- M a c las Heart Study, alandmark investigation of Dal the interrogated researchers The terol. havealso of effect lowering the LDL choles naturally mutations occurring in soughtCenter inDallas to determine whether Universitythe of Texas Southwestern Medical reduce LDL-cholesterol levels. it its encodes loses function, might therefore Breakingblood. ity, raising level the of LDL cholesterol inthe familiesin the enzyme’s increased the activ ‘bad’ — cholesterol. The mutations uncovered an enzyme that regulates levels of LDL — or tion gene inthe (LDL) cholesterol and harboured who amuta levels lethal oftially low-density lipoprotein French families with members had who poten researchersin 2003,when investigated three becoming reality. The breakthrough came for astrong tackling disease heart chance of has given vision this of future avaccine-led editing technologies such as CRISPR–Cas9 and blossomingdiscovery the of genome- m i l l Sensing possibilities, the investigators at a n

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could could A l l r i g - - - - - h t s 8 MARCH 2018 | VOL 2018 | NATURE 555 | MARCH 8 S23 | r e genetic sense. “They would dramatically be genetic “They sense. tions, on people have who had less luck inthe mutation in gene enable editing will researchers to confer a University of Pennsylvania Philadelphia. in variation and risk the of at disease heart the Kiran Musunuru, studies who human genetic of as them having won genetic the lottery,” says riskin the of ischaemic disease heart which was associated with an 88%reduction mutations inalmost 3%of Americans, African of a different, larger population similarly found mutations inherited two of one broken copy of about 2% of participants African-American had levels of LDL cholesterol. discovered They that DNA from 128 participants the with lowest the sample, blood a researchers the vided sequenced datathe from about 3,600individuals pro who nic groups of United the States. After combing ticipants recruited represent three the main eth in 6,000adults County. living inDallas The par cardiovascular out carried health from 2000–02 s e r Musunuru that thinks next inthe 20 years, v THE FUTUREOFMEDICINE e d . PCSK9 PCSK9 , or muta other beneficial 2 , resulting from one . Afollow-up study OUTLOOK 3 . “I think . “I think - - - - from and amain blood is the also engine of cholesterol — it cholesterol LDL tackle clears liver an is also excellent place from which to of cells than it inmost does other tissues. The cient organ, inthe editing agreater proportion and CRISPR–Cas9 the effi tool is especially is straightforward to deliver genes to liver, the CRISPR Therapeutics, inCambridge; also it Therapeutics inRichmond, and California, in Cambridge, Massachusetts, Sangamo therapy for companies such as Editas Medicine The liver is apreferred target organ of gene THE GATEKEEPER ORGAN condition known as hypercholesterolaemia). targetedto help people cholesterol with high (a become a reality within 5 years — initially reduce risk the of cardiovascular could disease approval.tory He that thinks gene therapy to first gene the (Glybera), therapy to gain regula development the vised of alipogene tiparvovec atpart uniQure in Amsterdam, where he super Netherlands. van Deventer played an important Forbionfirm Capital Partners inNaarden, the Deventer, operating at partner investment and work long inthe term, says Sander van delivering gene editing safe, can be effective restthe of lives,” their he enthuses. protected against attack heart and stroke for human, it would work,” Musunuru says. convinced that if we gave therapythis to a humanthe switched gene be can also off. “I’m (ref. 5). The inshowing team succeeded that CRISPR–Cas9 payload to target human to contain human liver cells, and tuned the lesterol LDL cho and inblood blood the a35–40% fall roughly level90% decrease in the of Pcsk9 in system directed against of an adenovirus containing aCRISPR–Cas9 liver with asingle silenced injection could be that more than of half many commentators have high. too deemed and costs about year, US$14,500per aprice that infusions of for drugs rest the of apatient’s life with statins. But treatment the involves regular by administered 21%, when in combination cut risk of the attack heart by 27%and stroke PCSK9 inhibitor, evolocumab (Repatha), can amount of cholesterol One blood. such inthe receptors and consequently reducing the increasing number the of LDL-cholesterol developed to inhibitbeen enzyme PCSK9, the Already, two therapies antibody-based have bind such cholesterol, its level rises. in blood the inside cell. the With fewer receptors available to ofsurfaces cellsthroughout body, the to move receptors for LDL cholesterol, found on the Baylor of College Medicine inHouston, Texas. says William Lagor, amolecular biologist at removal of excess cholesterol from body,” the lipid synthesis. liver is gatekeeper “The the for S24 | NATURE | S24 2018 VOL | MARCH 555| 8 OUTLOOK The approach is “absolutely plausible, even In 2014, Musunuru and his team showed The enzyme produced by areOthers more bullish. Technologies for 4 . Next, amouse used engineered they THE FUTUREOFMEDICINE Pcsk9 Pcsk9 genes mouse inthe PCSK9 . This to led a causes causes PCSK9 ©

