Circulation Research

REVIEW Therapy for the Heart Lessons Learned and Future Perspectives

Antonio Cannatà, Hashim Ali, Gianfranco Sinagra, Mauro Giacca

ABSTRACT: While clinical celebrates its first successes, with several products already approved for clinical use and several hundreds in the final stages of the clinical approval pipeline, there is not a single gene therapy approach that has worked for the heart. Here, we review the past experience gained in the several cardiac gene therapy clinical trials that had the goal of inducing therapeutic in the ischemic heart and in the attempts at modulating cardiac function in . Critical assessment of the results so far achieved indicates that the efficiency of cardiac gene delivery remains a major hurdle preventing success but also that improvements need to be sought in establishing more reliable large animal models, choosing more effective therapeutic , better designing clinical trials, and more deeply understanding cardiac biology. We also emphasize a few areas of cardiac gene therapy development that hold great promise for the future. In particular, the transition from gene addition studies using -coding cDNAs to the modulation of using small RNA therapeutics and the improvement of precise gene editing now pave the way to applications such as cardiac regeneration after and gene correction for inherited that were unapproachable until a decade ago.

Key Words: gene editing ◼ heart failure ◼ microRNA ◼ nanoparticles ◼ regeneration Downloaded from http://ahajournals.org by on May 7, 2020

t was 1989 when Steven Rosenberg and his team at and a few more hundreds are in the final stages of the the National Cancer Institute in Bethesda performed clinical approval pipeline.2 Given the rapid pace of prog- Ithe first approved genetic modification in humans.1 ress of gene therapy product development, the FDA has These investigators used a retroviral vector to genetically recently (January 2020) released 6 new guidelines for mark the lymphocytes that were retrieved from the tumor the Chemistry, Manufacturing, and Control information mass of a patient with melanoma before their reinfusion for human gene therapy investigational new drug appli- into the same patient. The purpose of the study was to cations (https://www.fda.gov/vaccines-blood-biologics/ prove that tumor infiltrating lymphocytes, once isolated biologics-guidances/cellular-gene-therapy-guidances). and expanded in vitro, maintain the capacity to home to These streamlined and standardized guidelines are the tumor and its metastasis. It was not only a key finding expected to further speed clinical application in this area. for immunotherapy but also the official birth of the field While gene therapy celebrates its clinical successes, of gene therapy. it is quite surprising that not a single application targets In the 30 years that followed, according to the Gene any of the diseases affecting the heart, while these rep- Therapy Clinical Trials Worldwide database (http://www. resent a most prevalent cause of morbidity, disability, abedia.com/wiley/, updated September 2019), ≈3000 and mortality in the world. This void reflects the many clinical trials have been approved, conducted, or initi- hurdles that are intrinsic to the development of gene ated in most fields of clinical medicine. Success has therapy in this area. taken a long time. Eventually, however, there are now This article reviews the past experience gained in car- seven products already approved by the Food and Drug diac gene therapy clinical trials and highlights the prob- Administration (FDA) or European Medicines Agency, lems that still need to be solved before success. It also

Correspondence to: Mauro Giacca, MD, PhD, King’s College London School of Cardiovascular Medicine and Sciences, The James Black Centre, 125 Coldharbour Ln, London SE5 9NU. Email [email protected] For Sources of Funding and Disclosures, see page 1409. © 2020 American Heart Association, Inc. Circulation Research is available at www.ahajournals.org/journal/res

1394 May 8, 2020 Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 Review

8 9 Fig- 1395 Despite Despite 6 May 8, 2020 Cardiac Gene Therapy Gene Cardiac AAV transgenes are transgenes are AAV 5 and Grimm and Büning. 7 10 ; cf. also later). Not different from other cell types, from other cell Not different ; cf. also later). . First generation adenoviral vectors, in which a which in vectors, adenoviral generation First 1A. 4 A vector family that is now considered the gold stan- gold the considered now is that family vector A Extensive experimentation in small and large animals in small and large Extensive experimentation their already satisfactory efficiency in transducing post- their already satisfactory efficiency in to signifi- amenable are still vectors tissues, AAV mitotic efficiency, cant improvement in terms of transduction and immune eva- tissue specificity, capacity, packaging capsid play key the AAV sion. In particular, AAV designing of field The processes. these all in roles has evolved from capsids with improved characteristics rationale design of selective cap gene mutants, to the generation of randomly mutated or shuffled libraries, are then selected and evolved in vivo for organ which specificity and liver de-targeting. Some of the gener- These ated variants display improved cardiac selectivity. and AAV-SASTG AAV2i8G9, include the AAV2i8, serotype 9 variants, or vectors some AAV chimeras, obtained through the screening of peptide display libraries or DNA-shuffled libraries (reviewed in study and Giacca by Zacchigna therapeutic angiogenesis in the 1990s angiogenesis in therapeutic at inject- aimed DNAamounts of naked ing large coding for plasmids the on based myocardium the into factors angiogenic could spontaneously that cardiomyocytes assumption DNA. extracellular internalize clinical tri- Double-blinded is largely this approach clearly indicated that als have - in study by Giacca and Zacchi unsatisfactory (reviewed gna Besides targeting cardiomyocytes, these capsid engi- can also be applied to endothelial neering approaches cells. A random peptide library display screening has delivery permit targeted motifs that revealed peptide into coronary artery endothelial cells using the AAV2 serotypes. or AAV9 expressed indefinitely. We have previously reviewed previously reviewed We have indefinitely. expressed of these vectors. the properties extensively therapeutic gene expression cassette substitutes the therapeutic gene expression to clinical tri- viral E1 and/or E3 regions, have advanced als for applications in both therapeutic angiogenesis and these vectors still retain a number heart failure. However, stimulates of which of adenoviral genes, the expression responses, which important inflammatory and immune concerns. and efficacy raise safety is based on the dard for cardiomyocyte gene transfer and nonpathogenic parvovirus AAV, small, defective, tropism of these especially because of the specific most notably car- vectors for postmitotic cells in vivo, vectors, for diomyocytes. Compared with adenoviral returns to baseline within transgene expression which weeks from administration, a few ure the nonpolar and hydrophobic nature of the cardiac sar- the nonpolar and hydrophobic a formidable barrier to negatively colemma represents nucleic acids. charged that at least 2 viral vector systems has instead indicated those based on at cardiac gene transfer, are effective (AAV); virus the adenoassociated and adenovirus This anecdote well reflects the This 3 clustered regularly interspaced short clustered regularly interspaced palindromic repeats dilated receptor epidermal growth factor fibroblast growth factor follistatin-like 1 factor granulocyte colony stimulating hypertrophic cardiomyopathy heart failure homologous recombination inhibitor-1c interleukin-6 myocardial infarction microRNAs nucleic acid locked long noncoding RNA myocardin-related transcription factor neuregulin platelet-derived growth factor protein-kinase A phospholamban protein phosphatase 1 RNA interference stromal cell–derived factor transcription activator-like effector nuclease vascular endothelial growth factor adenoassociated virus adenoassociated cyclase adenylate antisense oligonucleotide

Efficiency of nucleic acid delivery essentially relies essentially delivery acid nucleic of Efficiency Nonstandard Abbreviations and Acronyms Nonstandard Abbreviations DCM EGFR FGF FSTL G-CSF HCM HF HR I-1c IL-6 MI miRNA LNA lncRNA MRTF NRG PDGF PKA PLN PP1 RNAi SDF TALEN VEGF AAV AC ASO CRISPR problems that this discipline had to face over the years in all fields. Cardiac gene therapy has not been immune from this issue. on 2 parameters, the gene vehicle and the route of induce Early attempts at gene therapy to administration. In the late 1990s, when gene therapy, after an initial In the late 1990s, when gene therapy, drawbacks, tremendous phase, had suffered excitement a gene therapy pio- a journalist interviewed Inder Verma, were the 3 major unsolved issues asking him which neer, answer was adamant: according to his opinion. Verma’s are only 3 problems in gene therapy: delivery, “There delivery and delivery”. DELIVERY, DELIVERY, DELIVERY DELIVERY, DELIVERY, presents 3 new areas (small RNA (small areas new 3 presents cardiac therapeutics, that hold great and cardiac gene editing) regeneration, promise for future application. Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 Cannatà et al Cannatà

Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Review the dystrophic phenotypeinmice. specific gene expression, which could efficiently correct enhanced,muscle- a syntheticpromoterwith400-fold which endedinthegenerationofAAV vectorscarrying similar approach to that performed in , search of such tissue-specific promoters can follow a 1396 (reviewed instudybyDomenger andGrimm been thesubjectofinvestigationoverlastfew years or endothelialcellspecificityanddetargettheliverhas thetic regulatory sequences that provide cardiomyocyte characteristics. The rationaldesignorselectionofsyn- ment pertains to the design of promoters with improved remains justanaspectofimprovedtissue-specifictropism. internalization andCapfunction.Thus, capsidengineering and genomeconcatemerization)thatextend beyondvirion conversion port, single-stranded to double-stranded DNA molecular steps(inparticular, de-capsidation,nucleartrans- efficacy, sincethisistheultimateoutcomeofanumber engineering does not necessarily translate into improved expanding therepertoireofavailablevectorsbycapsid bypass surgery, orafterpercutaneouscatheterizationtoreach theleftventricle,followedbytransendocardialdelivery. coronary sinus,ontheleftsidepanel;orintramyocardial,rightpanel,througheitherdirectinjectionafterminithoracotomyduring reach theheart.These includeinjectionintothecoronaryarteryasduringstandardpercutaneousinterventionorretrograde adenoviral oradenoassociatedvirus[AAV]-based vectors).The approximate sizeofthedelivery vehicleisindicated.B,Maindeliveryroutesto orgenetransductionusing A, Schematic representationofthemaindeliverystrategiesforcardiacgenetherapy(injectionnakedplasmidDNA Figure 1.Deliveryvehiclesandroutesforcardiacgenetherapy. Cannatà etal A secondareaamenabletoAAV vector improve- In thisregard,however, itneedstobepointedoutthat May 8,2020 12 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 DOI: Circulation Research.2020;126:1394–1414. 11 ). The application by either FDA or EMA, or both. application by either or FDA EMA, catheters have been testedandapproved for human is lessinvasiveandfeasible instablepatient.Several performed inseveralroutinecardiaccatheterizations, ous approach, which isarelativelycommonprocedure related tocardiacsurgery. Conversely, thepercutane- dial access;however, itisfraughtwiththeproblems dial approach is straightforward in terms of myocar - transendocardial delivery(Figure 1B).The transepicar - lar injection using a percutaneous catheter, to achieve lowed by transepicardial delivery, or by intraventricu- through asurgicalaccess, that is,mini-thoracotomy fol- is thedirectintramyocardialinjection. This canbe either mon andeffective technique forcardiacgenetransfer gene therapyistherouteofadministration.Amostcom- important variableaffecting overallefficacyofcardiac Myostar (Biologics DeliverySystems,Diamond Bar, CA), San Francisco, CA), MyoCath(Bioheart, Inc,Sunrise,FL), monly useddevicesarethe Helix (BioCardia,Inc,South be used to inject gene therapy products. The mostcom- catheters were indicated for cell therapy, they might also In additiontogenetransfer efficiency, asecondmost Cardiac GeneTherapy 13 While these Review

