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T H E C C B Y - N C - N D L I C E N S E ( h t t p : / / c r e a t i v e c o mm o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / ) .

STATE-OF-THE-ART REVIEW

Therapeutic Genome Editing inCardiovascular Diseases

David M. German, MD, MPH,a Shoukhrat Mitalipov, PHD,a,b Anusha Mishra, PHD,a Sanjiv Kaul, MDa

SUMMARY

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A variety of genetic cardiovascular diseases may one day be curable using gene editing technology. Germline genome

editing and correction promises to permanently remove monogenic cardiovascular disorders from the offspring and

subsequent generations of affected families. Although technically feasible and likely to be ready for implementation in

humans in the near future, this approach remains ethically controversial. Although currently beset by several technical

challenges, and not yet past small animal models, somatic genome editingmay also be useful for a variety of cardiovascular

disorders. It potentially avoids ethical concerns about permanent editing of the germline and allows treatment of already

diseased individuals. If technical challenges of Cas9-gRNAdelivery (viral vector immune response, nonviral vector delivery)

can be worked out, then CRISPR-Cas9 may have a significant place in the treatment of a wide variety of disorders in which

partial or complete gene knockout is desired. However, CRISPR may not work for gene correction in the human heart

because of low rates of homology directed repair. Off-target effects also remain a concern, although, thus far, small animal

studies have been reassuring. Some of the therapies mentioned in this review may be ready for small clinical trials in

the near future. (J Am Coll Cardiol Basic Trans Science 2019;4:122–31) © 2019 The Authors. Published by Elsevier on

behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

S ince the successful sequencing of the humangenome more than 15 years ago, there hasbeen an explosion of knowledge regarding the

genetic contributions to common cardiovascular dis-eases, as well as advancement of the understandingof monogenic cardiovascular disorders. Althoughthis knowledge has allowed for the development ofpotent pharmaceuticals and better risk stratificationfor cardiovascular diseases, the rapid developmentof CRISPR-Cas9 techniques in the past 5 years maydramatically change the outlook for novel therapiesin cardiovascular disorders, including those previ-ously thought untreatable.

CRISPR, which stands for “clustered regularlyinterspersed short palindromic repeats,” refers to amechanism that evolved in bacteria to identify and

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m the aKnight Cardiovascular Institute, Oregon Health and Science

bryonic Cell and Gene Therapy, Oregon Health and Science University, Po

ve no relationships relevant to the contents of this paper to disclose.

authors attest they are in compliance with human studies committe

titutions and US Food and Drug Administration guidelines, includin

ormation, visit the JACC: Basic to Translational Science author instruction

nuscript received August 27, 2018; revised manuscript received October

remove foreign DNA from their genomes, using anRNA guide and CRISPR-associated (Cas) nuclease (1).It was quickly recognized that such systems have theincredible ability to facilitate precise editing of genesin both mature and developing organisms. Althoughthe introduction of CRISR-Cas9 has the potentialto revolutionize the mechanistic understanding ofcardiovascular diseases and aide in the developmentof novel pharmacological therapies, it is the prospectof genome modification in humans that wouldtransform how cardiovascular diseases are treated.

Before the latest development of gene editingtechniques, a few approaches that allowed genetargeting in animals existed (e.g., the generation of“knockout” mice). However, these techniques weretime consuming, technically challenging, and

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25, 2018, accepted November 15, 2018.

AB BR E V I A T I O N S

AND ACRONYM S

DMD = Duchenne muscular

dystrophy

DSB = double-stranded break

HCM = hypertrophic

cardiomyopathy

NHEJ = nonhomologous end-

joining

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inefficient. CRISPR has revolutionized gene targetingbecause it allows for production of double-strandedDNA breaks at precise, user-directed locationswithin the genome, thus facilitating editing of achosen segment of DNA in an efficient manner (1).

