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Calcium Up-Regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease Phase 2b (CUPID 2): a Randomised, Multinational, Double-Blind, Placebo-controlled Trial

Barry Greenberg, Javed Butler, G. Michael Felker, Piotr Ponikowski, Adriaan A. Voors, Akshay S. Desai, Denise Barnard, MD; Alain Bouchard, Brian Jaski, Alexander R. Lyon, MD, PhD; Janice M. Pogoda, Jeffrey J. Rudy, Krisztina M. Zsebo

Affiliations: UCSD Sulpizio Cardiovascular Center, La Jolla, CA, USA (Prof B Greenberg MD, Prof D Barnard MD); Stony Brook University, Stony Brook, NY, USA (Prof J Butler MD); Duke University School of Medicine, Durham, NC, USA (Prof G M Felker MD); Wroclaw Medical University and Military Hospital, Wroclaw, Poland (Prof P Ponikowski MD); University of Groningen, Groningen, Netherlands (Prof A A Voors MD); Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA, USA (A S Desai MD); Cardiology, PC, Birmingham, AL, USA (A Bouchard MD); San Diego Cardiac Center, Sharp Memorial Hospital, San Diego, CA, USA (B Jaski MD); Royal Brompton Hospital and Imperial College London, London, UK (A R Lyon MD); Celladon Corporation, San Diego, CA, USA (J M Pogoda PhD, J J Rudy BS); Santa Barbara, CA, USA (K M Zsebo PhD)

Corresponding Author: Barry Greenberg, MDDistinguished Professor of MedicineDirector Advanced Heart Failure Treatment ProgramUCSD Sulpizio Cardiovascular Center9444 Medical Center Dr., #7411La Jolla, CA 92037-7411Phone: 858-657-5267Email: bgreenberg@ucsd.edu

Key Words: Gene transfer therapy, heart failure, SERCA2a

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Funding statement: The clinical study, data analyses, and manuscript

support were funded by Celladon Corporation.

Summary

Background Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) activity

is deficient in the failing heart. Correction of this abnormality by gene

transfer may improve cardiac function. CUPID 2 investigated the clinical

benefits and safety of gene therapy through infusion of adeno-associated

virus 1 (AAV1)/SERCA2a in heart failure patients with reduced ejection

fraction.

Methods CUPID 2 was a phase 2b, multinational, double-blind, placebo-

controlled study of high-risk ambulatory patients with New York Heart

Association class II-IV symptoms, ischemic or non-ischemic aetiology, and left

ventricular ejection fraction ≤0·35. The study was conducted at 67 clinical

centres and hospitals in the United States, Europe, and Israel. Patients were

randomised 1:1 via an interactive voice and web response system to receive

a single intracoronary infusion of 1x1013 DNase-resistant particles of

AAV1/SERCA2a or placebo. Randomisation was stratified by country and by 6

minute walk test distance. Patients were followed for ≥12 months. The

primary efficacy endpoint was time to recurrent events (hospitalization,

ambulatory worsening heart failure treatment) analysed using a joint frailty

model to account for multiple, correlated events within subjects. Primary

efficacy endpoint analyses and safety analyses were performed on all treated

patients. The trial was registered with clinicaltrials.gov, number

NCT01643330, and is now closed.

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Findings Between July 9, 2012 and February 5, 2014, 1558 patients were

screened and 250 were enrolled; 121 were infused with AAV1/SERCA2a and

122 with placebo. Compared with placebo, AAV1/SERCA2a did not improve

the primary endpoint (128 recurrent events versus 104 recurrent events;

hazard ratio 0·93; 95% CI 0·53—1·65; p=0·81). No safety issues were noted.

Interpretation CUPID 2 was the largest gene transfer study performed in

heart failure patients to date. Despite promising results from earlier studies,

a single intracoronary infusion of AAV1/SERCA2a at the dose tested did not

improve the clinical course of heart failure patients with reduced ejection

fraction.

Funding Celladon Corporation.