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M a partial fix’). Musunurupartial suggests familial therapeutic gene ‘Benefits editing from a (see trophy are likely first to to from benefit be conditions such as Duchenne muscular dys genetic disorders”. Debilitating single-gene provenbeen safe patients inthe with grievous common after genome diseases editing has greater be will enthusiasm for human for trials have universal approval but that thinks “there pill and we we are think protected,” he warns. “It is way the human the mind works. Take a der efforts to help people to help themselves. therapy for individuals at risk would high hin interventions,” he says. He worries that agene for a population than focusing on high-cost ing lifestyle may much be more effective Netherlands,in the goes further. “Chang ologist at University Medical Centre Utrecht yet,” he says. Karel Moons, aclinical epidemi want to treat haven’t people who got adisease tobarrier negotiate. “You don’t necessarily says Lagor. But there a philosophical is also feasible”, from atechnological point of view, c m Musunuru accepts that not does idea the i coils in dystrophin can be deleted without coils indystrophin canbedeleted without benefit from apartialfix.Some ofthe he estimatesthat80%ofpatients could functional dystrophin. its lengthcaneliminatetheproduction of Olson saysthatmutationsanywhere along containing 79codingsections,orexons, and the genethatencodesdystrophin, islong, analogous tothecoilsofaspring. 20 or so repetitive sections,whichare Dystrophin’s central portioncomprises a shockabsorberinmusclecells( membrane-associated protein thatactsas lie defectsindystrophin, along approach.” CRISPR seemsanideal to themutatedgene. root causeofthedisease, approach istogothe “The onlyreasonable Medical CenterinDallas. of Texas Southwestern biologist attheUniversity it,” saysEricOlson,amolecular has resisted everytherapy appliedto in peoplewiththedisorder. “Thisdisease muscle failure istheleadingcauseofdeath progressive weakening ofmuscles;heart- 1 in 3,500 boysandmen,causesthe gene therapy. Theconditionaffectsupto in thevanguard ofdiseasestargetedby single-gene disorder thatwillprobably be Duchenne musculardystrophy isa l l a At the core of the condition At thecore ofthecondition Rather than correcting specific mutations, Rather thancorrecting specificmutations, n

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in low-income countries with less-well-funded approach for useful people as particularly being weeks for rest the of their lives.” He the views patients have few to antibodies take every is that it would given be just once, whereas strongest rationale for using genome editing and first candidates the be for therapy. “The benefit from genome editing, says Musunuru, most would severe disease receive greatest the to removeblood LDL with Those particles. the against PCSK9 and frequent cleaning of the requirealso treatment with antibodies directed statins, but is this often not enough. might They changes to lifestyle their and as diet, well as take faulty would make alot of to out knock sense the hood — in are who those worst affected. “It premature attacks — sometimes heart inchild LDL-cholesterol levels from causing birth, The associated mutations in families — as asimilarly place to logical start. disorder characterized three inthe French hypercholesterolaemia — the LDL-cholesterol community about thisnewtechnology.” is enormousexcitement inthe Duchenne clinical consequences,” saysOlson. “There muscle disease.There isnoescapingthe years. “Duchenneisthemostdevastating trials inpeoplecanbeginthenext five 90% ofnormaldystrophin levels has usedthetechniquetorestore upto the strategy inmice. Olson’sresearch group study. Hislabisnowevaluating thesafetyof protein,” saysRenzhiHan,wholedthe still read out,youmake apartiallyfunctional of thenormallevelisthoughttoimprove the mutations,” Olsonsays. truncated protein. “Oneeditcanbypassall The shortenedgenewillmake aworking, that containmutationscanberemoved. means thatsectionsofDNAwithin destroying theprotein’s function.This r by aviral vector e s Han and others are optimistic that Han andothersare optimisticthat People hypercholesterolaemia with can make Dystrophin production aslow5% e r v e PCSK9 muscle function; Olson thinks that muscle function;Olsonthinksthat d . a CRISPR–Cas9 system delivered a CRISPR–Cas9systemdelivered reaching 15% would bring reaching 15%wouldbring defective portionof gene patients,” inthose he says. major clinical benefits. In major clinicalbenefits.In simply by slicing out a simply byslicingouta 2017, researchers atthe 14 of mice by up to 40%, of micebyupto40%, Ohio State University in Ohio StateUniversityin levels in the heart muscle levels intheheartmuscle dystrophin-expression Columbus blewpast . “So long as the gene can . “Solongasthegenecan that target, restoring that target,restoring Dmd 15 PCSK9 . DMD using using