Using the abilities of the CRISPR-Cas9 system re-quires introduction of a Cas9 nuclease and a segmentof guide RNA (gRNA) into the cell(s) of interest, eitherthrough direct injection or via viral or nonviralvectors. In practice, the Cas9 nuclease most oftencomes from Streptococcus pyogenes (SpCas9), butStaphylococcus aureus Cas9 (SaCas9) is alsofrequently used because of its smaller size (thus,ease of entry into smaller vectors). The gRNA isapproximately 100-nucleotides long and contains a20-nucleotide protospacer sequence that hybridizesto complementary DNA, as well as a shorter 2 to 6nucleotide protospacer-adjacent motif (PAM) thatfacilitates binding of host DNA to the Cas9 nuclease(Central Illustration). It is the customization of theprotospacer that allows for Cas9 nuclease to create aspecific double-stranded break (DSB) in host DNA atuser-specified sites (1). However, the PAM sequence isnecessary for host DNA recognition and binding byCas9, as well as subsequent hybridization with theprotospacer region of the gRNA and eventual DNAcleavage (2). Thus, it represents another key elementrequired for target sequence specificity. For example,Cas9 from S. pyogenes recognizes a 50-NGG-30 PAMsequence, the requirement of which significantlylimits target specificity while also increasing theprobability of off-target activity. Theoretically, such aspecific PAM requirement could limit the therapeuticpotential of the technology. However, Cas9 enzymesfrom other bacterial species (e.g., Streptococcus ther-mophilus) (3,4), as well as those obtained via proteinengineering (5), bind to a variety of PAMs withenhanced nucleotide specificity and fewer nonspe-cific bindings or cleavage. These new Cas9 versionsare likely to render concerns over PAM specificityobsolete in the near future.

After cleavage by CRISPR-Cas9, DSBs can berepaired by 1 of 2 endogenous mechanisms. Nonho-mologous end-joining (NHEJ) is the dominant mech-anism in nonproliferating cells, including humanmyocardial cells, but it is error prone because theaffected DNA segment is reconstructed without use ofa DNA template (Figure 1). This method is highlyvulnerable to production of insertion-deletion muta-tions (indels), and, in general, would not be anacceptable component of genome editing when thedesired outcome is replacement of a dysfunctionalgene with a functional one. However, if knockout isall that is desired, production of indel mutations via

NHEJ may be a reasonable therapeuticapproach, even in somatic cells.

Homology-directed repair (HDR) is thesecond but much less frequent means bywhich DSBs can be fixed in somatic cells.HDR machinery rebuilds the DSB site usingeither homologous chromosomal DNA oran exogenous template DNA strand (a single-stranded oligodeoxynucleotide [ssODN])(Figure 1). The clinical applicability of CRISR-

Cas9 in humans is hampered, in part, by the lowrates of HDR compared with NHEJ in somatic geneediting.

In theory, genome editing techniques can beapplied to both developing embryos and intact matureorganisms to either produce loss-of-function in a genewith deleterious downstream effects (knockout) orto restore function to a mutated gene (knock in). Insomatic genome editing, genetic modifications arecreated in differentiated cells in a developing or adultorganism, which allows for treatment of establisheddisease or for disease prophylaxis in those geneticallyat-risk. Cas9 and gRNA are typically delivered in vivoby a viral vector; adenovirus, adeno-associated virus(AAV), and lentivirus have been used most commonlyin animals, and each has relative advantages anddisadvantages.

Germline genome editing is the process of trans-forming embryonic or germ cell DNA and can besuccessfully accomplished ex vivo via direct co-injection of Cas9 and gRNA with sperm into humanoocytes (6). It allows for complete and permanenttransformation of the human genome, affecting allsubsequent generations.

THERAPEUTIC APPLICATIONS

One of the promises of genome editing is that it willallow for treatment of monogenic diseases thatcurrently have either ineffective or minimallyeffective therapies. In cardiovascular medicine,heritable cardiomyopathies, such as hypertrophiccardiomyopathy (HCM), dilated cardiomyopathy, andDuchenne muscular dystrophy (DMD), as well asheritable arrhythmic disorders, vasculopathies suchas Marfan’s syndrome, and infiltrative diseases suchas transthyretin amyloidosis, are potential candidatesfor clinical applications of germline genome editingtechniques (Central Illustration). Editing of the germ-line in these types of disorders, in which a single genemutation is responsible for disease manifestation, iscapable of permanent correction of the disorder indescendants of affected individuals or those carryinga deleterious mutation.

