JACC: BASIC TO TRANSLATIONAL SCIENCE VOL.4,NO.7,2019

ª 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/).

PRECLINICAL RESEARCH

Gene Therapy Rescues Cardiac DysfunctioninDuchenneMuscular Dystrophy Mice by Elevating Cardiomyocyte Deoxy-Adenosine Triphosphate

a b c d,e Stephen C. Kolwicz, JR,PHD, John K. Hall, PHD, Farid Moussavi-Harami, MD, Xiolan Chen, PHD, d,e b,d,e e,f,g, b,e,g, Stephen D. Hauschka, PHD, Jeffrey S. Chamberlain, PHD, Michael Regnier, PHD, * Guy L. Odom, PHD *

VISUAL ABSTRACT

Kolwicz, S.C. Jr. et al. J Am Coll Cardiol Basic Trans Science. 2019;4(7):778–91.

HIGHLIGHTS

rAAV vectors increase cardiac-specific expression of RNR and elevate cardiomyocyte 2-dATP levels. Elevated myocardial RNR and subsequent increase in 2-dATP rescues the performance of failing myocardium, an effect that persists long term.

ISSN 2452-302X https://doi.org/10.1016/j.jacbts.2019.06.006 JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Kolwicz, Jr., et al. 779 NOVEMBER 2019:778– 91 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice

We show the ability to increase both cardiac baseline function and high workload contractile performance in ABBREVIATIONS aged (22- to 24-month old) mdx4cv mice, by high-level muscle-specific expression of either microdystrophin AND ACRONYMS or RNR. mDys = microdystrophin Five months post-treatment, mice systemically injected with rAAV6 vector carrying a striated muscle-specific CK8 regulatory cassette driving expression of microdystrophin in both skeletal and cardiac muscle, exhibited the = miniaturized murine creatine kinase regulatory greatest effect on systolic function. In comparison, mice treated with rAAV6 vector carrying RNR that expresses cassette exclusively in cardiac muscle not only exhibited greatly improved baseline systolic function but also improved CMV = cytomegalovirus diastolic function. cTnT = cardiac troponin T Importantly, vector-directed overexpression of RNR did not impair cardiac reserve during increased physiological dADP = deoxy-adenosine demand in aged mdx4cv hearts. diphosphate

dATP = deoxy-adenosine SUMMARY triphosphate

DMD = Duchenne muscular Mutations in the gene encoding for dystrophin leads to structural and functional deterioration of cardiomyocytes dystrophy and is a hallmark of cardiomyopathy in Duchenne muscular dystrophy (DMD) patients. Administration of re- mdx = mouse muscular dystrophy model combinant adeno-associated viral vectors delivering microdystrophin or ribonucleotide reductase (RNR), under rAAV = recombinant adeno- muscle-specific regulatory control, rescues both baseline and high workload-challenged hearts in an aged, DMD associated viral vector mouse model. However, only RNR treatments improved both systolic and diastolic function under those con- RNR = ribonucleotide fi ditions. Cardiac-speci c recombinant adeno-associated viral treatment of RNR holds therapeutic promise reductase for improvement of cardiomyopathy in DMD patients. (J Am Coll Cardiol Basic Trans Science 2019;4:778–91) © 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/).

uchenne muscular dystrophy (DMD) and its from contraction-induced injury (3–7). In addition to D milder and allelic form, Becker muscular a structural or mechanical role, the DGC also serves dystrophy (BMD), are the most frequent as a scaffold for cytoplasmic and membrane- muscular dystrophies, occurring once in w5,000 associated signaling proteins and ion channels male births, and are due to mutations in the dystro- (8–11). The complete absence of dystrophin results phin gene (1). DMD patients typically die due to car- in drastic reductions of all DGC components (12–14). diac and respiratory muscle failure; thus, Together, an absence of dystrophin and reduction in maintenance of adequate function in both cardiac theDGCcomponentscausesmembranedestabiliza- and skeletal muscle is critical for optimal DMD ther- tion and permeability defects that lead to myofiber apy. The primary function of dystrophin is to provide degeneration, repeated cycles of degeneration/regen- a structural role by mechanically linking the subsar- eration, and the gradual replacement of muscle fibers colemmal cytoskeleton to the extracellular matrix with fibrotic, connective, and adipose tissue. through the dystrophin-glycoprotein complex (DGC) In contrast, some in-frame deletions, truncations, (2). This linkage transmits the forces of contraction and missense mutations lead to reduced dystrophin to the extracellular matrix and protects muscles expression associated with milder phenotypes. These

From the aMitochondria and Metabolism Center, University of Washington, Seattle, Washington; bDepartment of Neurology, University of Washington, Seattle, Washington; cDivision of Cardiology, Department of Medicine, University of Washington, Seattle, Washington; dDepartment of Biochemistry, University of Washington, Seattle, Washington; eWellstone Muscular Dys- trophy Specialized Research Center, University of Washington, Seattle, Washington; fDepartment of Bioengineering, University of Washington, Seattle, Washington; and the gCenter for Cardiovascular Biology, University of Washington, Seattle, Washington. *Drs. Regnier and Odom contributed equally to this work and are joint senior authors. This work was supported by grants from the National Institutes of Health (NIH): R01 HL111197 (to Dr. Regnier and Hauschka), R01 HD048895, K08 HL128826 (to Dr. Moussavi-Harami) and R01 HL128368 (to Dr. Regnier) and 1U54AR065139-01A1 (to Drs. Hauschka, Chamberlain, Regnier, and Odom). Dr. Kolwicz was funded by the American Heart Association 14SDG18590020. Dr. Chamberlain has received personal fees from Solid Biosciences, SAB. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ institutions and US Food and Drug Administration guidelines, including patient consent where appropriate. For more infor- mation, visit the JACC: Basic to Translational Science author instructions page.