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Introduction

Despite advances in treatment, morbidity and mortality remain unacceptably

high for patients with heart failure (HF)1 and new approaches for improving

outcomes are needed. Identification of derangements in key pathways that

regulate cardiac function has provided potential novel targets for gene

therapy, and evidence that vectors such as adeno-associated viruses (AAVs)

can deliver genes of interest to cardiomyocytes, resulting in sustained

transgene expression in the heart, has stimulated interest in gene transfer as

a strategy for treating HF. The sarco/endoplasmic reticulum Ca2+ ATPase

(SERCA2a) regulates cardiomyocyte contraction and relaxation by

transporting Ca2+ from the cytosol into the sarcoplasmic reticulum during

diastole.2,3 A deficiency of SERCA2a is related to HF progression.4,5 Correction

of this deficiency has been shown to favourably affect calcium flux and

improve the function of cardiomyocytes derived from failing hearts. Gene

transfer of SERCA2a has also been shown to improve cardiac performance

and survival in experimental models of HF.4,5 Recently, we reported that a

single intracoronary infusion of recombinant AAV serotype 1 (AAV1)

delivering the SERCA2a gene to the heart had favourable effects in patients

with advanced HF in a pilot study.6,7 On the basis of these promising results,

the Calcium Up-Regulation by Percutaneous Administration of Gene Therapy

in Cardiac Disease Phase 2b (CUPID 2) study was designed to further assess

the effects of AAV1/SERCA2a therapy on clinical outcomes in a larger group

of patients with moderate to severe HF and reduced ejection fraction.8

METHODS

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Study design

The CUPID Phase 2b trial (CUPID 2; NCT01643330) was a multinational,

double-blind, placebo-controlled, randomised study designed to investigate

whether gene transfer therapy with SERCA2a improved outcomes in patients

with HF and reduced ejection fraction. The study design has been published.8

The study was conducted at 67 centres and hospitals in the United States

(US), Europe, and Israel according to the principles of the International

Conference on Harmonisation Guideline on Good Clinical Practice and the

principles of the World Medical Association Declaration of Helsinki. All

relevant Institutional Review Board and Institutional Bio-Safety Committee

approvals were obtained at each site. Manufacturing information is provided

in the Appendix (p 4).

Participants

Eligible patients were between 18 and 80 years of age with a diagnosis of

stable New York Heart Association (NYHA) class II-IV chronic HF due to

ischemic or non-ischemic cardiomyopathy and left ventricular ejection

fraction ≤0·35 on optimal tolerated stable medical therapy for at least 30

days prior to randomisation. In response to a lower than anticipated pooled

event rate during the early period of the trial, a protocol amendment

designed to increase risk for future HF events was initiated after enrolment of

101 patients. This amendment required eligible patients to have elevated N-

terminal pro–B-type natriuretic peptide (NT-proBNP) (>1,200 pg/mL, or

>1,600 pg/mL if atrial fibrillation was present) or HF-related hospitalization

within 6 months of enrolment into the study. Patients were required to have

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<1:2 or equivocal anti-AAV1 neutralizing antibody (NAb) titres at screening.

Exclusion criteria included cardiac surgery, percutaneous coronary

intervention, valvuloplasty, or intravenous (IV) therapy for HF within 30 days

prior to screening. A comprehensive list of exclusion criteria has been

published.8 All patients provided written informed consent.

Randomisation and masking

Following screening, patients were randomised in parallel in a 1:1 ratio to

receive either 1x1013 DNase resistant particles (DRP) AAV1/SERCA2a or

placebo. Randomisation was conducted through a fully validated and

controlled interactive voice and web response system provided by Almac

Clinical Technologies. Randomisation was stratified by country and the ability

to walk between 150 and 425 meters or outside of these distances on the 6

minute walk test (6MWT). A blinded kit was shipped to the investigative site

following randomisation. All patients and physicians were blinded to

treatment assignment, and the company that conducted randomisation was

not involved with other facets of the trial.

Procedures

Drug was administered a single time to each patient. On day 0, before

infusion of the investigational product, coronary angiography was performed

to determine the strategy for administering AAV1/SERCA2a and to confirm

that at least one coronary artery had Thrombolysis in Myocardial Infarction

(TIMI) flow grade 3. Infusion of the investigational product was tailored to the

patient and multiple infusion scenarios were possible depending on the

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extent and distribution of coronary artery stenosis, collateralization patterns,

and anatomic variations. During the single administration of drug, operators

were instructed to provide delivery using at most three infusions according to

the distribution of left ventricular blood flow.8 The overall goal was to achieve

homogeneous delivery to the myocardium with two-thirds of the dose to the

anterolateral and one-third to the posterolateral myocardium. It was

recognized that multiple coronary infusion scenarios were possible based on

occlusive disease and collateralization patterns and investigators received

instruction regarding perfusion options at the time their sites were activated.