CENTRAL ILLUSTRATION The Different Genome Editing Approaches (Germline and Somatic)and the Potential Cardiovascular Conditions They Could Treat

German, D.M. et al. J Am Coll Cardiol Basic Trans Science. 2019;4(1):122–31.

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FIGURE 1 Types and Frequencies of DNA Repair in Somatic Cells After Gene Deletion by CRISPR

Homolog template is almost never used, and externally provided template is used infrequently. Almost all the repair is performed using NHEJ.

CRISPR ¼ clustered regularly interspersed short palindromic repeats; DSB ¼ double-stranded break; HDR ¼ homology-directed repair;

NHEJ ¼ nonhomologous end-joining.

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For many disorders, germline genome editing isunlikely to find a major role in treatment and pre-vention because of the interplay of genetic andenvironmental factors in contributing to diseasemanifestation. For complex disorders such as coro-nary artery disease and atherosclerosis, somaticgenome editing may eventually play a role in treat-ment. In addition, somatic genome editing may alsobe useful for post-natal treatment of monogenic dis-orders, especially because clinical application ofgermline genome editing is likely to remain ethicallymore controversial than somatic genome editing forsome time. Nonetheless, with close to 7,500 mono-genic disorders affecting approximately 780 millionpeople, germline editing has the theoretical potentialfor permanently eliminating these diseases.

HYPERTROPHIC CARDIOMYOPATHY. HCM is a dis-ease of cardiac muscle that results in ventricularhypertrophy and has a propensity for arrhythmias,syncope, and heart failure. Ventricular outflow tractobstruction and associated systolic anterior motion ofthe mitral leaflet and resulting mitral regurgitation, aswell enhanced myocardial stiffness from fibrosis, areaccompanying findings. A variety of sarcomeric genemutations have been implicated in the disorder.Mutations in MYBPC3 account for approximately one-third of all HCM in humans, as well as a significantnumber of cases of inherited dilated and non-compaction cardiomyopathy (7). Ma et al. (6) recentlydemonstrated the successful correction of a MYBPC3

mutation in human germ cells using CRISPR-Cas9.They microinjected recombinant Cas9 protein withgRNA and ssODN DNA into human zygotes producedby fertilization of healthy donor oocytes with spermfrom a male donor who was heterozygous for MYBPC3mutation. Most (66.7%) of the embryos injected inthis way exhibited a homozygous wild-type geno-type, as opposed to 47.4% of control embryos. How-ever, 24% of the embryos exhibited mosaicism, and9.3% had a persistent heterozygous mutant genotype.The mosaicism was attributed to the inability ofCRISPR to correct all mutant genes after cell divisionoccurred.

When the investigators co-injected Cas9 withsperm into M-phase oocytes, 72.4% of the 68 result-ing embryos exhibited a homozygous wild-typegenotype, and there were no mosaic or mutantembryos. The absence of mosaicism was attributedto gene correction before the fertilized egg starteddividing. The remaining 27.6% of embryos wereuniformly heterozygous for the wild-type allele anda NHEJ-mediated repair. In addition, genomesequencing of CRISPR-Cas9�targeted blastomeresfailed to reveal significant off-target effects. Thestudy demonstrated, for the first time, that CRISPR-Cas9 could be used to abolish disease-causing muta-tions in human embryos and that modifications to thetiming of Cas9 injection during embryogenesisresulted in significant increases in HDR efficiency(Figure 2) (6). Furthermore, in mouse embryos, it wasshown that adding RAD51 significantly increased the

FIGURE 2 Types and Frequencies of DNA Repair in Germline Cells After Gene Deletion by CRISPR

Unlike somatic cells, the externally provided template is not used, and NHEJ is used only one-third of the time. The homolog template (from

the normal parent) is used for repair in two-thirds of cases. This form of repair is exclusive to the germline. Methods are being developed to

increase homolog repair to 100% to obviate the need for pre-natal genetic diagnosis before in vitro implantation of the corrected embryo.

Abbreviations as in Figure 1.