Manuscript received February 27, 2019; revised manuscript received June 20, 2019, accepted June 20, 2019. 780 Kolwicz, Jr., et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice NOVEMBER 2019:778– 91

pathologies are largely curtailed in mouse (mdx)and developed pressure (LVDevP). Although mdx4cv mice canine (cxmd) models of DMD following the vector- treated with mDys appeared to normalize LVDevP, this mediated delivery of muscle-specificexpressionof did not result in a significant increase in RPP. Upon highly functional miniaturized versions of dystro- further evaluation of cardiac function, the pressure- phin, microdystrophin (mDys) (15–24).Inmdx mice, volume relationship revealed that systolic pressure muscle pathology is milder than in humans, with the response with increased preload was significantly exception of the diaphragm; however, the dystrophic improved with the treatment of either RNR or mDys. phenotype worsens with increasing age including the However, only RNR treatment resulted in significant development of cardiac dysfunction (25–32). Admin- improvements in diastolic functional parameters, istration of recombinant adeno-associated viral returning them to values that were similar to wild- (rAAV)–mediated mDys therapy in mdx mice preceding type (WT) control hearts. As a further assessment of the onset of cardiomyopathy is highly car- cardiac function, we tested hearts using a high dioprotective (33–35). However, when mdx mice are workload challenge protocol. Both RNR and mDys treated with mDys at a late stage of cardiomyopathy, treatments improved systolic function in mdx4cv such as would be the case for a number of DMD pa- hearts without compromising cardiac reserve. These tients, a full rescue of the dysfunctional cardiac positive results suggest that targeted expression of phenotype is not achieved (30,35–37). RNR within the myocardium can significantly improve contractile performance in an advanced-age SEE PAGE 792 model of DMD cardiomyopathy and may have thera- We have developed a cardiac function–enhancing peutic implications for DMD patients. gene therapy approach that targets myosin in con- METHODS tractile filaments by overexpressing the enzyme ribonucleotide reductase (RNR). RNR converts aden- ANIMAL EXPERIMENTS. osine diphosphate (ADP) to deoxy-ADP (dADP), which Male WT C57Bl/6J (The mdx4cv is rapidly converted to deoxy-adenosine triphosphate Jackson Laboratory, Bar Harbor, Maine) and (dATP) in cells. In numerous in vitro studies, we have (generated in-house) mice were used for these studies shown that dATP increases cross-bridge binding and (17). All animals were experimentally manipulated in cycling, resulting in stronger, faster contraction and accordance with the Institutional Animal Care and faster relaxation (38–45).Wehavealsoreportedthat Use Committee of the University of Washington. dATP improves contractile properties of myocardium Experimental mice were administered vector at 22 to from end-stage human heart failure (HF) in vitro (42) 24 months of age via the retro-orbital sinus with a m and dogs with end-stage idiopathic dilated cardio- 200- l bolus injection in Hanks balanced saline so- 14 myopathy (46). In normal rodent muscle, we reported lution at a dose of 2 10 vg/kg. All mice were fi that increases in cardiomyocytes and cardiac function housed in a speci c-pathogen free animal care facility occur with as little as w1% of the ATP pool in the using a 12-h light/12-h dark cycle with access to food dATP form (40,47). Similarly, rAAV-mediated de- and water ad libitum. livery of RNR under cardiac-specificregulatorycon- VECTOR PRODUCTION. rAAV genomes containing trol resulted in enzyme overexpression exclusively in the CK8 regulatory cassette (expressed exclusively in cardiomyocytes and significantly improved left ven- skeletal and cardiac muscle) and the human codon tricular function without adverse cardiac remodeling optimized (GenScript) mDys (DR2-15/DR18-22/DCT) in normal and infarcted rodent hearts (48).Ourdata (24), followed by the rabbit beta-globin poly-adeny- indicated that dATP could rescue the preload lation (pA) signal, were generated using standard responsiveness of failing hearts, suggesting restora- cloning techniques. The rAAV genomes containing tion of the abnormal Frank-Starling Law of the Heart the cardiac muscle–specific cTnT455 regulatory that often occurs in HF. cassette, the codon optimized human RNR transgene In the current study, we compare the relative flanked by 100-bp untranslated regions, and the rab- therapeutic capacity of CK8-driven mDys or cardiac bit beta-globin pA were generated as previously troponin T (cTnT)–driven RNR, via intravenously described (48).The“dead” rAAV genomes or administered rAAV vectors in an advanced-age, DMD promoter-less firefly luciferase followed by the hu- cardiomyopathy mouse model. We show a restoration man growth hormone (hGH) pA (kindly provided by of myocardial workload as indicated by rate pressure J.S.C., University of Washington, Seattle, Washington) product (RPP) for baseline function in mdx4cv mice were used to generate the control rAAV genomes. The treated with RNR. This outcome was primarily resulting constructs were cotransfected with the attributed to the normalization of left ventricular pDG6 packaging plasmid into HEK293 cells to JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Kolwicz, Jr., et al. 781 NOVEMBER 2019:778– 91 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice

generate rAAV vectors carrying serotype 6 capsids, Heart cross-sections (10 mm) were co-stained with which were harvested, enriched, and quantitated as antibodies raised against alpha 2-laminin (Sigma, St. previously described (49). Louis, Missouri; rat monoclonal, 1:200), the hinge-1 VECTOR GENOME QUANTIFICATION. Total DNA was domain of dystrophin (alexa488 conjugated MAN- extracted from flash-frozen tissue samples with Tri- EX1011b, Developmental Studies Hybridoma Bank, Reagent (MRC Inc., Cincinnati, Ohio), according to University of Iowa, mouse monoclonal, 1:200), the manufacturer’s instructions. All real-time polymerase human RRM1 (Abcam, Cambridge, United Kingdom; chain reaction (PCR) reactions were performed on a rabbit monoclonal, 1:200), and the human RRM2 QuantStudio 3 Real Time PCR System (Applied Bio- (Abcam, rabbit monoclonal, 1:200). Conjugated sec- systems, Foster City, California) in a total volume of ondary antibodies (Jackson Immuno, Goat anti- 15 ml, consisting of 5 mlsampleDNA,10.0mlTaqMan Rabbit) were used at a 1:500 dilution. Slides were Universal PCR Master Mix (Applied Biosystems), mounted using ProLong Gold with DAPI (Thermo fi 0.2 mM of each primer, and 0.1 mMTaqMancustom Fisher Scienti c)andimagedviaaLeicaSPVconfocal probe (Applied Biosystems). Reaction conditions were microscope. Confocal micrographs covering a major- 50 Cfor2min,95 Cfor10min,and40cyclesof[95 C ity of the heart left ventricular muscle sections were for 15 s followed by 60 C for 1 min]. Each sample was acquired and montaged via the Fiji toolset (ImageJ) analyzed in triplicate for concentration of total mu- and InDesign (Adobe, San Jose, California). For his- ’ rine genomes and of total vector genomes. For vector tology, Masson s trichrome staining was used to fl m genome detection by quantitative PCR, the primers examine heart cross-sections. Brie y, 10- mmuscle ’ used to amplify either the rAAV6-cTnT455-RNR or cryosections were sequentially stained in Wiegerts – rAAV6-CK8-mDys, or rAAV6-Dcytomegalovirus(CMV)- iron hematoxylin (10 min), 1% Ponceau acetic acid Luc (control vector) were unique to each vector. For (5 min), and 1% aniline blue (5 s). the RNR vector, the amplicon spanned from the distal WESTERN BLOTTING. Radioimmunoprecipitation region of the cTnT promoter, continuing into the analysis buffer supplemented with 5 mM ethyl- proximal RNR1 subunit. For the mDys vector, the enediaminetetraacetic acid and 3% protease inhibitor amplicon was contained within the CK8 regulatory cocktail (Sigma, Cat# P8340), was used to extract cassette, whereas the amplicon for the control vector muscle proteins for 0.5 h on ice with gentle agitation resided within the hGH pA. hGH primers included: every 10 min. Total protein concentration was deter- 50-CACAATCTTGGCTCACTGCAA-30,50-GGAGGCTGAG mined using Pierce BCA assay kit (Fisher Scientific, GCAGGAGAA-30;TaqManprobe:50-6FAM- CTCCGCC Kent, Washington). Muscle lysates from WT, control TCCTGGGTTCAAGCG-MBGNQ-30;CK8RCprimers: mdx4cv,andtreatedmdx4cv (30 mg) mice were dena- 50-CCCGAGATGCCTGGTTATAATT-30,50- CGGGAACAT tured at 99C for 10 min, quenched on ice, and sepa- GGCATGCA-30;TaqManprobe:50-6FAM-CCCCCCAAC rated via gel electrophoresis after loading onto ACCTGCTGCCTCT-MBGNQ-30; cTnT455-RNR1 primers: Criterion 4–12% Bis-Tris polyacrylamide gels (BioRad). 50-CCCAGTCCCCGCTGAGA-30,50-AGGTTCCAGGCGCT Overnight protein transfer to 0.45 mm polyvinylidene GCT-30; and TaqMan probe: 50-6FAM-ACTCATCAATG difluoride membranes was performed at constant 43 TATCTTATCATG-MBGNQ-30. Results were presented volts at 4CinTowbin’s buffer containing 20% relative to DNA content in each 5-ml DNA tissue sample to methanol. Blots were blocked for 1 h at room tem- determine vector genomes per ng DNA. perature in 5% non-fat dry milk for 1 h before over- TISSUE PROCESSING AND IMAGING ANALYSIS. Tis- night incubation with antibodies raised against the sues were collected and analyzed 5 months hinge-1 region of dystrophin (Developmental Studies post-administration of vectors and compared with Hybridoma Bank, University of Iowa, 1:300), age-matched male control vector (rAAV6-DCMV-Luc) anti-RRM1 (Abcam, rabbit monoclonal, 1:1,000), anti- injected mdx4cv and WT mice. Hearts were either snap RRM2 (Abcam, rabbit monoclonal 1:1,000), and frozen in liquid nitrogen or were embedded in optimal anti-GAPDH (Sigma, Rabbit polyclonal, 1:50,000). cutting temperature compound (VWR International, Horseradish-peroxidase conjugated secondary anti- Bridgeport, New Jersey) and flash frozen in liquid body staining (1:50,000) was performed for 1 h at room nitrogen cooled isopentane for histochemical or temperature before signal development using Clarity immunofluorescence analysis. The snap frozen sam- Western ECL substrate (BioRad) and visualization ples were further processed by grinding to a powder using a Chemidoc MP imaging system (BioRad). under liquid nitrogen in a mortar kept on dry ice QUANTIFICATION OF CARDIAC dATP. Approxi- for subsequent extraction of nucleic acid and protein. mately 25 mgofflash frozen, freshly ground ventricle 782 Kolwicz, Jr., et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice NOVEMBER 2019:778– 91