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health-care systems: “I do not see daily pills or ANGPTL3 work by different mechanisms, The pace of innovation in gene editing has monthly injections as being a realistic approach in principle they should be additive,” says created an aura of optimism, particularly in the developing world.” But although a one-off Musunuru. Lagor agrees, adding that there are around the treatment of people with genetic treatment should be cheaper, drug companies also economic upsides. “It is likely that the cost disorders who have few or no other options. “It could be tempted to charge a high price, on of targeting two genes, or perhaps even three makes sense to begin therapeutic efforts with the basis that it achieves the same effect as do or four, would be the same as for one gene.” such diseases, even if the understanding of all decades of expensive antibody-based drugs. potential risks is imperfect,” says Liu. But there is For now, Musunuru says that we need REASONABLE OPTIMISM the potential for the technique’s use in the clinic to work out the safest way to perform Before gene-editing therapy can become to spread beyond these testing grounds. van gene editing in people — not necessarily routine, two main safety concerns must be Deventer has successfully lowered LDL choles- CRISPR–Cas9 — and also the best way for it addressed. First, off-target effects can occur terol in mice by silencing apolipoprotein B-100 to be delivered. Regulatory approval for a clini- when the RNA molecule that guides the Cas9 using a method called RNA interference13; he cal trial would then be required, which could cutting enzyme into position misidentifies its sees great potential in using the microRNAs that take a few years to achieve. complementary sequence of DNA, resulting in underpin the technique and, eventually, gene cuts being made in the wrong place. Second, editing to address heart disease. “ANGPTL3, STACKING TARGETS the cellular machinery that repairs the double- PCSK9 and APOC3 Since the discovery of PCSK9, other variants in strand breaks created in the DNA during gene “The strongest are targets not easily genes that alter the risk of cardiovascular disease editing might make an unexpected deletion rationale for addressed by small mol- have emerged. Some affect triglycerides, the or addition. Such mishaps could lead to the using genome ecules or antibodies,” he main component of fat in the body; high levels development of cancer. And although a con- editing is says. And the one-off of triglycerides in the blood are a known risk siderable degree of risk might be acceptable that it would nature of gene-editing factor for heart disease. Apolipoprotein C-III for seriously ill patients with no other option, be given just treatments cuts down on inhibits the breakdown of triglycerides by preventive gene therapy must clear a higher bar. once.” issues with patients not enzymes; a mutation in APOC3, the gene that “If the vaccine is being envisioned for the gen- following advice about encodes it, was discovered in a population of eral population, then it needs to be essentially when to take a drug — a perennial problem Amish people in the United States in 2008 100% safe,” says Musunuru, “at least to the same concerning people on long-term medication. (ref. 6). The 5% of the group who were carri- degree as the infectious-disease vaccinations “If you are talking about cardiovascular ers had lower levels of LDL cholesterol, higher that are routinely given to infants and children.” disease as a global health threat, which it levels of high-density lipoprotein (HDL) — or A new technology from chemical biologist undoubtedly is, then protecting the entire ‘good’ — cholesterol and lower levels of tri- David Liu’s laboratory at Harvard University population is what we need,” says Musunuru. glyceride in the blood, all of which might reduce in Cambridge, Massachusetts, has therefore Lifestyle changes are important, but a sub- the risk of cardiovascular disease. A similar excited those in the gene-editing field. Liu stantial portion of the risk of heart failure pattern has also been found in people