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chances of HDR repair. This high incidence of HDRseems to be inherent in embryos and is probablymeant to prevent spontaneous mutations fromgermline transmission. This method, in combinationwith pre-implantation genetic diagnosis, may beuseful clinically in the prevention of transmission ofHCM and other monogenic disorders.

Somatic genome editing might be feasible in HCMas well, because the development of cardiac hyper-trophy, myocardial fibrosis, and symptomatic diseaseis generally a gradual process. Mearini et al. (8)administered nonmutant Mybpc3 cDNA to Mybpc3knockout mice (without use of CRISPR-Cas9) via anAAV vector (specifically AAV9, the most cardiotropicAAV serotype) and found that this therapy success-fully increased expression of functional cMyBP-C (tow60% of wild-type levels). This prevented cardiachypertrophy and cardiac functional impairment inyoung mice (8). Although AAV vectors are generallytoo small to package SpCas9, use of other Cas9enzymes may allow for testing of somatic genomeediting of this disease. Several other naturallyoccurring and engineered Cas9 enzymes are smallerin size, which may facilitate such viral delivery (5).However, it must be realized that HDR is uncommonin somatic cells (Figure 2, top panel); thus, genecorrection in the human myocardium may not bepossible.DUCHENNE MUSCULAR DYSTROPHY. DMD is arelatively common X-linked disease that leads toprogressive skeletal muscle weakness and fatal

cardiomyopathy. It is caused by mutations in theDMD gene that codes for dystrophin. The gene is long,with 79 exons, and mutations anywhere along thelength of the gene can cause the entire protein to bedysfunctional. Because of the heritability of the dis-order and the lack of effective therapies, DMD is anappealing candidate for germline genome editing.

Investigators injected Cas9, gRNA targeting exon23, and template ssODN DNA into zygotes of micewith a nonsense Dmd mutation and implanted themodified zygotes into female mice. Sequencing ofDmd exon 23 in these corrected mice revealed mosa-icism in most animals, although a majority of themshowed improvement in muscle function even whenonly a subset of cells had functional dystrophin thatwas restored through either NHEJ or HDR (9). Modi-fications of methods to include nuclease and gRNAinjection into M-phase oocytes with sperm, as wasdone by Ma et al. (6), might further improve theseresults.

Achieving effective in vivo genome editing of car-diac muscle seems a more formidable task because ofthe difficulties of delivering Cas9 and gRNA to cardiactissue. However, in a mouse model of DMD, El Refaeyet al. (10) showed that systemic administration ofS. aureus Cas9 and gRNA in an AAV vector led torestoration of the DMD reading frame, and thus,expression (in w40% of cardiac muscle fibers), andultimately to improvements in cardiac myofiber ar-chitecture, cardiac fibrosis, and papillary musclecontractility. Recently, Amoasii et al. (11) showed an

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increased expression of cardiac and skeletal muscledystrophin in a dog model of DMD after treatmentwith intravenous AAV9 that contained Cas9 andgRNA. These findings demonstrated that, in casessuch as DMD, partial effectiveness also has a signifi-cant potential to improve symptoms and clinicaloutcome. Additional studies will be required toevaluate long-term safety and efficacy in larger ani-mals, and ultimately, humans.

OTHER NONISCHEMIC CARDIOMYOPATHIES. A widevariety of additional causes of nonischemic cardio-myopathy will likely one day be treatable via eithergermline or somatic genome editing. For example,phospholamban regulates intracellular calcium con-centrations through its inhibitory actions on sarco-plasmic reticulum calcium�adenosine triphosphatase(SERCA2), and mutations in the gene for phospo-lamban (PLN) have been identified as a cause ofdilated nonischemic cardiomyopathy (12). Kanekoet al. (13) performed germline genome editing viaCRISPR-Cas9 to knockout the PLN gene in a mousemodel of severe heart failure (calsequestin [CSQ]overexpressing mice). Compared with control heartfailure mice, PLN knockout mice survived longer andhad improved cardiac size and function (13).