cardiac tissue was used for direct quantification of significance was tested at the p < 0.05 level. Statis- intracellular dATP using the high-performance liquid tical analyses were completed using Prism 7.0 chromatography (HPLC)–with tandem mass spec- (GraphPad Software, San Diego, California). trometry (MS/MS) method previously described (50). RESULTS Briefly, samples were extracted 1 to 3 days before measurement using a 50% methanol solution. The IMPROVEMENTS IN BASELINE CARDIAC FUNCTION supernatant was stored at -20C until ready for in- IN VECTOR-TREATED MDX4CV HEARTS. As depicted jection into the HPLC-MS/MS system. A Water’sXevo- 4cv in Figure 1, 22- to 24-month-old mdx mice were TQ-S mass spectrometer coupled with a Water’s administered 1 of 3 treatments: rAAV6-cTnT455- RNR Acquity I-Class HPLC was used for the analysis (Mil- 4cv (referred to as mdx þRNR); rAAV6-CK8- mDys ford, Massachusetts). Monitoring in negative mode 4cv (referred to as mdx þ mDys), or rAAV6-DCMV-Firefly via electrospray ionization was used to acquire MS/ Luciferase control vector (referred to asmdx4cv)ata MS ions. dATP concentrations were quantified with dose of 2 1014 vg/kg. By the end of the 20-week standards and normalized to tissue weight. treatment period, both mdx4cvþRNR and mdx4cvþ LANGENDORFF ISOLATED PERFUSED HEART mDys mice showed improvements in survival rates EXPERIMENTS. 4cv Ex vivo cardiac function was compared to mdx mice, although this did not reach assessed in Langendorff isolated heart preparations statistical significance (Supplemental Figure 1). At the as previously described (47,48,51). Hearts were end of 5 months, an extensive evaluation of ex vivo perfused at a constant pressure of 80 mm Hg with a cardiac function using the Langendorff isolated heart fi modi ed Krebs-Henseleit buffer supplemented with preparation was performed. The isolated heart tech- glucose and pyruvate. The perfusate contained nique allows for the direct assessment of inherent (mmol/l): 118 NaCl, 25 NaHCO3, 5.3 KCl, 2.0 CaCl2,1.2 myocardial function without the confounding effects MgSO4, 0.5 ethylenediaminetetraacetic acid, 10.0 of neuro-humoral or other systemic variables. An glucose, and 0.5 pyruvate, equilibrated with 95% O2 additional cohort of age-matched, untreated C57BL6 and 5% CO2 (pH 7.4). Temperature was maintained at mice (WT) was used as comparison control. At base- 4cv 37.5 C throughout the protocol. Left ventricular (LV) line, RPP was significantly decreased in mdx hearts fi function was monitored via a water- lled balloon duetoanapproximate20%decreaseinLVDevP inserted into the LV and connected to a pressure (Supplemental Figures 1A and 1B). RNR-treated transducer. LV systolic pressure (LVSP), end diastolic mdx4cv mice exhibited a restoration of RPP pressure, heart rate (HR), and minimum and (p ¼ 0.056) primarily due to a normalization of maximum rate of pressure change in the ventricle LVDevP (Supplemental Figures 2A and 2B). Although 4cv ( dP/dt) were obtained from the attached data mDys-treated mdx hearts appeared to normalize acquisition system (PowerLab, ADInstruments, Colo- LVDevP,thisdidnotleadtoasignificant improve- rado Springs, Colorado). After 5 min of stabilization, ment in RPP (Supplemental Figures 2A and 2B). hearts were equilibrated for 10 min at spontaneous Both þdP/dt and –dP/dt, an index of ventricular fi w HRs and then xed at a HR of 450 beats/min with an contractility and relaxation, respectively, were electrical stimulator (Grass Technologies, Warwick, decreased 30% in mdx4cv hearts (p ¼ 0.061). The þdP/ Rhode Island). Pressure-volume relationships (i.e., dt was similar to control in both RNR-treated mdx4cv Frank-Starling curves) were assessed by gradually and mDys-treated mdx4cv hearts. However, only RNR- 4cv increasingthevolumeoftheLVballoon.Aftera5-min treated mdx hearts showed –dP/dt values similar to recovery period, the perfusate was changed to an control levels (Supplemental Figures 2C and 2D). identical buffer as above except for the addition of POSITIVE CHANGES IN FRANK-STARLING MECHANICS IN 4.0 mmol/l CaCl2 to simulate a high workload chal- VECTOR-TREATED MDX4CV HEARTS. lenge for 20 min. To evaluate further systolic and diastolic function in vector- STATISTICAL ANALYSIS. All values are reported as treated mdx4cv hearts, we examined the pressure- means\ SEM.Starlingcurvesandhighworkload volume relationship (i.e., Frank-Starling mechanism) function were analyzed by 2-way repeated measures in the isolated perfused heart preparation. The LVSP analysis of variance followed by pairwise compari- response to increased preload was significantly 4cv 4cv sons using Tukey’s alpha adjustment method. Other improvedinbothinmdx þRNR and mdx þmDys endpoint data were analyzed via 1-way analysis of hearts compared to mdx4cv (Figure 2A). However, only variance or Student’s t-tests as appropriate. Kaplan- RNR treatment improved the diastolic response in Meier methods were used to analyze survival curves mdx4cv hearts, to levels similar to WT (Figure 2B). and compared using the log-rank test. Statistical Both contractility and relaxation (i.e., þdP/dt and JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Kolwicz, Jr., et al. 783 NOVEMBER 2019:778– 91 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice

FIGURE 1 Overview of Experimental Design for the Treatment of Aged (22-24 Month) mdx4cv Mice

(A) Graphic representation of rAAV6 vectors used in the present study. The ribonucleotide reductase vector contains the human cDNA for the RRM1 and RRM2 subunits whose expression is driven by the cardiac specific (cTnT455) regulatory cassette (RC). The human microdystrophin (DR2-R15/DR18-R22/DCT) vector has expression driven by the CK8 muscle-specific RC. The control vector used in the present study carries the firefly luciferase transgene whose CMV early promoter/enhancer RC has been deleted. (B) Outline of animal enrollment, vector administration, and experimental protocols implemented following a treatment period of 5 months. cDNA ¼ complementary DNA; CMV ¼ cytomegalovirus; ITR ¼ inverted terminal repeat; dATP ¼ deoxy-adenosine triphosphate; p2A ¼ porcine teschovirus-1 2A; pA ¼ poly-adenylation; qPCR ¼ quantitative polymerase chain reaction; rAAV6 ¼ recombinant adeno-associated viral vector 6; RC ¼ regulatory cassette; WT ¼ wild-type.

4cv –dP/dt, respectively) were impaired in mdx baseline function but did not impair the response to a compared to age-matched control subjects (Figures 2D short-term physiologic increase in cardiac work and 2E). Both mdx4cvþ RNR and mdx4cvþ mDys hearts (47,48). To verify that the improved systolic and dia- had significantly elevated þdP/dt values above stolic function in RNR-treated mdx4cv hearts at base- mdx4cv (Figure 2D). Interestingly, treatment of mdx4cv line was not associated with an inability to respond to hearts with RNR also significantly improved –dP/dt an increased energetic demand, we stressed hearts values (Figure 2E). All told, these data suggest that with a combination of high calcium and elevated heart both RNR and mDys treatment can improve systolic rates, via pacing stimulation. As shown in Figures 3A 4cv 4cv function in mdx hearts. However, only the RNR and 3B, mdx hearts had a blunted response to the treatment corrected diastolic dysfunction in increased workload as both LVDevP and RPP were mdx4cv hearts. w25% to 30% lower than WT hearts. In addition, both þdP/dt and –dP/dt were impaired in mdx4cv AUGMENTED RESPONSE TO INCREASED CARDIAC relative to WT hearts (Figures 3C and 3D). Systolic pa- WORKLOAD IN TREATED MDX4CV HEARTS. We pre- rameters in mdx4cvþmDys hearts were effectively viously reported that RNR overexpression in trans- improved and similar to age-matched WT hearts for genic or vector-treated mouse hearts elevated the entire duration of the workload challenge 784 Kolwicz, Jr., et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice NOVEMBER 2019:778– 91

FIGURE 2 Pressure-Volume Relationships in Isolated Perfused Hearts From Vector-Treated mdx4cv Mice

Hearts were isolated from mdx4cv mice treated with control vector (mdx4cv,n¼ 6), ribonucleotide reductase (mdx4cvþ RNR, n ¼ 6), or microdystrophin (mdx4cvþ mDys, n ¼ 5). Age-matched, nondiseased, nontreated wild-type (WT) mice were used as control subjects (n ¼ 8). The pressure-volume relationship (i.e., Frank-Starling mechanism) was evaluated by gradually increasing the volume of the left ventricular (LV) balloon. Hearts were paced at 450 beats/min throughout the protocol. (A) Left ventricular systolic pressure (LVSP). (B) LV end-diastolic pressure (LVEDP). (C) LV developed pressure (LVDevP, the difference between systolic and diastolic pressures). (D) Positive (pos) rate of pressure change was calculated by the first derivative of the ascending LV pressure wave (þdP/dt), and is used as an index of ventricular contractility. (E) Negative (neg) rate of pressure change calculated by the first derivative of the descending LV pressure wave (–dP/dt), and is used as an index of ventricular relaxation. * p < 0.05 mdx4cv versus WT; # p < 0.05 mdx4cvþ RNR versus mdx4cv; † p < 0.05 mdx4cvþ mDys versus mdx4cv.