). 33 30 4 165 31 1397 trials; 28 Over 20 25 A similar fate 34 VEGF121.10NH May 8, 2020 Cardiac Gene Therapy Gene Cardiac GV 2. While REVASC ); Figure 2. While ); and by transendocardial by and ); AGENT and AGENT-2 were 34 33 35 ) and in one small study aimed at plasmid efficacy by the administra- 29 ; via mini-thoracotomy in a Phase 165 32 cDNA under the control of the strong 121 and KAT [Kuopio Angiogenesis Trial] [Kuopio and KAT NORTHERN [NOGARevas- Angiogenesis 27 26 , one of the master regulator of the angiogenesis , one of the master Compared with plasmid DNA, much more effective Compared with plasmid DNA, more effective much 165 CMV enhancer/promoter, named Ad CMV enhancer/promoter, reported durable improvements in cardiac function cardiac in improvements durable reported perfusion, of increased proof of objective a lack despite clinical applications in the late 1990s in the late clinical applications 2000s and early plasmids of injection intramyocardial the entailed have VEGFencoding growth factor)- (vascular endothelial A (commercial name: BIOBYPASS), which was used in which (commercial name: BIOBYPASS), artery dis- with coronary in patients a series of studies into the myocardium dur- vector was delivered ease. The an open-labeled, ing coronary bypass artery grafting in noncontrolled trial Someare largely results from these applications positive had an open-labeled designed and anecdotal, as these the placebo strong in angiogenic is notoriously effect randomized, double-blinded, placebo- therapies. When naked plasmid DNAcontrolled trials with were injection to show a major positive impact performed, these failed or symptoms (the EUROINon either clinical outcome - JECT1, the NOVA trial was prematurely terminated due to a trial was prematurely terminated the NOVA - sponsoring company’s portfolio decision, after no differ ence was detected in the treated patients. cDNA delivery vehicles for cardiac gene therapy are the first adenovi- vectors based on adenovirus. One of - a vector express ral vectors to be used clinically was ing the VEGF-A (the VIF-CAD study plasmid Additional clinical studies with intramyocardial based factor, delivery are ongoing for hepatocyte growth on a previous, small clinical Phase I experimentation. process that is triggered and maintained by secreted maintained by is triggered and process that induc- cells and later acting on endothelial factors first capillaries. of the newly formed ing maturation tion of G-CSF (granulocyte colony stimulating factor). increasing VEGF-A ). Unsatisfactory results were also reported for a Figure 2). Unsatisfactory results were also reported bi-cistronic plasmid encoding both FGF2 VEGF-A and (premature termination) also occurred for the AGENT clinical trial program, which (Angiogenic Gene Therapy) the potential benefit of the intra- was aimed at exploring expressing vectors 5 adenovirus an of delivery coronary FGF4, named Ad5FGF-4. randomized, double-blinded, placebo-controlleddouble-blinded, randomized, in trials stable angina. AGENT-3 and -4 tri- patients with chronic als were planned to determine the efficacy and safety II, randomized, nonplacebo, controlled trial in no-option patients, performed at multiple clinical sites in North America (the REVASC trial [Randomized Evaluation of VEGFAngiogenesis] for cularization Therapy: Assessment by RadioNuclide Assessment Therapy: cularization Imaging], process (reviewed in study by Giacca and Zacchigna process (reviewed delivery using the NOGA system in 2 studies performed trial in Europe (the NOVA 18–20 The only The as this pro- 14–17 21 22 The site of injection can be The 13 Vein system anatomy, presence of presence anatomy, system Vein 23,24 New blood vessel formation in adult life occurs New blood vessel formation in adult life An alternative approach to anterograde coronary infu- An alternative approach Over 30 taken cell therapy clinical studies have transepicardial, percutaneous, FDA-approved percutaneous, transepicardial, device, the Santa Vascular, (Medtronic System Delivery TransAccess the cardiac venous be advanced into can Rosa, CA), a and, using approach vein a femoral system through by intravascular ultrasound, needle guided trans-vascular treatment advanced to deliver the desired effectively layer. from the epicardial The birth of gene therapy for cardiovascular disorders birth of gene therapy for cardiovascular disorders The in the mid 1990s coincided with a series of attempts at inducing therapeutic angiogenesis in patients with applica- coronary or peripheral artery disease. These vessel blood new that notion the by fueled were tions formation is a cytokine-driven process and that the over- of some of the angiogenic cytokines was expression animal in ischemia of conditions acute solve to sufficient after over 150 clinical trials, there is not models. Yet, therapeutic a single successful application to achieve angiogenesis in humans. vessels, a through capillary sprouting from preexisting GENE THERAPY CLINICAL STUDIES INDUCETO CARDIAC THERAPEUTIC ANGIOGENESIS sion is retrograde infusion through the vein system. This vein system. This sion is retrograde infusion through the is commonly used in cardiac surgery to which approach, can increase transduction cardioplegia, deliver effective fashion. However, a transepicardial in especially efficacy, bothof occlusion balloon requires it artery coronary the is proportional to and the respective vein, and its efficacy time. occlusion the advantage of intracoronary administration, advantage of intracoronary and Stiletto (Boston Scientific, Natick, MA). Natick, (Boston Scientific, and Stiletto cedure is routinely performed for left heart catheteriza- cedure is routinely intervention. tion and invasive percutaneous coronary employed for treatment delivery 2 main techniques The occlusion of are either infusion after complete balloon a sub or nonocclu- the coronary flow or infusion during rate is relatively sive procedure. Despite the complication is that the the main disadvantage of this approach low, flow. coronary the with compete to has treatment infused using be achieved Improvements in this procedure might retrograde, instead agents increasing permeability or by of anterograde, vector administration. identified preoperatively by different imaging techniques techniques imaging different by preoperatively identified the procedure by either electro- and confirmed during mapping using the NOGAmechanical system (Biosense ultrasound or intravascular CA), Diamond Bar, Webster, 3-dimensional cardiac imaging. 2-dimensional and cardiac resynchronization devices, and a relatively high cardiac resynchronization might hinder the risk of coronary sinus or vein rupture widespread applicability of this technique. Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 Cannatà et al Cannatà

Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Review 1398 Coronary ArteryDisease];NCT01550614 cardial Ischemia inPatients With StableAnginaDueto for Myo- trial [Efficacy andSafety of Ad5FGF-4 ASPIRE tradename ofGenerx,wasdeliveredintracoronary(the vector, underthe patients inwhomthesameAd5FGF4 in11 assessed myocardialperfusionusingSPECT studies wasanapplicationin6centersRussia,which currently notreported.Finally, afollow-upoftheAGENT The recruitmentstatus ofthistrialis NCT00438867). toris Who AreNotCandidatesforRevascularization]; AWARE trial[Angiogenesis in Women With Angina Pec- angina who are not candidates for revascularization (the the same vector was planned in women with refractory Based onthesefindings,anadditionalclinicalstudywith dial perfusion,specificallyinpostmenopausalwomen. with improvements of surrogateendpointsmyocar- wasassociated indicated thattreatmentwithAd5FGF-4 the trialswasstopped. unlikely toreach significance,andfurtherenrolmentin ing nosafety concerns,showedthatdifferences were whileindicat- interim reviewofthedatafromAGENT-3, patients. After >500patientswereoverallrecruited,an in selected doses in larger populations of of Ad5FGF-4 Cannatà etal mentioned KAT trial, which alsocontainedanarm with advantage ofadenoviralvectors. Onewasthealready trial; NCT02928094). is stillplannedtostartrecruitment in2020(theAFFIRM patients withrefractoryangina, which wasfiledin2016, clinicalstudyin320 reported yet.AfurtherPhase III run between2012and2016;noresultshavebeen A subsequent pooled analysis from AGENT-3 and-4 A subsequentpooledanalysisfromAGENT-3 Additional experimentations havecontinued totake May 8,2020 37 ). This study 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 DOI: Circulation Research.2020;126:1394–1414. 36

performed inChina(reviewed instudybyWang etal trialsexpressing hepatocyte growthfactor,of PhaseII to bestartedin2020(NCT01757223) andaseries increased at1yearfromtreatment(theKAT301 trial docardial injections;myocardialperfusionwasreported transen- the ischemic myocardiumbyNOGA-mediated fer revealedamodestbut significantimprovement of cardial adenovirus-hepatocyte growthfactorgenetrans- report on 15 patients who received percutaneous endo - isoforms of 3different VEGF-A trialbasedontheexpression also includeaPhaseI/II After asmall,open-labelclinicalstudy, α (hypoxia-inducible factor). expressing HIF-1 intramyocardial administrationofanadenoviralvector PhaseI study reportedthesafety ofthe cebo-controlled, www.regenheart.eu/study-design). Finally, asmall,pla- centers intheEuropeanUnion(NCT03039751; https:// cathetersystem,which willbeconductedat6 a NOGA using studywithadenoviraldeliveryofVEGF-D Phase II multicenter domized, double-blinded,placebo-controlled, This is currently followed by the ReGenHeart trial, a ran- sesses angiogenicactivity. ΔNC), which pos- (VEGFD mature formofVEGF-D esis trialaimedatdeliveringaproteolyticallyprocessed, randomized,controlled,angiogen - of arecentPhaseI/IIa first-generation adenoviralvectorwasalsoatthebasis dence ofmajoradverseeventsinthelongterm. however, nosignificantdifference inmortalityorinci- A patients receivinganadenovirusexpressing VEGF- 165 Ongoing clinicalstudiesbasedonadenoviralvectors . 28 This studyreportedimprovementinperfusion; endothelial growthfactor-A. indicatesvascular the trialname.VEGF-A the indicationoftherapeuticgeneand alongwith or adenoviralvectors,bottom), top method used(nakedplasmidDNA, grouped accordingtothedelivery trials fortherapeuticangiogenesis, The figuresummarizesthemainclinical angiogenesis. Figure 2. 40 The vectorwasdeliveredto Clinical trialsfortherapeutic 43 viamini-thoracotomy, Cardiac GeneTherapy 44 amorerecent 42 38,39 31 41 A ). ). Review