DYSLIPIDEMIA AND ATHEROSCLEROTIC CARDIO-

VASCULAR DISEASE. Lipid metabolism is an attrac-tive target for somatic genome editing for severalreasons. Editing of genes involved in the develop-ment of dyslipidemia has the potential to affectnot only individuals with monogenic disorders (e.g.,familial hypercholesterolemia) but also the largepopulation of individuals without monogenic lipiddisorders who have established atherosclerotic dis-ease or elevated risk for cardiovascular events. Inaddition, although progress in somatic gene editinghas thus far been hampered by challenges in genedelivery to tissues of interest, the liver is one partic-ular tissue where success has already been achieved.For instance, AAV vectors have been used success-fully in human trials for gene transfer and treatmentof hemophilia A and B (14,15), and additional pre-clinical studies have used them for somatic genomeediting for lipid disorders.

Proprotein convertase subtilisin/kexin type 9(PCSK9) became a novel therapeutic target for pre-vention of atherosclerotic cardiovascular diseasewhen it was observed that loss-of-function mutationsin PCSK9 were associated with reduced low-densitylipoprotein cholesterol and reduced risk forcoronary heart disease, with no clear adverse clinicalconsequences (16). Pharmacological inhibition ofPCSK9 in vivo also dramatically lowers low-density

lipoprotein cholesterol and reduces cardiovascularevents in subjects with an elevated baseline risk (17).Ding et al. (18) showed that when Cas9 and gRNAtargeting Pcsk9 was delivered in vivo to mice via anadenovirus vector, approximately 50% of the Pcsk9alleles in the liver tissue were successfully edited,with a wide variety of loss-of-function indels gener-ated via NHEJ (Figure 1). No significant off-targeteffects were seen. Furthermore, this experimentresulted in substantially (w90%) lower levels ofplasma PCSK9 levels and total plasma cholesterol(35% to 40% reduction) in the edited mice (18).Similar results were achieved using mice with trans-planted human hepatocytes using an adenovirusvector (19). Although these studies used adenovirusvectors to deliver Cas9 and gRNA to the liver,adenovirus is not a suitable vector for use in humantherapies because of its substantial immunogenicity.Because of its size, S. pyogenes Cas9 cannot be pack-aged in smaller, less immunogenic vectors such asAAV. However, Ran et al. (20) produced knockout ofPcsk9 in a mouse liver using an AAV vector packagedwith S. aureus Cas9, which resulted in w95% decreasein blood PCSK9 and w40% decrease in blood choles-terol levels with no major off-target effects. Thesestudies provided proof-of-concept that it might soonbe possible to immunize patients against atheroscle-rotic cardiovascular disease by using somatic genomeediting to permanently lower plasma lipid levels.Additional work will need to ensure these methodsare safe and effective in humans.

Although some of the results of these earlyinvestigations in somatic genome editing in cardio-vascular disease have been encouraging, most ofthese studies have depended on NHEJ-mediated DSBrepair (Figure 1). Because HDR tends to be inefficientand to only operate in proliferating cells, these tech-niques would not be useful for producing gain-of-function genome edits in mature cardiac or vasculartissues. In addition, off-target effects have the po-tential to be more damaging if NHEJ is used (poten-tially producing activating or inactivating indelmutations in proto-oncogenes or tumor suppressorgenes, or producing loss of function mutations inother essential genes).

One promising technique that may be able toovercome this limitation is in vivo base editing(21,22). Base editors are CRISPR-Cas9 systems thathave been modified to alter single base pairs ratherthan induce DSBs. For example, base editor 3 (BE3)is a S. pyogenes Cas9 mutated to induce only asingle-strand break, and it is attached to a cytosinedeaminase domain that exchanges a cytosine base atthe nick site for a uracil base. This modified Cas9

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enzyme then removes the corresponding guaninebase, and the result is a C-G pair replaced with aU-A pair; the uracil is ultimately permanentlyreplaced with thymine. Chadwick et al. (23) used anadenovirus vector and BE3 to show that this methodcould also be used to disrupt mouse Pcsk9, and againfound significant reductions in plasma PCSK9 andcholesterol levels in treated mice.