(Figures 3A to 3C). Measures of systolic function The expression of mDys appeared to be saturated 4cv significantly increased in mdx þ RNR hearts during relative to full-length dystrophin levels, with both the initial half of the high workload protocol and being properly localized to the sarcolemma of car- 4cv remained w15% higher than mdx (Figures 3A to 3C). diomyocytes. We also evaluated general muscle his- Interestingly, –dP/dt values tended to be elevated topathology and potential differences in myocardial only in mdx4cvþ RNR hearts during the physiologic fibrosis by Masson trichrome staining, and observed challenge (Figure 3D). These data show that both RNR no discernable difference between treated or un- and mDys treatments improve systolic function in treated mdx4cv mice (Figure 5). In addition, neither mdx4cv hearts without compromising cardiac reserve. RNR nor mDys treatment significantly altered body Combined with the baseline and pressure-volume weight, heart weight, or the heart weight–to–body relationship assessments, our data show that, in weight ratio (Supplemental Figure 3). Western blot- addition to the systolic enhancements, RNR has an ting was performed to determine the extent of rAAV added benefit of improving diastolic function. vector 6–mediated RNR and mDys protein expression RNR AND mDys TRANSDUCTION, EXPRESSION, AND profiles in ventricular tissue (Figure 6). We observed CARDIOMYOCYTE LOCALIZATION. To evaluate the mDys protein expression in ventricular tissue that localization of RNR and mDys protein within the hearts approached levels similar to WT mice, whereas both of mice, we performed immunofluorescence imaging. human RNR subunits were found to be elevated to As shown in Figure 4,theRNRsubunit(Rrm1)was comparable levels within ventricular tissue robustly expressed in ventricles of RNR-treated mice. (Figure 6A). To evaluate the relative proportions of JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Kolwicz, Jr., et al. 785 NOVEMBER 2019:778– 91 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice

FIGURE 3 The Response of Vector-Treated mdx4cv Mice to High Workload Challenge in Langendorff Isolated Heart Preparations

Hearts were isolated from mdx4cv mice treated with control vector (mdx4cv,n¼ 6), ribonucleotide reductase (mdx4cvþ RNR, n ¼ 6), or microdystrophin (mdx4cvþ mDys, n ¼ 5). Age-matched, nondiseased, nontreated wild-type (WT) mice were used as control subjects (n ¼ 4). All hearts were perfused with a glucose-pyruvate buffer containing high calcium (4.0 mmol/l) to simulate a high workload challenge for 20 min. Hearts were paced at 450 beats per minute throughout the protocol. (A) LVDevP (the difference between systolic and diastolic pressures). (B) Rate pressure product (RPP, the product of LVDevP and HR). (C) Positive rate of pressure change calculated by the first derivative of the ascending LV pressure wave (þdP/dt), is used as an index of ventricular contractility. (D) Negative rate of pressure change calculated by the first derivative of the descending LV pressure wave (–dP/dt), is used as an index of ventricular relaxation. * p < 0.05 mdx4cv versus WT; # p < 0.05 mdx4cvþ RNR versus mdx4cv; † p < 0.05 mdx4cvþ mDys versus mdx4cv. Abbreviations as in Figures 1 and 2.

dATP concentrations within ventricular tissue, we DISCUSSION performed HPLC-MS/MS analysis on ground ventric- 4cv 4cv ular tissue from mdx and mdx þ RNR mice. The In the present study, we used a novel gene therapy concentration of dATP within the ventricular tissue approach that targets myosin in the contractile fila- 4cv obtained from mdx mice treated with RNR ments of cardiomyocytes via overexpression of the (0.57 0.22 pmol dATP/mg) was approximately RNR enzyme to rescue cardiomyopathy in a DMD 4cv 10-fold higher relative to mdx control subjects mouse model. RNR overexpression results in elevated (0.05 0.02 pmol dATP/mg) (Figure 6B). For adult WT, dATP, which can be used by cardiac myosin (in place we previously reported an average dATP value of 0.02 of ATP), and increases cross-bridge binding and pmol/mg tissue with a SD of 0.01 (50). Additionally, cycling, resulting in stronger, faster contraction and cardiac vector genome data was comparable relative faster relaxation (38–45,52). We developed an rAAV to the vector dose administered (Figure 6C). vector that over-expresses RNR selectively in heart 786 Kolwicz, Jr., et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice NOVEMBER 2019:778– 91

FIGURE 4 Cardiac Transduction Following Intravenous Delivery of Ribonucleotide Reductase or Microdystrophin

At 5 months after vector administration, cryosections were prepared and immunostained with antisera against dystrophin or ribonucleotide reductase. A considerable level of protein is detected for each ribonucleotide reductase subunit-1 (human specific) as indicated by immunofluorescent staining (red) localized primarily within the cytoplasm of cardiomyocytes with occasional perinuclear accumulation (upper panel). On the lower panel, the robust level of expression for dystrophin in WT and in aged mdx4cv mice treated with AAV6-CK8-mDys (laminin staining, inset image). Abbreviations as in Figures 1 and 2.