who carry has developed a technique that uses a modi- and stroke comes from the genome. “You the mutation in Crete, Greece. fied CRISPR–Cas9 system to alter individual don’t need to choose between medicine and Musunuru is optimistic that knocking out a pairs of bases in cells without having to break lifestyle. You should be doing both,” says Liu, gene called ANGPTL3 can reduce levels of LDL the DNA double strand10. His team was able to citing people with diabetes, who fare best when cholesterol and triglycerides. He was part of a chemically change the DNA base cytosine (C) they take medication and adjust their lifestyle. team that reported in 2010 on three genera- into uracil (a base found in RNA), which the cell “To vaccinate large numbers of people, that tions of a family with mutations in ANGPTL3 later replaced with thymine (T). In 2017, Liu’s is some way off,” says Musunuru. But gene edit- and that had no history of heart disease and team created another tool that could rearrange ing could reset the odds for those who didn’t had low levels of cholesterol and triglycerides an adenine (A) so that it resembled a guanine win the genetic lottery, he predicts. “One way in the blood7. In 2017, three family members (G), and then hoodwinked the cell into fixing or another, genome editing is going to underlie who had a complete loss of function of the pro- the complementary strand of DNA to make the a host of new types of cardiovascular therapies tein encoded by ANGPTL3 were examined8. edit permanent, therefore changing an A•T pair over the next 25 years.” ■ “As far as we can tell, they are substantially into a G•C (ref. 11). protected against cardiovascular disease, but “Base editing is as big a development as the Anthony King is a freelance science writer suffer no harmful consequences whatsoever,” original introduction of CRISPR–Cas9 to the based in Dublin. says Musunuru. At least 1 in 300 people has genome-editing field,” says Musunuru. “It’s 1. Abifadel, M. et al. Nature Genet. 34, 154–156 (2003). a broken copy of ANGPTL3, which has been totally changed how I’ve been thinking about 2. Cohen, J. et al. Nature Genet. 37, 161–165 (2005). shown to reduce the risk of ischaemic heart tackling cardiovascular disease — in a positive 3. Cohen, J. C., Boerwinkle, E., Mosley, T. H. Jr & Hobbs, disease by roughly one-third9. way.” He is planning to test Liu’s A-to-G base H. H. N. Engl. J. Med. 354, 1264–1272 (2006). 4. Ding, Q. et al. Circ. Res. 115, 488–492 (2014). Another potential target is the gene LPA, editor in mice to see how well it works. 5. Wang, X. et al. Arterioscler. Thromb. Vasc. Biol. 36, which encodes lipoprotein (a). High levels of Gene-editing researchers have embraced 783–786 (2016). lipoprotein (a) are a main risk factor for heart targeted base editing to install precise changes 6. Pollin, T. I. et al. Science 322, 1702–1705 (2008). 7. Musunuru, K. et al. N. Engl. J. Med. 363, disease and stroke, yet no treatments have been without the uncertainty that accompanies a 2220–2227 (2010). approved by regulators such as the US Food double-strand break. The technique has been 8. Stitziel, N. O. et al. J. Am. Coll. Cardiol. 16, and Drug Administration specifically to lower used in labs to correct genes in yeast, plants, 2054–2063 (2017). 9. Koschinsky, M. & Boffa, M. Expert Opin. Ther. Targets its levels. “This really is an ideal candidate zebrafish, mice and even human embryos. 18, 747–757 (2014). for disruption with a liver-directed CRISPR A proof-of-concept study by Alexandra 10. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & gene-editing approach,” says Lagor. Initial Chadwick, a postdoctoral researcher in Musun- Liu, D. R. Nature 533, 420–424 (2016). 11. Gaudelli, N. M. et al. Nature 551, 464–471 (2017). candidates for the treatment would be people uru’s lab, delivered a base editor into the livers 12. Chadwick, A. C., Wang, X. & Musunuru, K. with extremely high levels of lipoprotein (a) of adult mice to disable Pcsk9, halving the level Arterioscler. Thromb. Vasc. Biol. 37, 1741–1747 who also have cardiovascular disease. of Pcsk9 and cutting LDL cholesterol by almost (2017). 12 13. Koornneef, A. et al. Mol. Ther. 19, 731–740 (2011). The most effective treatments will probably one-third . Musunuru adds that he has prelimi- 14. Refaey, M. et al. Circ. Res. 121, 923–929 (2017). disrupt several of these genes at once to pro- nary results showing base editing of Angptl3 in 15. Amoasii, L. et al. Sci. Transl. Med. 9, eaan8081 vide the greatest benefit. “Since PCSK9 and mice using Liu’s C-to-T method. (2017).

©2018 Mac millan Publishers Li mited, part of Spri nger Nature. All ri ghts reserved. ©2018 Mac millan Publishers Li mited, part of Spri nger Nature. All ri ghts8r eMARCHserved. 2018 | VOL 555 | NATURE | S25