These techniques were extended to modification ofANGPTL3 (angiopoietin-like 3), which codes for aprotein that regulates blood lipid levels. Loss-of-function mutations in ANGPTL3 are associated withreduced low-density lipoprotein cholesterol and tri-glycerides, as well as a reduced risk for coronary heartdisease (24,25). Chadwick et al. (26) again used BE3,introduced via an adenovirus vector, to producecytosine-to-thymine nonsense mutations in Angptl3in mice. Treated mice had a 31% decline in plasmatriglycerides and a 19% decline in plasma cholesterol.There were no significant signs of off-target muta-genesis (26). Although the current usefulness of base-editing techniques is limited to generating specificpoint mutations largely confined to the generationof knockouts, and further limited by challenges inpackaging larger components into small viral vectors,future advances may ameliorate some of these prob-lems. In addition to PCSK9 and ANGPTL3, somaticgenome editing may be useful in addressing cardio-vascular risk carried by LPA, APOB, or other geneticmutations. Germline genome editing may also beuseful in treating heritable monogenic dyslipidemias.

AGE-RELATED CLONAL HEMATOPOIESIS. Somaticgenome editing has the additional potential ofproviding treatment for diseases that are only nowbeginning to be understood. Clonal hematopoiesis(of indeterminate potential, CHIP), which is alsoknown as age-related clonal hematopoiesis (ARCH),has recently become widely appreciated as a possiblecontributor to cardiovascular disease. It refers to theclonal amplification of hematopoietic cells due tothe accumulation of somatic mutations that confercompetitive advantages to these cells at the expenseof other cell types (27). Its prevalence increases withage, and it is associated with increased risk foratherosclerotic cardiovascular disease, stroke, anddeath (28). Although mutations in a large variety ofdriver genes have been implicated in the develop-ment of ARCH, TET2 and DNMT3A are believed toplay a major role.

Sano et al. (29) used lentivirus vectors carryingCas9 and gRNA to inactivate Tet2 and Dnmt3a (viaindel mutations) in mouse bone marrow cells andthen implanted those cells into irradiated mice.

Mice with Tet2 or Dnmt3a inactivation displayeda greater decline in cardiac function as well asincreased cardiac size and fibrosis when challengedwith angiotensin II infusion compared with thosewithout inactivation of those genes. In addition,inactivation of both genes led to increased expressionof pro-inflammatory cytokines (29). These resultsfurther implicated these genes in ARCH�mediatedcardiovascular disease, and potentially suggest afuture therapy. If methods can be developed to reli-ably knock in somatic gene mutations, it may bepossible to reverse driver gene mutations using avector that targets hematopoietic stem and/or pro-genitor cells. Consequently, somatic genome editingmay be a useful approach to treating ARCH inhumans, thus reducing cardiovascular risk. Theseapproaches may be preferable to bone marrowtransplantation.

TRANSTHYRETIN CARDIAC AMYLOIDOSIS. One of thechallenges of somatic genome editing has beendefining the optimal method for delivery of theCRISPR-Cas9 components to the cells of interest. Viralvectors are not optimal for this purpose. Finn et al. (30)demonstrated that, under the right conditions,nonviral vectors may be able to successfully deliversomatic genome editing tools to desired tissues.Transthyretin (TTR) cardiac amyloidosis is an infiltra-tive disease of cardiac muscle that results from depo-sition of abnormal pre-albumin (transthyretin) proteinand is caused by either heritable TTR gene mutationsor accumulation of wild-type transthyretin. It may bean attractive candidate for somatic genome editingbecause most transthyretin is produced in the liver.

Finn et al. (30) administered a lipid nanoparticlepackaged with Cas9 mRNA and gRNA targeted tothe Ttr gene in mice and found a significant (>97%)drop in serum TTR levels. It is unclear whetherthis approach will translate into disease phenotyperescue or prevention. Additional studies will beneeded to investigate the effects on cardiac tissueand to determine whether the lipid nanoparticlevector can be used successfully for other conditions.Other nonviral vectors (e.g., microbubbles) that canbe selectively destroyed in the tissue of interest byultrasound to introduce the vector locally also holdpromise (31).