muscle via inclusion of a modified cardiac-specific metabolism. More recently, a declination in cardiac enhancer/promoter derived from the human cTnT function was not observed until 12 months of age (57) gene, which facilitates increases in myocyte contrac- and abnormal in vivo pressure-volume dynamics tion and cardiac performance in normal rodent hearts were observed in 22-month-old mdx mice (58). as well as in infarcted rodents and pig hearts (48,53). Treatment of young male mdx mice with rAAV6 vec- Importantly, we previously showed that dATP- tor delivering cytomegalovirus (CMV) promoter/ producing cells deliver it to surrounding myocar- enhancer driven microdystrophin did not correct the dium by diffusion through gap junctions (54),such impairments in þdP/dt, –dP/dt, or LV systolic pressure that only a minority of cardiomyocytes needs to be when assessed at 5 months of age via hemodynamic transduced to have global benefits within the heart. analysis (35). However, an improvement was noted for We now show that using this vector technology leads the preload recruited stroke work in mdx mice treated to a clear benefit of improving cardiac function in an with mDys compared to untreated mdx or WT control advanced-age model of DMD cardiomyopathy. subjects (35). Early manifestations of impaired cardiac dysfunc- In our current study, 27-month to 29-month-old tion in mdx mice are generally not reported. Khair- mice were subjected to ex vivo assessment of cardiac allah et al.(55) observed a decline in LV function function using the Langendorff isolated heart prepa- in isolated perfused hearts that coincided with ration, which is a century-old methodology with decreased fatty acid oxidation and increased glucose several advantages and limitations (59).The oxidation. However, perfused heart function in mdx perfusate used is similar to, but not the same as mice was maintained at 8 months, despite significant blood, and situations of physiologic challenge (e.g., reductions in phosphocreatine levels and free energy Frank-Starling and high calcium) may exaggerate the availability from ATP hydrolysis (56).Theauthors in vivo situation. However, the procedure remains a surmised that young adult mdx hearts, akin to DMD simple and reproducible experiment that allows for patient hearts, experience right ventricular dilation, interrogation of cardiac physiology in the absence of LV diastolic deficits, and abnormal energy confounding systemic variables and serves as a viable JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Kolwicz, Jr., et al. 787 NOVEMBER 2019:778– 91 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice

FIGURE 5 Heart Histologic Staining of mdx4cv Mice Suggests No Morphologic Alterations From RNR Therapy

Representative full-view photomicrographs of Masson trichrome staining of the hearts from mdx4cv mice displaying control vector (DCMV), and rAAV6-treated with either RNR or mDys from mdx4cv mice. Similarly, a 20 enlarged view of the corresponding images (*) is shown. Abbreviations as in Figures 1 and 2.

tool to perform cardiac phenotyping in preclinical In addition to the evaluation of basal cardiac per- studies (60). Under baseline conditions and at spon- formance, we tested cardiac reserve with a combina- taneous heart rates, we observed a significant tion of high calcium plus elevated HRs and confirmed 4cv reductioninRPPinmdx hearts. Decreased systolic asignificantly blunted response in 27-month to and diastolic performance in mdx4cv hearts also 29-month-old mdx4cv hearts. Consistent with a pre- existed while examining the length-tension relation- vious report in young mdx mice (35),expressingmDys ship (i.e., Frank-Starling mechanism). Treatment of in aged mdx4cv hearts significantly improved systolic mdx4cv mice with either the RNR or the mDys vector performance during increased physiologic demand to restored systolic pressure development without alevelsimilartoage-matched healthy control affecting spontaneous HRs at baseline. Both of the subjects. We previously reported that transgenic or treatments restored the disrupted Frank-Starling vector-directed overexpression of RNR did not impair response, particularly for systolic function. The RNR cardiac reserve during increased physiologic demand treatment also had beneficial effects on the diastolic in young healthy hearts (47,48). Herein, we report properties of the mdx4cv myocardium. This strongly that RNR over-expression in mdx4cv hearts normalizes supports our previous studies where we observed a both the systolic and diastolic response to an tendency for overexpression of RNR to improve increased cardiac challenge. myocardial diastolic response in young, healthy, Approximately two-thirds of DMD mutations are transgenic mice (47),micetreatedwithAAV(48),and deletions that span 1 or more exons, often leading to in a porcine HF model (53). large deletions clustering around 2 mutational 788 Kolwicz, Jr., et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice NOVEMBER 2019:778– 91

FIGURE 6 Protein Expression Levels for mDys, RNR, and Resultant Vector Genomes Due to rAAV6-Mediated Gene Transfer

(A) RNR and mDys protein expression detection as revealed by immunoblotting of cardiac whole tissue lysates using either RRM1, RRM2, or antidystrophin antibody. (B) HPLC-MS/MS intracellular [dNTP] quantification from methanol extracted cardiac tissue. (C) qPCR analysis of vector genomes from cardiac tissue revealed similar vector genomes being represented for all treated cohorts. dNTP ¼ deoxynucleotide triphosphate; HPLC-MS/MS ¼ high-performance liquid chromatography–tandem mass spectrometry; qPCR ¼ quantitative polymerase chain reaction; other abbreviations as in Figures 1 and 2.