53 1399 - ). Fol 54,55 homeosta- These posi- These 2+ 56,57 May 8, 2020 Cardiac Gene Therapy Gene Cardiac AAV1.SERCA2a viral 13 The result of this study, this study, result of The 49 reuptake by overexpressing reuptake by overexpressing 2+ The results of this trial showed trial this of results The 58 ) and the SERCA-LVAD study (Sar- study SERCA-LVAD the and ) 51 After experimental success of I-1c gene experimental After 52 Unfortunately, this treatment failed to improve this treatment Unfortunately, 50 A third approach to improve cardiac function in HFin function cardiac improve to approach third A by A second proposed approach for HFA second proposed approach gene therapy is tive results prompted a randomized, double-blinded, placebo-controlled, Phase II clinical study that assessed - of 5 doses of an adenoviral vector express the safety - admin investigation name RT-100) (Ad5.hAC6, ing AC6 intracoronary. istered that this treatment partially ameliorated systolic function, Phase A larger, HF. mostly in patients with nonischemic sis by blocking the maladaptive response due to altered sis by blocking β- stimulation. Signal transduction G from these receptors is mediated by a heterotrimeric (adenylate cyclase) located protein that activates an AC which on the cytosolic side of the receptor complex, in turn acti- which to cAMP, catalyzes conversion of ATP has shown that vates PKA. experimentation Preclinical (AC6) cyclase adenylate of 6 isoform of overexpression on failing hearts. has a beneficial effect which showed reduction in HF showed which sig- hospitalization and in nificant improvement and functional both symptoms IIb, Phase to a larger, paved the way parameters, ran- trial, CUPID2double-blinded, multicenter domized, study (NCT01643330). with trial enrolled 250 patients This - of both and nonisch ischemic HF, stable symptomatic ejection fraction, with severely depressed emic etiology, fashion to receive intracoronary infu- randomized in a 1:1 sion of either placebo or 1×10 or an AAV vector with a chimeric AAV2/AAV8 capsid, AAV2/AAV8 vector with a chimeric or an AAV and vasculature blood the traverse to reported was which muscle while selectively transduce cardiac and skeletal de-targeting the liver and the lungs (BNP116 - placeboeither of infusion intracoronary receive differ or of AAV1.SERCA2a.ent doses particles. of a constitutively active form of I-1c, which binds PP1. which of a constitutively active form of I-1c, of this intervention is to increase the ultimate effect The levels of the SERCA2aproper β-adrenergic restore and stimulation. transfer in rodents, large animal work was performed in rodents, transfer vector an AAV9 by the intracoronary administration of clinical outcomes. This discouraging finding also halted also finding discouraging This outcomes. clinical studies using the same vec- 2 other related clinical of AAV1. efficacy evaluating the AGENT-HFtor, study, SERCA2aventricular assist devices left in patients with (NCT01966887 lowing this experimentation, a small, Phase I clinical study lowing this experimentation, entailing intracoronary infusion of in 2020, is scheduled BNP116.sc-CMV.I1c of NAN-101) in (under the name 12 patients with NYHA Class III HF (NCT04179643). is to restore the normal Ca gene transfer coplasmic/Endoplasmic Reticulum Ca2+-ATPase/Left coplasmic/Endoplasmic Reticulum Ca2+-ATPase/Left of the Assist Device), assessing the potential Ventricular remodeling in subjects with HF same vector to modify LV Both studies were prematurely termi- (NCT00534703). nated in 2016. aimed at modulating Ca ). A A 3). A Phase I/II trial 47 and citations therein). and citations 46 ATPase is downregulated ATPase 2+ Based on the results of this of the results on Based 48 45 reuptake by the sarcoplasmic reticu- 2+ ATPse SERCA2a, is inhibited by which ATPse handling in cardiomyocytes, since this is is handling in cardiomyocytes, since this 2+ 2+ Three of the proposed gene therapy strategies have gene therapy strategies have of the proposed Three on the extensive trial, was based first, and more The When these experimentations are analyzed collec- are analyzed these experimentations When One main target for intervention in HF is the modu- -adrenergic stimulation. Blockade of I-1c (inhibitor- -adrenergic stimulation. Blockade de-phosphorylated PLN (phospholamban). The main main de-phosphorylated PLN (phospholamban). The kinase phosphorylating PLNis the in cardiomyocytes cAMP monophosphate)-dependent (cyclic adenosine of control the PKA is under (protein-kinase A), which β 1c) binding to PP1 (protein phosphatase 1) leads to increased PLN and thus increased SERCA2a 2 decades, a number of Over the last activity. the have explored preclinical studies in animal models by modulating these mechanisms of beneficial effect cardiac function in HF. to improve gene transfer (Figure 5); a summary clinical experimentation reached of the main HF gene therapy clinical studies is shown . in the Table of the SERCA2atransfer cDNA, based on the observa- tion that this sarcoplasmic Ca in failing hearts and that restoration of its levels improves heart function in bothand swine. mice Development of novel therapies for heart failure (HF)Development of novel are epidemic this condition has reached sorely needed, as population 2% of the adult proportions, now affecting years than 75 worldwide and over 10% of those older Teerlink and by Metra study (see GENE THERAPY CLINICAL STUDIES FOR HEART FAILURE cardiac function compared with a matched control group control with a matched compared cardiac function follow-up. at a 6-month for therapeutic clear that gene therapy it seems tively, raised it expectations the fulfilled not has angiogenesis high- success well of clinical Lack over 20 years ago. results animal translating in difficulties intrinsic the lights - be attributed to a number of differ into patients and can are critically discussed later. ent reasons, which study, the CUPIDstudy, - trial ( Upregulation by Percu With in Patients taneous Administration of Gene Therapy Cardiac Disease; NCT00454818) was the first clinical CUPID to treat HF. gene therapy attempt to use AAV was a Phase II, blinded, randomized, placebo-controlled, multicenter study enrolling 39 patients with symptomatic severely reduced ejection fraction, and implantable HF, of sudden prevention primary for defibrillator cardioverter were randomized into 4 arms to cardiac death. Patients key factor for Ca pivotal in ensuring normal cardiac function (Figure cardiac normal pivotal in ensuring followed these experimental findings, showing safety of findings, showing safety followed these experimental the expressing vector AAV1 an of infusion intracoronary SERCA2ahuman cDNA. lum is the Ca Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 lation of Ca Cannatà et al Cannatà

Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Review depolarization ofthecardiomyocyteplasmamembraneinducesopeningL-type voltage-dependentCa coding for SERCA2a, AC6, withtheindicationofvectorusedandnameclinicalstudies(initalic). orI-1c(rectangulargreen boxes), coding forSERCA2a, inhibited byI-1c(inhibitor-1c).The cartoonshowsthe3 mainclinicalapproaches tomodulatethesepathwaysinheartfailurebytransfering thecDNAs (proteinphosphatase-1),which inactivation.Thiscardiac cycle.Conversely, is ismainlycarriedoutbyPP1 leadstoSERCA2a dephosphorylationofPLN the sarcoplasmicreticulumbyATPase andinparteliminatedoutsidethecellbyNa SERCA2a, of asmallquantityCa C, and subsequent contraction. In the relaxation phase, RyR2 is inhibited by the FKBP12.6protein. The releasedCa C, andsubsequentcontraction.Intherelaxationphase,RyR2isinhibitedbyFKBP12.6protein. (1) theL-type Ca located onthecytosolicsideofreceptorcomplex, which catalyzesconversionofATP tocAMP. which This phosphorylates inturnactivatesPKA, β (protein-kinaseA),which isunderthecontrolofβ adenosine monophosphate)-dependentPKA (cyclic phosphorylation blocks thisinhibition.The incardiomyocytes(followedbypumpactivation)isthecAMP mainkinasephosphorylatingPLB while inhibitsSERCA2a, (phospholamban).Initsnonphosphorylatedform,PLB pumpiscontrolledbyassociationofthisproteinwithPLN SERCA2a 1400 of AAVbone vectors, segments oftheprotein,which canfit intotheback- icked byafusionproteinoftheintracellular C1andC2 that adenylylcyclasetype6 (AC6) activity canbemim- Recent experimental work indicates NCT03360448). ment plansandstrategybeingre-evaluated(FLOURISH; however, itwaslaterwithdrawnduetoclinical recruit- clinicaltrialwasplannedtobeginenrollment in2018; III Cannatà etal The figureshows3currentgenetherapyapproaches targetedtocardiac excitation-contraction couplingthroughthecardiomyocyteCa Figure 3.Clinicaltrialsforheartfailure. receptor. Massive entryofCa FKB12.6; and (3) PLB, blocking its inhibitory activity on SERCA2a. These modificationsamplifytheefficacyofCa blocking and(3)PLB, itsinhibitoryactivityonSERCA2a. FKB12.6; through thedelivery ofgenescodingfor extracellular safety ofgenedelivery. -adrenergic receptorswiththeirligandsactivatesanassociated,heterotrimericGprotein,which inturnleadstoactivationofanAC (adenylatecyclase) An alternativemannertosustain afailingheartis May 8,2020 2+ channels, thusdeterminingfurtherCa 2+ intothecytosol;thisinturndeterminesreleaseofCa 59,60 2+ intothecytosoltriggersbiochemical couplingbetweenactinandmyosin,which ismediatedbyCa possibly improving efficacy and 2+ 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 DOI: Circulation Research.2020;126:1394–1414. entryeach depolarizationcycle;(2)RyR2,causingdissociation oftheinhibitoryprotein factor)-1⍺ VEGF-B, models,mostnotably effective in largeanimalHF a numberofcytokinesandgrowthfactorshasproved factors thatexert abeneficialfunction.Overtheyears, end point,despite reportingimprovements inselected failedtomeet its primarycomposite NCT01643590) study(STOP-HF; studyPhaseII placebo-controlled JVS-100. plasmid,named diomyopathy usinganaked DNA clinical experimentation inpatientswithischemic car- 2+ from the stores through the RyR2 ryanodine fromthesarcoplasmicreticulumstoresthroughRyR2ryanodine 61,62 65 . 64 However, a randomized,double-blind, Treatment⍺ withSDF1 S100A1, -adrenergic stimulation.Inparticular, engagementsof + / Ca 2+ 63 exchanger (NCX). The activityofthe and SDF (stromal cell–derived andSDF 2+ is in part re-convoyed into isinpartre-convoyedinto 2+ release and re-uptake every releaseandre-uptakeevery 2+ channels, withpermitentry reached PhaseI Cardiac GeneTherapy 2+ binding to troponin bindingtotroponin 2+ cycle.The Review 1401 and using AAV and using AAV 68 May 8, 2020 Cardiac Gene Therapy Gene Cardiac amount of information was learned, which can guide was learned, which amount of information future development in this area. Efficiency of Gene Delivery A foremost problem of gene therapy remains the efficiency As anticipated, plasmid trans- of in vivo nucleic acid delivery. concerns; is simple and not fraught with major safety fection the efficiency of naked DNA uptake by cardio- however, In addition, measurable levels of myocytes remains poor. are only maintained for the first couple ofgene expression weeks after naked plasmid injection, a condition that might This seems exert a therapeutic effect. not be sufficient to particularly relevant for therapeutic angiogenesis, since in bothexperimentation genetic models A third A third 67 A parallel clinical study of the the of study clinical parallel A 66 - plasmid in patients with crucial limb isch same SDF1⍺ NCT02544204) also failed. emia (STOP-PAD; While the overall clinical outcome of the cardiac gene the overall clinical outcome While large still a therapy applications seems unsatisfactory, CLINICAL TRIALS FOR GENE THERAPY OF CARDIAC DISORDERS—THE LESSONS WE LEARNED groups of patients. of groups Molecules and methods for the delivery of RNAFigure 4. Molecules and methods for the delivery of therapeutics. and of a phosphorothioate bond nucleotide (upper left) lower part of acid (LNA) (PS, upper right). The A, Chemical structure of a locked-nucleic extremities of the oligonucleotide nucleotides are positioned at the 2 LNA-modified in which gapmer, the panel shows the structure of an LNA to allow RNase with the target RNA H accessibiltity to the central part once the duplex is formed. B, Chemical structure of the main cationic and formation (poly(ethylenimine) [PEI], and lipid nanoparticle formation. C, Main polymers used for polyplexes neutral lipids used for lipofection poly- representation of DNA-nanoparticle D, Schematic structures. [PLL], poly(lactic-co-glycolic acid) [PLGA]). L-lysine Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 Phase I/II trial with the same plasmid administered failure heart with patients in delivery retrograde by (RETRO-HF; was initiated in 2014 but NCT01961726) has not reported yet. Cannatà et al Cannatà

Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Review to thevector. liver diedbecauseofasystemicinflammatoryresponse disorderofthe trial forthetreatmentofararemetabolic whenayoungpatientrecruitedingenetherapy 1999, concerns. This problembecamedramaticallyapparentin immune and inflammatory responses, which raise safety adenoviral vectors. These vectors, however, elicit robust kine stimulationisrequiredtoformastablevasculature. tem for the transcriptional or posttranscriptional control of tem forthetranscriptional orposttranscriptional controlof cological treatmentatthetime ofvectoradministration (or itsimmunogenicitycanbeeasilycontrolledbypharma- that canbeinjected. of theseproblems,which defactolimitthedoseofvector temporally coincidentwiththeprogressiveappreciation trials)were of theapplications(amongwhich theAGENT trials forcardiacgenetherapy, thedecisiontostopsome to vector delivery were reported in the adenovirus-based 1402 vectors indicatesplatelet-derivedgrowthfactor. See text forfurtherexplanation. PDGF cardiomyocytes tocontroltheirproliferation. The lowerpartofthefigureshowscytokinesandothermoleculesactingfromoutsidethesecells. andthemainsignaltransductionpathwaysactinginside The upperpartofthefigurelistsaseriescellcycleproteins,microRNAs, showntostimulatecardiomyocyte proliferationandcardiacregeneration. Figure 5.Molecules,genes,andRNAs Cannatà etal deleterious overtime. when continuousangiogenic factorexpression mightbe is notthecase,forexample, oftherapeutic angiogenesis, permanent expression ofthetransgeneisdesirable.This However, theuse ofthesevectorsisonlyindicatedwhen A mostefficientmannerforgenetransfer isbyusing In contrast to adenovirus, AAV is poorly immunogenic 69 May 8,2020 hasshownthatprolonged(severalweeks)cyto- 70 Despitenospecificsafety issuesrelated 69,72,73 Unfortunately, noefficientsys- 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 DOI: Circulation Research.2020;126:1394–1414. 71 ). ). this turnedouttobe43copiespermicrogramtotalDNA transplantation ormechanical supportdeviceimplantation, samples fromtreatedpatientswhodeceasedorunderwent wasassessedincardiac DNA amount ofAAV1.SERCA2a trials.When the intheCUPID cDNA ery oftheSERCA2a extensive cardiomyocytetransduction,such wasthedeliv- proteins aremorelikelytosucceedthanthoserequiring transfer, applicationsbasedontheproductionofsecreted study byZacchigna etal gene expression invivoiscurrentlyavailable(discussed act asdecoys for serumneutralizingantibodies. considered detrimentalfortransduction, theycouldstill respectively). While90%, empty capsids are commonly study(25%versus compared withthe original CUPID trial was the reported lower amount of emptyviral vectors CUPID2 inthe hampered efficacyofAAV1-SERCA2a having asizeof≈ lium (paracellularpermeability)is inefficient forparticles invasive procedure,extravasation fromtheintactendothe- trials,isaminimally nary injection,such asintheCUPID acts intracellularly. Whilethat SERCA2a vector intracoro- cells weights≈ (as a reference, DNA from1×10 (as areference, DNA Given the still relative inefficiency of cardiac gene Given thestillrelativeinefficiencyofcardiacgene An additional,delivery-related factorthatmighthave 6 µg).This was definitelytoolowconsidering 20 nmdiameter, such asAAV virions. 6 ). 6 mononucleated diploid mononucleateddiploid Cardiac GeneTherapy 74 50

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1403 and 79 Safe Neutral Neutral reported Recruiting Functional Results not in symptoms improvement Main Findings Slight increase in LVEF, no changes no changes LVEF, Analogous con- May 8, 2020 72 Cardiac Gene Therapy Gene Cardiac dosing events) cardiac events cardiac events clinical outcomes LVEF changes and changes LVEF functional changes Primary End-Point Functional: 6 MWD exercise, BNP, echo, echo, exercise, BNP, Composite: symptoms, Time to recurrent event Time Functional: 6MWD and MLWHFQ at 4 mo post- Number of major adverse Number of major Number of major adverse (hospitalization/worsening heart failure/cardiovascular questions the root of this assumption. However, the recognition that no cells the recognition that However, 4 78 12 12 12 12 12 12 77 Up, mo Follow- in patients with an implanted left ventricular n/a 52% 48% 100% 100% 100% 100% 165 Ischemic In the therapeutic angiogenesis studies, the lack of clin- the lack In the therapeutic angiogenesis studies, In terms of gene combinations, at the end of 2019, n/a 55% 82% 68% III/IV 100% 100% 100% NYHA ical success might be consequent to the complexity of the complexity ical success might be consequent to the requires a precisely dosed vascularization process, which factors different of combination scheduled temporally and acting on both endothelial cells and vessel mural cells, as bothof generation the requires as well networkcapillary a 30than less No larger conductance vessels. and different factors are known to take part in physiological vessel for- of any one mation; thus, it is possible that the expression of these factors alone might not be sufficient to trigger or VEGF-Aexample, a power- is For process. complete the and sprouting; ful inducer of endothelial cell proliferation in response to this the vasculature that is formed however, factor alone is leaky and not functional. in either the bone marrow or the heart can act as true cardiac stem cells citations therein). The delivery of gene cocktails, obviously, obviously, delivery of gene cocktails, citations therein). The difficulties of clinical translation. increases the technical trial a Phase I clinical in of enrollment the completion was announced for a triple gene therapy (INXN-4001, NCT03409627) entailing the retrograde coronary sinus S100A1, SDF1infusion of a plasmid expressing α and VEGF-A therapy was the promotion of cardiac homing of bothhoming of cardiac promotion was the therapy bone marrow–derived and cardiac stem cells to the sites injury. of ischemic siderations also apply to the combination of VEGF and hepatocyte growth factor (see study by Gerritsen assist device, either as a bridge to transplant or destination n/a n/a 33% 28% 31% 25% 23% LVE F Mean infusion infusion infusion infusion Method injection injection injection Delivery Intracoronary Intracoronary Intracoronary Intracoronary Coronary sinus Intramyocardial Intramyocardial Intramyocardial for HF gene 17 72 93 12 56 39 243 No. of Patients study placebo- placebo- placebo- controlled controlled controlled open label open-label randomize, Open label multicenter, multicenter, Open-label, randomized, randomized, Prospective, Randomized, Double-blind, Double-blind, Study Design dose-escalation dose-escalation Double-blinded, - most of the large animal experi 75,76 HF n/a Trial Trial AC6 Name CUPID RETRO- CUPID 2 NAN-101 STOP-HF I-1-c AC6 Gene SDF-1 SDF-1 SDF-1 Main Clinical Studies of Gene Therapy for Heart Failure for Studies of Gene Therapy Main Clinical SERCA2A SERCA2A 6MWD indicates 6-min walk distance; BNP, B-type natriuretic peptide; LVEF, left ventricular ejection fraction; and MLWHFQ, Minnesota Living with Heart Failure Minnesota Living with Heart Failure left ventricular ejection fraction; and MLWHFQ, B-type natriuretic peptide; LVEF, 6MWD indicates 6-min walk distance; BNP, I II I / II II I / II IIb I Phase Table. Questionnaire. A third set of considerations relates to the efficacy of of the therapeutic gene. In some instances, the choice one of the example, the gene itself is problematic. For of action of SDF1proposed mechanisms ⍺ Choice of Therapeutic Gene Choice of Therapeutic Inappropriate Animal Models Inappropriate outcome the from learned lesson important second, A trials is the likely of several of the gene therapy clinical preclinical the available of of most inappropriateness clinical stud- models. All the therapeutic angiogenesis in results preclinical convincing very on based were ies humans. However, small and large animals, to then fail in to mimic a chronic there is no adequate preclinical model usually develops on This HF. as human condition such coronary disease or heart the basis of either extensive year-long periods, muscle dysfunction and develops over of multiple comorbidities, most nota- often in the context few, a Despite mellitus. diabetes and hypertension bly notable exceptions, mentations have been performed in young and healthy animals and the induced disease has been commonly of short duration. Similar considerations also apply to the available models of HF caution in large animals. Thus, in translating large animal findings to must be exercised patients, especially when the detected treatment effects are statistically significant but biologically miniscule, as, is the case for several plasmid treatments. for example, Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 Cannatà et al Cannatà

Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Review skeletal muscle,however, failtodosointhe heart. are veryefficientatdrivinganangiogenicresponseinthe cardiac ischemia, which wasshowntoprovidebenefitinpigswith chronic (myocardin-relatedtranscriptionfactor-A), MRTF-A confounding factor. etiology. This variability might represent an important ischemic andnonischemicological conditions,ofboth HF itself comprises a variegate set of path- definition of relevant, itisbecomingprogressivelyapparentthatthe evidence indeed indicate that cardiomyocyte loss is a major evidence indeed indicatethatcardiomyocyte lossisamajor ficient tosustainglobalorgan function.Numerouslinesof myocytes survivedinadamaged heartmightnotbesuf- to addresstheproblemthat thetotalnumberofcardio- improving individualcardiomyocyte function,however, fail All the available gene therapy approaches are aimed at diac endotheliumbiologyisclearlyneeded. growth factordelivery. Abetterunderstandingofthecar- which might explain thelack of efficacy ofangiogenic impaired at mountingan efficient angiogenic response, it mightbeconceivedthatcardiacendothelial cells are 1404 angiogenesis. occurs bydirectarteriogenesisandnotthrough tion (MI) vessels formation in the heart after myocardial infarc- sive angiogenesis,issuppressed.Additionally, collateral organs wherethegrowthoftumors,which requireexten - a poorlyangiogenicenvironment.Itisoneofthefew still scatteredevidenceindicatesthattheadultheartis ties atinducingtherapeuticangiogenesis.Different but biology, inparticular, relatedtotheunexpected difficul- that wearestillmissingsomespecificaspectofcardiac learnedlessons,itseemspossible Besides theabove Are We MissingSomething? (≈20% ofpatients were in NYHA classII enrolledpatientswhohadlowerNYHAclasses CUPID2 or IV,tomatic patients in NYHA functional class while III comprisedseveralsymp- the inclusioncriteriainCUPID mightbepartiallyattributedtothefactthat with CUPID trialcompared For example, thefailureofCUPID2 clinical trials might also relate to the type of study design. A componenttothefailureofsomegenetherapy Study Design a peripheralarterydiseasegenetherapystudy, α hasfailedtoimproveclinicalendpointsin ever, HIF-1 drive afunctionalangiogenicresponse.Ofthese,how- response toischemia mightbebetterpositionedto α. SDF1 the efficacyof remain thepoorefficiencyofplasmidgenetransfer and therapy. Majorunknownsofsuch anapproach, however, Cannatà etal in largeanimalswithco-morbidities. Conceptually similarconsiderations alsoapplytoHF. Finally, upstream transcriptional regulators of the May 8,2020 82 Finally, AAV vectorsexpressing VEGF 81 stillawaitsconfirmationfromstudies 49,50 ). Even more 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 DOI: Circulation Research.2020;126:1394–1414. 80 83 while Thus, ular tachycardia, gene therapyofcatecholaminergic polymorphicventric- their clinicaltranslation,withafew exceptions such as sinus node dysfunction—are not considered here, as lation, or the generation of a biological pacemaker for delivery ofdominantnegativeproteinsforatrialfibril- allele-specific gene silencing in dominant arrhythmias, gene therapy for arrhythmogenic disorders—for example, mentation. Other very interesting applications, such as already enteredorisrapidlyapproaching clinicalexperi- a rapidpaceoverthelastfew yearsandinwhich ithas gene editing—inwhich genetherapyhasprogressedat therapeutics,cardiacregeneration,and noncoding RNA In thischapter, wefocusonto3therapeuticareas— GENETIC THERAPIES IN THE PIPELINE 25% ofthetotalnumberthesecellsinleftventricle diomyocytes dieinafew hours,representingasmuch as cause ofHF. whenoften1billioncar- This isclearafterMI, logical conditions. normaland patho- networkinthe heartinboth miRNA sive reviewsareavailablecovering thefunctionof hundreds ofproteinstofine-tunecellfunctions.Exten- simultaneously control the production of several tens or can (miRNA) ognition ispromiscuous,each microRNA to partiallycomplementarysequences.Sincetargetrec- by base pairing regulate abundance of target mRNAs that These are21 to23nt-long,double-strandedRNAs encodedfromthehumangenome. mature miRNAs information for1917annotatedprecursorand2654 Base 22.1;http://www.mirbase.org/index.shtml) reports (lncRNAs). coding RNAs andlongnon- (miRNAs) one of2classes,microRNAs of interest for gene therapy essentially fall into ing RNAs developing innovativetherapeuticapproaches. Noncod- pavestheway to by avastseriesofnoncodingRNAs thelial cells,andresidentmacrophages)arecontrolled function (includingcardiomyocytes,fibroblasts,endo- The discoverythatvirtuallyallaspectsofcardiaccell Therapeutics forHeartDisease Noncoding RNA have beenpublishedrecently. reviews on these experimental gene therapy applications cardiac regenerationisspecificallydiscussedlater. cardiomyocyte number. Howgenetherapycanstimulate the problemoflimitingcardiomyocytelossorincreasing by Bragaetal but alsooccursinmostcardiacdiseases(reviewedstudy >100 000. inhumans,rangingfrom <20 number oflncRNAs >200-nt. There isabroadrangeofestimates forthe moleculesof a classoffunctional,nontranslated RNA The latest release of the miRBase database (miR- The latest release of the miRBase 93 Again, most aspectsofcardiac functionand 85 ). None of the available therapies address ). Noneoftheavailabletherapiesaddress 86 90–92 remainsmorechallenging. Excellent LncRNAs areinsteaddefinedas LncRNAs 87–89 Cardiac GeneTherapy 000 to 84

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1405 118,119 120 One of these 123 May 8, 2020 Cardiac Gene Therapy Gene Cardiac 121,122 Some of the drawbacks of charged lipoplexes can lipoplexes Some of charged of the drawbacks A more significant progress in this area was however The delivery of small double-stranded RNA small double-stranded delivery of The - therapeu lipid nanoparticles was used to develop patisiran, the first the market reach therapeutic to RNA interference in August 2018 in both United States and Europe. the lowers the hepatic levels of transthyretin for the Patisiran treatment of hereditary transthyretin-induced amyloido- sis, a rapidly progressive and often fatal disease caused be overcome by the use of neutral lipids, which show lipids, which be overcome by the use of neutral clearance from improved biodistribution and reduced of these neutral lipids is the circulation. An example In Vivo the commercial preparation MaxSuppressor RNA-LANCEr II—composed of 1,2-dioleoyl-sn-glycero- squalene oil, polysorbate 20, and an 3-phosphocholine, was used to form a neutral lipid emul- antioxidant—which sion to deliver various miRNAs in the cancer and cardio- vascular fields. cationic lip- the introduction of ionizable represented by as 1,2-dioleoyl-3-dimethylammonium-propane, ids, such and of methods to load nucleic acids into small (<100 nm diameter) lipid nanoparticles with neutral surface positively ionizable cationic lipids are These charge. at low pH and are thus efficient to encapsulate charged when nucleic acids. Subsequently, negatively charged the pH is raised to physiological values, the surface of the lipid nanoparticles becomes neutral. tics, such as miRNAs as tics, such and siRNAs, take advantage can various uptake using their intracellular from facilitating of lipid-mediated A first generation vehicles. chemical based on nucleic reagents was (lipofection) transfection molecule of a cationic lipid by mixtures acid entrapment as 1,2-di-O-octadecenyl-3-trimethylammonium(such - propane or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-tri of charge positive the methyl-sulfate, methylammonium of DNA), neutralizes the negative charge - formu which as 1,2-di-(9Z- co-lipid helper (such lated with a neutral octadecenoyl)-sn-glycero-3-phosphoethanolamine); the very popular, include lipoplexes Figure 4B. These of formulation 3:1 (a reagent Lipofectamine commercial 1,2-di-(9Z-octadecenoyl)- and the cationic lipid DOSPA and its derivatives. sn-glycero-3-phosphoethanolamine) with transfection cell for tools efficient are Lipoplexes siRNAs or miRNAs their large particle in vitro; however, imparted by size (often >1 µm) and the positive charge clearance, toxicity, the cationic lipid results in rapid plasma systemically. and inflammation once administered first miRNAThe mimic tested in a clinical trial was miR- lipid vehicle, called 34a, formulated in a pH-dependent MRX34,patients with and delivered in a Phase I trial to lipid cancers. The liver cancer or other advanced primary named Smarticle, was composed of amphoteric carrier, at physiological lipids having an overall anionic charge tumor environ- pH, while becoming cationic in the acidic was terminated ment. Development of the drug, however, due to severe immune-related side effects.