INHERITED ARRHYTHMIC DISORDERS. Channelo-pathies and other inherited arrhythmic disorders areanother area of potential therapeutic application ofgenome editing. Although CRISPR-Cas9 is alreadyproving useful in the characterization of ion channelprotein function and drug�gene interactions ininduced pluripotent stem cell�based cell cultures,

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genome editing will also likely provide the opportu-nity to treat these rare disorders.

PRKAG2 syndrome is a rare familial disorder char-acterized by abnormal glycogen storage, ventricularpre-excitation, recurrent arrhythmias, and cardiachypertrophy. It is caused by mutations in the PRKAG2gene coding for a regulatory subunit of the adenosinemonophosphate-activated protein kinase (AMPK)(32). Xie et al. (33) showed that post-natal correctionof the disorder was achieved in mice via a singleadministration of Cas9 and gRNA in an AAV9 vector,with significant reduction in left ventricular wallthickness, decreased myocardial glycogen content,normalization of the QRS width and PR intervals, andimprovements in myofibril organization and ventric-ular function. Further work is required to translatethese results to different types of PRKAG2 mutationsand to larger animals to better define the correctedphenotype.

Long QT syndrome (LQTS), which predisposesindividuals to life-threatening cardiac arrhythmias,would potentially be an attractive candidate forgermline and somatic genome editing. Rare cases ofLQTS are caused by mutations in genes that code forcalmodulin (CALM1, CALM2, and CALM3), a proteinthat binds calcium and interacts closely with L-typecalcium channels in cardiomyocytes. When this pro-tein is over-expressed, it causes action potentialprolongation. CRISPR interference is a novel methodof modulating gene expression without permanentlymodifying the genome; gene expression is modifiedby using dCas9 (“dead” Cas9, which lacks endonu-clease activity) along with a transcriptional activatoror suppressor. Limpitikul et al. (34) cultured car-diomyocytes from induced pluripotent stem cellsderived from a patient with a disease-causing CALM2mutation, and demonstrated that these cells recapit-ulated the cellular phenotype of LQTS. Treatmentwith CRISPR interference significantly lowered levelsof CALM2 mRNA, and calmodulin protein, and alsoreduced action potential duration. In addition, theseinvestigators showed that expression of CALM1 orCALM3 could also be selectively reduced in this way(34). It remains to be seen whether this approachmight be effective in vivo or applicable to othervariants of LQTS or other cardiac conditions.

MARFAN SYNDROME. Base editing techniques maybe one way of more precisely correcting pathogenicsingle nucleotide mutations with perhaps fewerconcerns about the inefficiencies of HDR and aboutoff-target effects. Marfan syndrome is a connectivetissue disorder associated with abnormalities inmultiple organ systems, but for which morbidity and

mortality are most closely tied to the risk of thoracicaortic aneurysm and aortic dissection. It is most oftencaused by autosomal dominant mutations in the geneFBN1, which codes for the extracellular matrix pro-tein fibrillin-1 (35). Zeng et al. (36) recently identifiedan individual with Marfan syndrome who was het-erozygous for a pathogenic T7498C mutation in theFBN1 gene and cultured human cells with an identicalmutation in FBN1. After those cells were transfectedwith gRNA and BE3, sequencing revealed that 10 of20 clones had been edited, 8 with a desirable C-to-Tcorrection and 2 others with unwanted base pairconversions. The investigators then tested the tech-nique in human embryos; zygotes produced viain vitro fertilization of donor oocytes with sperm fromthe same Marfan syndrome patient were subse-quently microinjected with BE3 and gRNA. In 100% ofthe 7 treated embryos, the FBN1 T7498C mutationwas corrected, compared with 50% of controlembryos, although there was 1 unwanted base con-version in 1 of the treated embryos. No unintendedcorrections were discovered via screening of potentialoff-target sites in corrected embryos (36). The resultsprovided proof-of-concept that base editing tech-niques might be applicable to the correction of path-ogenic gene mutations in the human germline,especially if problems of off-target conversions couldbe more thoroughly resolved.