hotspots; the most common spanning from exons 45- DMD-related cardiomyopathy usually occurs 55 resulting in removal of a central portion of the rod during middle to late adolescence, but the clinical domain inclusive of disrupting the neuronal nitric presentation is deceptive, as patients are wheelchair- oxide synthase (nNOS) localization domain. The sec- bound and are not required to perform increased car- ond most common hotspot spans from exons 3-19, and diac workload. The diversity of cardiac phenotype in removes the majority of the amino-terminal actin- DMD suggests many levels of pathogenic mechanisms. binding domain 1 which is essential for dystrophin The pathologic heterogeneity of the DMD ventricular function. When such deletions interrupt the reading myocardium is a consequential result of myocardial frame, the resultant mutated protein usually ex- atrophy (65,66), compensatory mechanisms leading to presses at extremely low levels and associates with cardiac remodeling with ensuing ventricular dilation the loss of ambulation at or before 12 years of age. and fibrosis (67,68). Absence of clear genotype- Even when such deletions produce an in-frame phenotype correlation in DMD probably results from partially functional truncated protein, such as occurs at least 4 secondary cellular processes including þ in BMD, dramatic phenotype diversity can occur. For aberrant intracellular Ca2 homeostasis, decreased example, in a previous study, BMD subjects whose nitric oxide–cyclic guanosine monophosphate path- out-of-phase deletions, toward the alignment of ways, mitochondrial dysfunction, and increased adjoining spectrin-like repeats, developed dilated reactive oxygen species, which individually or cardiomyopathy at nearly a decade earlier than in collectively influence the clinical phenotype (69).As patients with in-phase deletions (61).Incontrastto an indication of the progressive nature of DMD car- large deletion mutations, there are reported cases diomyopathy, the estimated overall incidence of where individual missense mutations lead to X-linked latent DCM is 25% by 6 years of age and 59% by 10 dilated cardiomyopathy (62–64).Inonecase,anovel years of age in DMD patients (70). As such, further missense mutation within exon 9 at nucleotide 1043 evidence suggests latency in cardiac dysfunction at resulted in a T279A amino acid change in a highly basal levels that becomes more pronounced with an conserved position of mutation. This mutation resul- induction of increased physiological demand. ted in a substitution of a beta-sheet for alpha-helical Consistent with these observations, an impaired structure, destabilizing the protein, and leading to response to beta-adrenergic stimulation by either the cardiac specific phenotype described by the au- dobutamine or isoproterenol can be detected in mdx thors (63). mice at 3 to 4 months of age (35,71).Exercisecan JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Kolwicz, Jr., et al. 789 NOVEMBER 2019:778– 91 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice

potentially be a double-edged sword for DMD, where be nonimmunogenic, supplemental RNR therapy in the enthusiasm for potential skeletal muscle benefits conjunction with mDys therapy might be beneficial for can be dampened by the insidious cardiotoxicity from DMD patients. Future studies are aimed at deter- training (72–74). With gene replacement therapy via mining the extent of functional restoration by rAAV-mediated mDys in current ongoing clinical trials, combining both RNR- and mDys-based therapies. it will be of interest to assess cardiac function to ACKNOWLEDGMENTS The authors thank James determine whether additional therapeutic support in Allen and Christine Halbert (Wellstone Muscular fi the form of increased contractility will be bene cial. Dystrophy Specialized Research Center, University of fl Relative expression assays by immuno uorescence Washington, Seattle, Washington) for assistance with and Western blotting indicate robust expression vector production, purification, and quality assessment. levels for the RNR subunits (RRM1 and RRM2) and m Dys in the vast majority of cardiomyocytes. How- ADDRESS FOR CORRESPONDENCE: Dr. Guy L. ever, intracellular variability in RNR protein detec- Odom, Department of Neurology, University of tion (via immunostaining) was observed for both Washington, 850 Republican Street, S248, Box RRM1 and RRM2, indicating that not all car- 358055, Seattle, Washington 98109. E-mail: godom@ diomyocytes were effectively transduced. Neverthe- uw.edu. OR Dr. Michael Regnier, Department of less, we previously have shown that gap junction Bioengineering, University of Washington, 850 transport of dATP from transduced cardiomyocytes to Republican Street, S180, Seattle, Washington 98109. adjacent cardiomyocytes occurs (54), which likely E-mail: [email protected]. accounted for the consistently elevated LV function of mice treated with the rAAV vector that over- PERSPECTIVES expresses RNR. This is in contrast to the anchored sarcolemma localization of mDys, which would occur COMPETENCY IN MEDICAL KNOWLEDGE: RNR over- only in transduced muscle cells. Additionally, expression represents an emerging therapy for improving car- because muscle-specificregulatorycassetteswere diac dysfunction via cardiac myosin activation in a variety of used for expression of therapeutic proteins, we can clinical situations. Currently, there are 3 ongoing clinical trials surmise that <1% of protein expression would be using rAAV-mediated variants of mDys that have shown broad generated from non-muscle cell types present in the promise in terms of safety, and in biomarker indicators sug- heart, suggesting that the functional benefitwastruly gesting pathologic improvements. If cardiac dysfunction is caused by cardiomyocyte transduction. As previously detected in the patients from these clinical studies, our studies stated, the cTnT regulatory cassette drives RNR in aged, mdx mice suggest that combinatorial RNR therapy may expression only in heart muscle, whereas the CK8 be beneficial. regulatory cassette drives mDys expression no selec- tively in both cardiac and skeletal muscle cells, but at TRANSLATIONAL OUTLOOK: Our novel strategy of vector- only very low background levels in other tissues. This mediated, cardiac-specific over-expression of RNR shows raises the possibility that the functional benefitof promising potential for rescuing both systolic and diastolic expressing mDys in skeletal muscles might increase function in aged, DMD mice even in the absence of addressing energetic demands on the heart, thereby partially the underlying dystrophin deficiency. Future studies investi- masking some of the mDys-derived cardiac functional gating the combined effects of mDys and RNR may disclose an benefits (37). Because RNR delivery to DMD mice of improved therapeutic strategy to provide structural and func- advanced age increased cardiac function in the tional improvement of DMD patient hearts. absence of RNR expression in skeletal muscle, and because over-expression of endogenous RNR should

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