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TherapeuticGenomeEditing:ProspectsandChallengesDavidBenjaminTuritzCox,RandallJeffreyPlatt,andFengZhang†BroadInstituteofMITandHarvard7CambridgeCenterCambridge,MA02142,USA;McGovernInstituteforBrainResearchDepartmentofBrainandCognitiveSciencesDepartmentofBiologicalEngineeringMassachusettsInstituteofTechnologyCambridge,MA02139,USAAbstractRecentadvancesinthedevelopmentofgenomeeditingtechnologiesbasedonprogrammablenucleaseshavesignificantlyimprovedourabilitytomakeprecisechangesinthegenomesofeukaryoticcells.Genomeeditingisalreadybroadeningourabilitytoelucidatethecontributionofgeneticstodiseasebyfacilitatingthecreationofmoreaccuratecellularandanimalmodelsofpathologicalprocesses.Aparticularlytantalizingapplicationofprogrammablenucleasesisthepotentialtodirectlycorrectgeneticmutationsinaffectedtissuesandcellstotreatdiseasesthatarerefractorytotraditionaltherapies.Herewediscusscurrentprogresstowardsdevelopingprogrammablenuclease-basedtherapiesaswellasfutureprospectsandchallenges.Oftheapproximately25,000annotatedgenesinthehumangenome,mutationsinover3,000geneshavealreadybeenlinkedtodiseasephenotypes(),andmoredisease-relevantgeneticvariationsarebeinguncoveredatastaggeringlyrapidpace.Now,duetosharpdropsinsequencingcost,thecompletionofthehumangenomeproject,andtheexponentialgrowthofhumangenomesequencingdatafromdiseasedindividuals,theroleofgeneticsinhumanhealthhasbecomeamajorfocusofresearch,clinicalmedicineandthedevelopmentoftargetedtherapeutics1.Theseadvancesinourunderstandingofthegeneticbasisofdiseasehaveimprovedourunderstandingofdiseasemechanismsandpointedtowardpotentialtherapeuticstrategies.However,despitevalidtherapeutichypothesesandstrongeffortsindrugdevelopment,therehaveonlybeenalimitednumberofsuccessesusingsmallmoleculestotreatdiseaseswithstronggeneticcontributions2.Emergingtherapeuticstrategiesthatareabletomodifynucleicacidswithindisease-affectedcellsandtissueshavepotentialfortreatmentofmonogenic,highlypenetrantdiseases,suchassevere-combinedimmunodeficiency(SCID),haemophilia,andcertainenzymedeficienciesduetotheirwell-definedgeneticsandoftenlackofsafe,effectivealternativetreatments.Twoofthemostpowerfulgenetictherapeutictechnologiesdevelopedthusfararegenetherapy,whichenablesrestorationofmissinggenefunctionbyviraltransgeneexpression,andRNAinterference(RNAi),whichmediatestargetedrepressionofdefectivegenesbyknockdownofthetargetmRNA(reviewedin3,4).Genetherapyhasbeenusedtosuccessfullytreatmonogenicrecessivedisordersaffectingthehematopoieticsystem,suchas†Towhomcorrespondenceshouldbeaddressed.Correspondenceshouldbeaddressedtozhang@broadinstitute.org..HHSPublicAccessAuthormanuscriptNatMed.Authormanuscript;availableinPMC2015July06.Publishedinfinaleditedformas:NatMed.2015February;21(2):121–131.doi:10.1038/nm.3793.AuthorManuscriptAuthorManuscriptAuthorManuscriptAuthorManuscriptSCIDandWiskott-Aldrichsyndrome,bysemi-randomlyintegratingfunctionalgenesintothegenomeofhematopoieticstem/progenitorcells5-7.RNAihasbeenusedtorepressthefunctionofgenesimplicatedincancer,agerelatedmaculardegenerationandtransthyretin(TTR)-amyloidosisamongothers,resultinginatherapeuticeffectinclinicaltrials(:NCT00689065,NCT01961921andNCT00259753).Despitepromiseandrecentsuccess,genetherapyandRNAihavelimitationsthatpreventtheirutilityforalargenumberofdiseases.Forexample,viralgenetherapymaycausemutagenesisattheinsertionsiteandresultindysregulatedtransgeneexpression6.Alternatively,RNAiuseislimitedtotargetswheregeneknockdownisbeneficial.Also,RNAioftencannotfullyrepressgeneexpression,andisthereforeunlikelytoprovideabenefitfordiseaseswherecompleteablationofgenefunctionisnecessaryfortherapy.RNAimayalsohavepoorspecificity,posingpotentialsafetyconcernsandsometimesdecreasingtheeffectivenessoftreatment8-10.Genomeeditingtechnologiesbasedonprogrammablenucleasessuchasmeganucleases(reviewedin11),zincfingernucleases(reviewedin12),transcriptionactivator-likeeffectornucleases(reviewedin13,14),andclusteredregularlyinterspacedshortpalindromicrepeat(CRISPR)-associatednucleaseCas9(reviewedin15)areopeningthepossibilityofachievingtherapeuticgenomeeditingindiseasedcellsandtissues,resultingintheremovalorcorrectionofdeleteriousmutationsortheinsertionofprotectivemutations.InthisReview,wewilldescribethedifferentnuclease-basedgenomeeditingtechnologies,themechanismsbywhichtheyproducegeneticchanges,considerationsfortheirusesintherapeuticsettingsandmajorchallengesthatwillneedtobeaddressedtorealizetheirclinicaltranslation.Althoughalargenumberofgenomeeditingtherapeuticeffortshavefocusedontreatmentofmonogenic,highly-penetrantdisorders,wewillalsodiscussintriguingtreatmentstrategiestoapplythisclassoftherapytodiseasessuchasviralinfectionsandcancer.GenomeEditingTechnologiesProgrammablenucleasesenableprecisegenomeeditingbyintroducingDNAdoublestrandbreaks(DSBs)atspecificgenomicloci.DSBssubsequentlyrecruitendogenousrepairmachineryforeithernon-homologousend-joining(NHEJ)orhomologydirectedrepair(HDR)totheDSBsitetomediategenomeediting.Todate,f