ETHICAL CONSIDERATIONS

Several ethical concerns must be considered withgermline gene editing because any intended andunintended changes would be transmitted to subse-quent generations. Mosaicism is also a cause of majorconcern. However, as shown by Ma et al. (6), mosai-cism can be largely prevented if CRISPR is introducedbefore cell division starts. Furthermore, detailedanalysis demonstrated no off-target effects. However,the gene edited in this case was only 4 base pairslong, and more off-target effects may occur whenediting larger genes (37). Whether these off-targeteffects are automatically recognized and correctedin the human embryo is not known. Consequently,more studies need to confirm the absence of off-target effects in human genome editing, and tofurther develop novel techniques such as base editingthat might allow for more precise mutation correctionin select cases.

One can argue that as long as we have pre-implantation genetic diagnosis we do not needgermline gene editing because we can select theunaffected embryos for implantation. However, thisargument does not hold for polygenic diseases,

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or when both parents are homozygous for a genevariant. It has also been argued that applications ofgermline gene editing could widen inappropriatelyand be used for purposes other than therapy. How-ever, we must address these concerns with effectivepolicies rather than prevention of the developmentof potential therapies. International consensus andtight regulation will be critical to ensure that thistechnology is only used for necessary treatmentpurposes rather than creating “designer” babies orproviding nonessential treatment. Indeed, recentreports suggest that CRISPR-Cas9 has already beenused to create babies in China, sparking an interna-tional debate and stressing the immediate importanceof setting up such regulatory systems (38). The earlierthese deliberations begin, the better prepared societywill be for such treatments when they becomeavailable.

Another concern is that the expense associatedwith germline gene editing and in vitro fertilizationwill result in the treatment only benefiting wealthypatients. Coverage by insurance could mitigate thisconcern, and governmental health care servicesshould take this possibility into account when insti-tuting future policies. In many instances, the cost ofgermline gene editing and in vitro fertilization wouldbe significantly less than the lifelong pharmacologicaltreatment of the condition, therefore making thesetreatments economically more attractive.

Finally, objections to germline gene editing onreligious grounds will continue to exist and peoplewith such objections can decline this therapy.Accordingly, the National Academy of Medicine hasrecommended that attempts to make gene therapysafe should continue (39).

CONCLUSIONS

It is rapidly becoming apparent that a wide varietyof cardiovascular diseases may one day be curableusing CRISPR-Cas9 or similar technology, includingmany that heretofore have been entirely untreatable.

Germline genome editing promises to permanentlyresolve monogenic cardiovascular disorders forthe offspring and subsequent generations of affectedindividuals. Although technically easier and likely tobe ready for implementation in humans in the nearfuture, this approach remains ethically controversial.Public debate and public policy determinations willneed to proceed rapidly to allow decisions to be maderegarding how and when these therapies may be usedclinically. In addition, further technical matters willneed to be more fully resolved, including those oflong-term risks, off-target effects, mosaicism, andapplicability to a wider variety of mutations andcardiovascular conditions.

Although currently beset by several technicalchallenges, and not yet past small animal models,somatic genome editing may also be useful for avariety of cardiovascular disorders. It potentiallyavoids ethical concerns about permanent editingof the germline and allows treatment of alreadydiseased individuals. If technical challenges ofCas9-gRNA delivery (viral vector immune response,nonviral vector delivery, size of the vector) can beworked out in large animals and humans, thenCRISPR-Cas9 may have a significant place in thetreatment of a wide variety of disorders in whichpartial or complete gene knockout is desired (e.g., forPCSK9 and DMD). More challenging (and perhapsunachievable) will be knock in via HDR or baseediting in nonproliferating cells. Off-target effectsremain a concern in somatic genome editing as well,although small animal studies thus far have beenreassuring. Some of the therapies mentioned in thisreview will be ready for small clinical trials in the nearfuture (perhaps soonest in high-risk patients withhereditary lipid disorders).

ADDRESS FOR CORRESPONDENCE: Dr. Sanjiv Kaul,Knight Cardiovascular Institute, Oregon Health &Science University, UHN 62, 3181 Southwest SamJackson Park Road, Portland, Oregon 97239. E-mail:kauls@ohsu.edu.

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KEY WORDS CRISPR, gene editing,germline gene correction