97 102 104 and 95 ; against Salamon 114 94 miR-21, - struc This ). 112 101 of miR-25 to 100 . These LNA- These 4A. 105 Systemic admin- 106 Salamon et al, Salamon 94 Several additional anti- Figure ; 111 ; antagonizing endothelial antagonizing ; 99 109 ). One of the most effective of effective ). One of the most 98 . 96 116,117 ). - to prevent cardiac hypertrophy or pathologi 96 Other applications of the anti-lncRNA anti- 103 whereas regional administration is effective in whereas regional administration is effective 115 107 and Lucas et al and Lucas ; inhibition of miR-199a-5p hypertrophy reverses ; against Chast both; against Chast and attenuates prevents ; inhibition of miR-146aof ; inhibition peripartum in a beneficial is 95 108 113 A number of laboratories taken advan- have already Antisense inhibition using ASOs also apply to ASOsapply using also inhibition Antisense Inhibition of endogenous miRNAInhibition and lncRNA levels On a final note, the clinical development of ASOsOn a final note, the clinical development of 110 containing oligonucleotides can be designed with a ste- containing oligonucleotides is segment a central LNA by which approach, ric block GapmeRs (LNA gaps non-LNA by flanked et al, Lucas et al Lucas istration of anti-miR-92a LNA ASOs enhances blood istration of anti-miR-92a LNA of damaged tissue vessel growth and functional recovery in mice miR-24 limits MI size by preventing endothelial apopto- sis Wisper counteracts MI-inducedWisper fibrosis and cardiac dys- function. pigs ture improves stability while still permits recruitment of ture improves stability while still permits RNAse for target RNAduplex H to the degradation. tage of microRNA- inhibition using ASOsdiffer with models. These experimental in various ent chemistries inhibition of miR-133, include, for example, lncRNAs. For example, GapmeR antisense oligonucle- antisense GapmeR example, lncRNAs. For lncRNAotides against the cardiac fibroblast-enriched Meg3 prevents cardiac fibrosis and diastolic dysfunc- tion or miR-32 of miR-29 to contrast cardiac fibrosis, and van Rooij and Olson and van Rooij and dysfunction are governed by one or multiple lncRNAs by one or multiple are governed dysfunction et al, in study by Hobuß (reviewed cal remodeling, of miR-34 to assist recovery after MI,cal remodeling, of miR-34 to assist recovery can be achieved by administering short, antisense oligo- short, antisense by administering can be achieved (ASOs) comple- deoxy-oligonucleotides or nucleotides avoid rapid degradation To mentary to their sequences. modi- uptake, a number of chemical and facilitate cellular - into the nucleic acid back fications can be introduced and Behlke bone (reviewed in study by Lennox or bases miR antisense oligonucleotide applications were recently miR antisense oligonucleotide applications reviewed in study by De Majo and De Windt. improve contractility in failing hearts. and fibrosis in in fibrosis and mice in HF these modifications now seems to be the inclusion of now seems to be the inclusion of these modifications the sugar nucleotides, in which nucleic acid (LNA) locked bondcontains an extra the 2’ and 4’ carbons connecting bridge) (2’-O,4’-C methylene for cardiovascular applications can take advantage of over 30-year development, antisense oligonucleotide over 100 Phase I clinical trials have been per- for which II Phases 25% have reached formed, of which or III; 6 antisense RNA therapies have already obtained approval for commercial use. TAC-induced pathological cardiac remodeling TAC-induced cardiomyopathy mouse model. Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 sense technology are reviewed in Hobuß et al, Hobuß et in reviewed are technology sense Cannatà et al Cannatà

Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Review the circulating exosomes tendtoaccumulate intheliver. isrelativelylow, and ture, asloadingefficiencyof miRNA tive material,such aslipids,proteins,andnucleicacids. cell-to-cell communicationthroughthetransfer ofbioac- to100nmextracellular vesiclesinvolvedin namely 30 advantage ofendogenousvesiclessuch asexosomes, geting. the exosome membrane, of specific ligands for cell tar- apeutics arereviewedinstudybyBocaetal. ther- delivery systemsfortheofnoncodingRNA remains theirtoxicity invivo.Otherclassesofnanoscale file. indicate thatthistherapyhasanacceptablesafety pro- NAs arecurrentlyundergoingclinicalexperimentation. andmiR- 8 lipidnanoparticlesformulationsofsiRNAs improves cardiac function after MI inpigs. improves cardiacfunctionafterMI spheres of>9µmdiameter promotes angiogenesisand encapsulated inpoly(lactic-co-glycolicacid)micro- the intracoronary administration of an antagomiR-92 vasculature. For example, recentevidenceindicatesthat be relevantorevendesirablewhenthetargetis plexes haveusuallylargesize,which howevermightnot 1406 intoexosomes miRNAs Methods canbedevisedfor theenrichment ofselected growth. (epidermal growthfactorreceptor)tosuppresstumor sion, loadedwithamiR-16mimicandtargetedtoEGFR of amutationsingenesthatcontrolbacterialcelldivi- nmindiameter,400 which areproducedasaresult TargomiRs. These arebacteriallyderivedminicellswith thelioma wereadministeredintravenouslywithmiR-16 trial carriedoutinAustralia,patientswithpleuralmeso- sented bytheuseofbiologicalcarriers.Inanopen-label mouse model. and an antagomiR to breast cancer cells in a miRNAs structurecomprising 2 fully deliveranRNA-triple-helix somes. PAMAM dendrimerswerereportedtosuccess- escape byactingasprotonspongesonceintheendo- poly(ethylenimine), dendrimersfacilitateendosomal with negativelycharged smallnucleicacids.Similarto ing cationicgroupsontheirbranches, which associate ure deliveryaredendrimers(Fig- interest forsmallRNA poly(lactic-co-glycolic acid) polymers, such aspoly(ethylenimine),poly-L-lysine and withsyntheticornatural orsiRNAs mulation ofmiRNAs cholesteroldisease whohaveelevatedLDL levels. ous injectioninpatientsathighriskforcardiovascular administeredasasubcutane- targetingPCSK9, siRNA In thecardiovascularfield,oneoftheseisinclisiran,an by mutationsinthetransthyretingene. Cannatà etal Finally, deliveryisrepre- aneweravenueformiRNA Another class of syntheticmolecules that areof An alternativetotheuseoflipidmixturesisfor- 131 4D). These are highly branched polymers contain- Another biological miRNA delivery system takes Anotherbiological miRNA 134 130 May 8,2020 These technologies, however, are still imma- Preliminary resultsfromthisphaseIclinicaltrial 128 Amajorlimitationofthesemolecules 133 andfortheinclusion,within 126 ; Figure 124 4C. These poly- At leastother 127 129 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 DOI: Circulation Research.2020;126:1394–1414. 125 123 132

at Notch-responsive promoters. adult hearts due to suppressive epigenetic modifications myocytes. gene transfer drives the expansion of neonatal cardio- dioverter defibrillatorbyAAV- oradenovirus-mediated et al, due tothedownregulation of itsc-ErbB2receptor. opment, butitlosesefficacy at1weekafterbirthinmice ispowerfulduringdevel- myocyte proliferation byNRG1 see studybyBragaetal (neuregulin-1; kin-6), Fstl1 (follistatin-like1),andNRG1 (platelet-derivedgrowthfactor),IL-6 (interleu- PDGF (fibroblastgrowthfactor)family,members oftheFGF eration during development andatbirth. These include cytokines areknowntostimulatecardiomyocyteprolif- exploited forregenerationpurposes(Figure 5).Afew control cardiomyocyte proliferation and can thus be made inunderstandingwhich arethemechanisms that tion throughtheproteinagrin. links theextracellular matrixtocardiomyocyteprolifera- tion during embryonic development. A fourth mechanism and divide(reviewedinstudybyBragaetal, stimulating adultcardiomyocytestoenterthecellcycle diac regenerationinadultmammalscouldbeobtainedby mander andfish. These observationssuggestthatcar- similar towhatoccursthroughouttheentirelife insala- after birthisrepaired through cardiomyocyte proliferation, mice andpigsindicatesthatcardiacdamageimmediately threshold tobeclinicallyrelevant.Evidenceinneonatal damage, however, to an extent that remains below the humans andmice.This percentageincreasesslightlyafter which wasestimatedintheorderof1%peryearboth with their very limited renewal capacity in adult organisms, myocytes bygenetransfer. Celllossintheheartcontrasts regeneration bystimulatingtheproliferation ofcardio- recently isbasedonthepossibilityofachieving cardiac A specifictherapeuticareathathasgainedmomentum Gene Therapy forCardiac Regeneration through regeneration insteadoffibrosis ing theinhibitoryMst1kinase repairmyocardialinjury Transgenic miceoverexpressing activatedYAP or lack- down knock requires intracellularco-stimulation(eg,byp38 ismodestand of cardiomyocyteproliferation byFGF1 prevent clinicaltranslation.For example, stimulation ing theinhibitors,however, withsomecaveats thatstill the positiveregulatorsofthesepathwaysorantagoniz- diac regenerationcanbeachieved byoverexpressing Hippo Three ofthese(theWnt/β-catenin, involves activation of atleastfoursignalingpathways. cellular environmentsurroundingcardiomyocytes.This myocyte replicationalsoresultsfrom sensing theextra - Over thelastyears,significantprogresshasbeen Modulation ofcardiomyocyteproliferation andcar- 135 142 140 Tzahor andPoss, ). Overexpression oftheNotch implantablecar- pathways)regulatecardiomyocyteprolifera- 143,144 However, thispathwayisswitched offin 136 85 andcitationstherein).Cardio- andUygurLee 141 145 Stimulationofcardio- 138 Cardiac GeneTherapy 147,148 Notch, 85 ; inaddition, Hashimoto Hashimoto 137 139 ). and 146

Review

156 1407 were 163 This cre- This 6 May 8, 2020 160–162 Cardiac Gene Therapy Gene Cardiac and miR34a 162 miR-294, a member of the miR- of the a member miR-294, ). 85 156 and the miR-17~92 cluster member cluster member and the miR-17~92 in all cases using AAV vectors, were all vectors, in all cases using AAV 164 121 The forced inhibition of these miRNAs forced inhibition The after 159 Of interest for therapeutic applications, miRNAs applications, therapeutic for interest Of regu- The efficacy of the antisense or sponge approach antisense or sponge approach efficacy of the The Expression of miRNAs using AAV vectors, however, however, vectors, miRNAsof Expression AAV using shown to exert a beneficial effect after after effect beneficial a rodents. in MI exert to shown sponges sequestering suppressive miR- Alternatively, vectors, as shown in using AAV NAs can be expressed miR-99/100and let-7 of case the miRNAs a for and lncRNAsinhibitory recently discovered, few in (reviewed study by Braga et al MI effects. regenerative in mice exerts miR-19a/19b, and the complementary strand miR-199-5p) was tested after MI cardiac regeneration infarction in pigs. While and improvement in cardiac function was clearly evident proliferation. Two important groups of these inhibitory of these inhibitory important groups Two proliferation. miRNAs family miRNAs, include the let-7 - sup which stem cell development and during press the participates which family, miR-15 and the differentiation, cell cycle from the of cardiomyocytes in the withdrawal after birth. late mRNA by base-pairing to stability and translation sequences. As a consequence, partially complementary target tens or hundreds of dif- these molecules can regulators transcripts, thus qualifying as broad ferent replication of biological functions, as is the of complex such cells connected electrically and structured highly inhibitory Endogenously expressed, as cardiomyocytes. miRNAs nucleic acids. can be silenced using antisense miR-15b against AntimiR LNAs 302 superfamily strictly depends on the levels of expression of the inhibi- strictly depends on the levels of expression tory ncRNAs their relative relevance in controlling and tempting strategy the regeneration phenotype. A more phenotype to cardiomyocytes is to impart a proliferative with small RNAs, irrespective whether the molecules physiology normal the in part take administered are that of miR- of the heart. In infarcted mice, the expression 199amiR-590a, or shown to stimulate cardiomyocyte proliferation and res- shown to stimulate cardiomyocyte proliferation toration of cardiac function after MI. is fraught with problems in view of clinical application. vectors persist indefinitely in the transduced cells, These of their transgenes cannot readily be while expression regulated by the currently available promoters. ates general safety issues for vectors expressing propro- issues for vectors expressing ates general safety genes and specific cardiac concerns, in light of liferative de-differentiate to partially cardiomyocytes, need, for the vectors deliver AAV to undergo replication. Additionally, the pri-miRNA is then processed by the gene, which RNA to eventually generate the machinery interference mature miRNA both Thus, miRNA duplex. strands (5p might which transfer, gene upon are produced 3p) and in one became evident This result in unwanted effects. large animal study so far conducted for cardiac regener- the miR-199a vector expressing an AAV6 ation, in which miR-199a-3pgene (encoding the pro-proliferative

, 156 WAF1/CIP1 or knock- 149 Several of these belonging to the 85 121 Recent work shows Collectively, these miR- these Collectively, 154 156,157 151 ) can stimulate cardiomyocyte pro- ) can stimulate cardiomyocyte ) or silencing of negative regulators ) or silencing of negative ; however, this remains poorly practical ; however, KIP2153 152 A second class of proproliferative miR- of proproliferative A second class 155 induce effective cardiac regeneration. cardiac regeneration. effective induce 85 122 150 or delivery of miR19a/19b 158 , and p57 KIP1 A very appealing alternative to achieve cardiac regen- A very appealing alternative to achieve Other microRNAs are instead known to be physiologi- Similar to all cell types, cell cycle regulation in car- cell cycle regulation cell types, all Similar to for therapeutic applications. proliferative eration by manipulating the cardiomyocyte potential is to resort to the microRNA network. A large body of evidence over the last decade indicates that a number of miRNAs control, either positively or negatively, the rate of cardiomyocyte proliferation. that bona be can instead replication fide cardiomyocyte cell delivery of multiple by the simultaneous achieved cycle activators miRNAs in 2 large screenings that ana- were identified lyzed 988 miRNAs encoded by the in bothcells. human and rodent same miRNA group, induce cardiomyocyte proliferation a in embryonic, postnatal, and adult hearts and exerts after MI.therapeutic effect includes sev- A third group miRNAs,eral unrelated the human miR- among which 199a-3p, was originally identified as one of the which proregenerative miRNAsmost effective in rodents p27 while acting as a tumor suppressor for several human malignancies. cally expressed in cardiomyocytes and to suppress their cally expressed diomyocytes is also governed by a series of positive and governed by a series of positive and diomyocytes is also on Cyclin/CDKconverge that regulators negative regu- of work has shown that the overexpression lation. Past (including E2F family members, cyclin positive regulators D1, cyclin D2 overexpression of YAP using an AAV9 vector an AAV9 using YAP of overexpression out of the Mst1 co-factor Salvador in mouse hearts with in mouse hearts Mst1 co-factor Salvador out of the post-MI HF However, genetic manipulation of the Hippo pathway for pathway Hippo the of manipulation genetic However, caution, to be taken with purposes needs therapeutic pathway role that this broad tumor suppressor given the for several cancers. exerts (eg, the cyclin-dependent kinase inhibitors p21 (eg, the cyclin-dependent NAs can be classified into one of three categories. The categories. NAs can be classified into one of three first corresponds to miRNAsexpressed that are highly to maintain in embryonic stem cells and are required include members of pluripotency of these cells. These the largely correlated miR-302~367 miR-miR-290 and share the same seed sequence. In mice, which family, reactivation of the miR-302-367 cluster induces cardiac regeneration. Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 NAs include a series of miRNAs regulating the cell cycle the miR- comprise These and involved in tumorigenesis. 17~92 cluster and its paralogues miR-106b~25 and of miR- cardiac expression miR-106a~363. Transgenic 17~92, liferation, in agreement with a series of studies in trans- in studies of series a with agreement in liferation, or single gene overexpression genic animals. However, cell at driving full is often ineffective downregulation results in mitotic replication and cytokinesis and often catastrophe and cell . Cannatà et al Cannatà

Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Review ing RNA therapeuticsremainshoweverexciting. ing RNA capacity ofcardiomyocytestoproliferate usingnoncod- eration couldbeachieved bystimulatingtheendogenous clinical experimentation. The conceptthatcardiacregen- efficacy andsafety inlargeanimals,beforemovingto therapeuticsandtest their formulation ofthesencRNA 1408 interspaced short palindromicrepeats[CRISPR]/Cas9 (theclusteredregularly Crick basepairingofaguideRNA ognising specificgenomic sequences through Watson- ates anormalgenomesequence. as atemplateforhomology directed repair, which recre- sequence the otheroneemployingan homologous DNA error-prone non homologous end joining pathway while introduction ofsmallinsertionsordeletionsthroughthe repair canactinessentially2manners,oneentailingthe machineryDNA damage to thatspecificsequence. Then, damage repair increases recruitmentofthecellularDNA sequencemarkedly correspondence ofadesiredDNA breaks in the the presence of a double-stranded DNA application stemsfromthelong-standingnotionthat has openedaroutetopossibleclinicalapplications.Their DNA breaks introduce site-specific, double-stranded cially inpostmitoticcells. editing hasbeenconsideredtooinefficientinvivo,espe- inherited mutations.For decades,however, precisegene gene sequencesinthegenomeaimingatcorrecting therapy. This wouldallowtoinsert,eliminate,ormodify gene editingremainsoneoftheultimategoals DNA mutationsthroughprecise The repairofspecific Gene EditingforInheritedCardiac Diseases late aregenerativeresponse. using acationiclipidformulationissufficienttostimu- mimics ormiR-590-3p cardial injectionmiR-199a-3p dence alreadyshowsthat,inmice,asingleintramyo- clinical cardiacregeneration.Currentexperimental evi- transiently opensanewtranslationalperspectivefor pigs developedfatalarrhythmias at longertimes. andtreatment,severalofthetreated one monthafterMI Cannatà etal liferation andde-differentiation inthelong term. followed bydysfunctionduetocardiomyocytehyperpro- however, cardiac regenerationwasobservedafterMI, cluster,mice overexpressing themiR-302~367 inwhich finding isalsoconsistentwithobservationsintransgenic hydrogel (reviewedinstudybyBragaetal ery of cholesterol-modifiedmimics using a miR-302b/c 19a/19b, ormiR-708 mimics,ortheintracardialdeliv- miR- the dailyintravenousadministrationofmiR302b/c, lipid deliveryreagentorusingthesameformulationfor administration ofmiR-19a/19bmimicsusinganeutral in mice, by the intramyocardial observed, again after MI Further studiesareclearlyneededtoimprovethe The discoveryofbacterial nucleases capableofrec- The useofprogrammableendonucleasesableto The possibilityofdeliveringtherapeuticnucleicacids May 8,2020 166 Consistentresultswere 167 85 ). 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 DOI: Circulation Research.2020;126:1394–1414. 122 165 This as miR-669a for sarcoglycan dysfunction as miR-669a MYBPC3 gene MYBPC3 mal models,forexample, byoverexpressing thenormal cases, standardgenetherapywasshownpossibleinani- skeletal muscle exon skipping in both dogs skeletal muscleexon skippinginboth shown remarkable efficiency in inducing cardiac and mutagenesishasalready lar, CRISPR/Cas9-mediated offers unprecedentedpossibilitiestothisfield.Inparticu- application ofthenewgeneeditingtechnologies now associated DCM, myosin regulatorylightchain—the MYL2protein. lar compartments. genesencodingforproteinsofdifferent cellu- in >40 erogeneous. Pathogenic variantshavebeenidentified mediated correctionofaLaminA/Cdefect to phenotypiccorrectionex vivo(eg,CRISPR/Cas9- pluripotent stemcell-derivedcardiomyocyteshaveled number of recent studies performed in patients’ induced would seemtobeanidealtherapeuticstrategy. Indeed,a are oftendominant.For alltheseconditions, geneediting in which ion channel and desmosome protein mutations erations alsoapplytoseveralarrhythmogenicdisorders, ease most often act in adominantmanner. Similar consid- then,themutationscausingdis- AAV vectors.InHCM cause thediseasearetoolargetofitintoconventional ofseveralthegenesthat thecDNAs cially inDCM, therapy facesimportanthurdles.This isbecause,espe- genes. in theMYH7andMYBPC3 of whichto the development of this condition, 80% are genic mutationsinsarcomericproteinshavebeenrelated dystrophy exons 50and52,respectively. carrying frameshiftmutationsinDuchenne muscular respectively). cardiomyopathy [HCM], andhypertrophic ticular, dilatedcardiomyopathy[DCM] genetic correctionofinheritedcardiomyopathies(inpar- For the heart, this holds aconcrete promise for the gene editingsystems,hasgivenmomentumtothefield. recognitionasinother system), insteadofprotein-DNA cardiomyopathy using a minigene-carrying vector therapy forDuchenne musculardystrophy-associated gene replacement.These include,asexamples, gene ing advantageofindirect approaches ratherthanstraight to avariableextent, usingtruncatedproteinsorbytak- more conventionalgenetherapyhasprovensuccessful, tion oftrans-splicing(forlaminA/Cmutations ase-mediate correctionofphospholamban [transcription activator-likeeffector nuclease] exonucle- the mutantand normalsequences,which canbe not end joiningrequireshigh efficiency discriminationof tion ofthedeleteriousallele throughnonhomologous can beproblematic.For adominantcondition,inactiva- oligonucleotides forexon skippingforDMD The genetic background of DCM isparticularlyhet- The geneticbackground ofDCM In both DCM and HCM, canonical replacement gene and HCM, DCM In both different patho- isconcerned,>1400 As farasHCM Translation ofthesefindingstothe clinic,however, 178 172 or a phosphomimetic variant of the 169 transductionofgenemodifiers(such In some DCM preclinicalmodels, InsomeDCM 177 Here,inonlyafew 168 Cardiac GeneTherapy 173 181 175 180 ) and induc- ). andpigs 171 orTALEN ortitin- 174 179 ). The 170 or 176

Review

1409 May 8, 2020 Cardiac Gene Therapy Gene Cardiac Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, A, Morgan K, Kasid Cornetta Moen R, P, Rosenberg SA, Aebersold SL. into humans– Gene transfer JC, Topalian Yang EM,Karson MT, Lotze with advanced melanoma, using tumor-infiltrat- immunotherapy of patients by retroviral gene transduction. N Engl J Med. ing lymphocytes modified doi: 10.1056/NEJM1990083032309041990;323:570–578. Med. 2019;381:455–464.J Engl N High KA, Roncarolo MG. Gene therapy. doi: 10.1056/NEJMra1706910 . 1999;153:68–70, 73. Time Jaroff L. Fixing the genes. Giacca M, Zacchigna S. VEGFGiacca M, Zacchigna gene therapy: therapeutic angiogen- Gene Ther. 2012;19:622–629. doi: esis in the clinic and beyond. 10.1038/gt.2012.17 adenoviral vectors. Gene therapy progress and prospects: St George JA. Gene Ther. 2003;10:1135–1141. doi: 10.1038/sj.gt.3302071 as vectors virus Adeno-associated M. Giacca L, Zentilin S, Zacchigna system. Circ Res. therapeutic and investigational tools in the cardiovascular 2014;114:1827–1846. doi: 10.1161/CIRCRESAHA.114.302331 and intracellular factors regulating cardio- S, Giacca M. Extra- Zacchigna Cardiovasc Res. 2014;102:312–320. life. in postnatal myocyte proliferation doi: 10.1093/cvr/cvu057 AAV in advances latest mighty: increasingly but Small H. Büning D, Grimm evolution. Hum Gene Ther. 2017;28:1075– design, and vector research, 1086. doi: 10.1089/hum.2017.172 JA, Kleinschmidt MD, R, Arap W, Pasqualini Weitzman F, Kaul Müller OJ, virus to M. Random peptide libraries displayed on adeno-associated Trepel . 2003;21:1040–select for targeted gene therapy vectors. Nat Biotechnol 1046. doi: 10.1038/nbt856 HA, Kleinschmidt M, Katus M, Trepel Hecker T, S, Korff K, Michelfelder Varadi 9 serotype AAV on displayed libraries peptide random Novel OJ. Müller JA, vectors. Gene Ther. for selection of endothelial cell-directed gene transfer 2012;19:800–809. doi: 10.1038/gt.2011.143 vectors-do not judge AAV Domenger C, Grimm D. Next-generation doi: Hum Mol Genet. 2019;28:R3–R14. its cover. a virus (only) by 10.1093/hmg/ddz148 HQ,Pham J, D, Boon H, Evens Tipanee MY, Rincon W, Tulalamba S, Sarcar muscle- Next-generation al. et M, Loperfido M, Keyaerts E, Samara-Kuko . 2019;10:492.directed gene therapy by in silico vector design. Nat Commun doi: 10.1038/s41467-018-08283-7 Catheter-based Siminiak T. Viles-Gonzalez JF, Martens TP, Sherman W, . 2006;3(suppldelivery of cells to the heart. Nat Clin Pract Cardiovasc Med doi: 10.1038/ncpcardio04461):S57–S64. JM. JJ, Leiden M, Lepore Biocompatibility of cardio- Palasis Marshall DJ, important an vectors: adenovirus with delivery catheters gene vascular Mol Ther. determinant of the efficiency of cardiovascular gene transfer. 2000;1:423–429. doi: 10.1006/mthe.2000.0059 ZX, Hinds KA, Pessanha Hill JM, RB, AJ, Raman VK, Thompson Dick Yu TR, Martin BJ, et al. Serial cardiac magnetic BS, Varney Guttman MA, stem cells. Circulation. resonance imaging of injected mesenchymal 2003;108:1009–1014. doi: 10.1161/01.CIR.0000084537.66419.7A CH, Dunnington DD,Esakof M, Maysky PR,JF, Symes Vale DW, Losordo Ashare AB, Lathi K, Isner JM. Gene therapy for myocardial angiogenesis: phVEGF165of injection myocardial direct with clinical results initial sole as Circulation. 1998;98:2800–2804.therapy for myocardial ischemia. doi: 10.1161/01.cir.98.25.2800 Milliken CE, Maysky M, Esakof DD, Symes PR, DW, Losordo Vale Isner JM. mapping to assess ventricular electromechanical Left JF, efficacy of phVEGF(165) for therapeutic angiogen- gene transfer doi: Circulation. 2000;102:965–974. myocardial ischemia. esis in chronic 10.1161/01.cir.102.9.965 A, Chronos NA, Henry Bartunek JJ, Zeiher AM, Terzic P, Dib N, Menasche M, et al; International Society Yacoub F, NS, Fernández-Avilés TD, Peters Recommendations for success- Research. for Cardiovascular Translational ful training on methods of delivery of biologics for cardiac regeneration: a Research. report of the International Society for Cardiovascular Translational doi: 10.1016/j.jcin.2009.12.013 JACC Cardiovasc Interv. 2010;3:265–275. 7. 1. 2. 3. 5. 6. 8. 9. 4. 17. 10. 11. 12. 13. 14. 15. 16. 18. 874764 “REANIMA” 2020 programme, Horizon Commission from the European 874764 Italy. Trieste, CRTrieste, continuous support of Fondazione along with the Disclosures None. REFERENCES

However, However, 184 - of modu the impossibility However, 182,183 In contrast to non homologous end joining gene cor- In contrast to non lating the resulting phenotype renders this method an phenotype renders this method an lating the resulting gene function investigation but a outstanding tool for for clinical application. still an immature approach directed repair requires the activa- rection by homology recombination machinery. tion of the cellular homologous of a patient with HCM the germline mutations Recently, 16 of the MYBPC3 in exon caused by a GAGT-deletion in human preimplantation embryos gene was corrected by CRISPR/Cas9 gene editing, using the normal allele repair. as a template for homology directed this requires cell cycling and is thus possible in ex vivo possible in ex this requires cell cycling and is thus This heart. the in cardiomyocytes in not but cells cultured of ways problem could be overcome by the identification recombina- the homologous of expression inducing of the vivo, to nudge cells in postmitotic in tion machinery or by repair, correction system toward homology directed replication, for coupling gene repair with cardiomyocyte the treatments described earlier. using one of example, Sources of Funding of Sources (ERC) Council work was supported by the European Research This Advanced “CuRE”; (BHF) by the British Heart Foundation Grant Programme Grant 787971 RG/19/11/34633 BHF and the King's College London Centre of Research Excellence grant RE/18/2/34213; “CardioReGenix” and by grants 825670 Acknowledgments A. Cannatà, H. Ali, G. Sinagra, and M. Giacca conceived the manuscript and wrote the text. Affiliations Centre of Research British Heart Foundation the King’s College London, From of Cardiovascular Medicine and Sciences, United Kingdom Excellence, School (A.C., H.A., M.G.); Department of Medical, Surgical and Health Sciences, Univer- Inter- Italy (A.C., G.S., M.G.); and Molecular Medicine Laboratory, sity of Trieste, (ICGEB), national Centre for Genetic Engineering and Biotechnology Trieste, Italy (H.A., M.G.). ARTICLE INFORMATIONARTICLE CONCLUSIONS 30over Despite therapy gene since passed have years application has was first conceived and no successful remains a young yet been developed for the heart, this In par- in its quiver. discipline with many additional arrows using studies addition gene from transition the ticular, protein-coding cDNAs- of gene expres to the modulation sion using RNAtherapeutics and the improvements in new avenues for this precise gene editing seem to offer in myocardial discipline, paving the way to applications infarction, heart failure, and inherited cardiac diseases until a decade ago. that were unapproachable straightforward with all gene sequences. In addition, addition, In gene sequences. all with straightforward haploin- be followed by should not allele inactivation shown that gene work has indeed Previous sufficiency. using in the heart in vivo can be achieved inactivation the required CRISPR/Cas9vectors to deliver AAV components. Circulation Research. 2020;126:1394–1414. DOI: 10.1161/CIRCRESAHA.120.315855 Cannatà et al Cannatà

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