An IV nitroglycerin infusion was started 10 to 25 minutes prior to infusion of

the investigational product to enhance uptake of AAV1/SERCA2a in

cardiomyocytes by increasing vasodilation of the capillary bed.9

During the 12-month active observation period, assessments of efficacy,

safety, and quality of life were undertaken at months 1, 3, 6, 9, and 12. Data

collection on clinical endpoints continued until the primary analysis data

cutoff was reached, which was when all patients completed the 12-month

active observation period and at least 186 adjudicated HF-related recurrent

events had occurred.

Outcomes

The primary efficacy endpoint was time to recurrent events, defined as

hospitalizations due to HF or ambulatory treatment for worsening HF. The

secondary efficacy endpoint was time to first terminal event, defined as all-

cause death, heart transplant, or durable mechanical circulatory support

device (MCSD) implantation. All primary and secondary endpoints were

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reviewed by a blinded clinical endpoints committee (Appendix p 3) and

adjudicated according to standardized definitions. Adjudication criteria for

these events, as well as detailed statistical methods, have been previously

described.8 Exploratory analyses included the effect of the investigational

product on change from baseline in NYHA class, exercise ability as assessed

by the 6MWT, quality of life as assessed by the Kansas City Cardiomyopathy

Questionnaire (KCCQ), and NT-proBNP.

Safety was assessed in all patients who received treatment with

AAV1/SERCA2a or placebo. Safety parameters included incidence and

severity of adverse events and time to cardiovascular-related death.

Post-treatment tissue and serum processing

During follow-up of patients enrolled in the study, participating centres were

instructed to try to obtain tissue samples from treated patients at the time of

cardiac transplantation, implantation of a MCSD, or at autopsy. The levels of

AAV1/SERCA2a were determined using methods previously described.7 In

addition, AAV1 NAb testing was performed in study patients using serum

collected at the 6 month follow-up visit.

Statistical analysis

Monte Carlo simulation using background rates and correlations similar to

those observed in CUPID 1 estimated that 186 recurrent events in 250

patients with a median follow-up time of 18 months would provide 80%

power at the 0·05 two-sided significance level to detect a recurrent event

hazard ratio (HR) of 0·55 using a joint frailty model.

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The intention-to-treat (ITT) analysis population was defined as all randomised

subjects.10 A modified ITT (mITT) analysis population was also pre-specified,

comprising only randomised patients who received study medication.10,11 The

primary analysis of the primary and secondary endpoints was done at the

primary analysis data cutoff using the mITT population ; secondary analyses

were done using the ITT population (all randomised patients) and additional

pre-specified populations (Appendix p 5). Treatment effects on the primary

and secondary endpoints were estimated simultaneously by a semi-

parametric joint frailty model12 implemented using the NLMIXED procedure13

in SAS (SAS Institute, Inc., Cary, NC). This model accounts for correlated

recurrent events within patients and the correlation between recurrent and

terminal events (i.e., informative censoring). The reference time point was

randomisation date for the ITT population and treatment date for the mITT

population and for the additional pre-specified populations (e.g. excluding

patients who had major protocol deviations and excluding patients who were

positive or equivocal for neutralizing antibodies). Primary and secondary

endpoints were graphically depicted using the mean cumulative function14

and the survival function (estimated by the PHREG procedure in SAS),

respectively. Sensitivity analyses using alternative models for both endpoints

were also performed.

The trial was registered with clinicaltrials.gov, number NCT01643330.

Role of the funding source

This trial, including patient management, data collection, and data analysis,

was funded by Celladon Corporation. Celladon also provided funding for

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manuscript and graphics support. The corresponding author had full access

to all data in the study and, with the support of the full author group, had

final responsibility for the decision to submit for publication.

Results

From July 9, 2012 through February 5, 2014, 1558 patients at 67 centres in

the US, Europe, and Israel underwent NAb prescreening for CUPID 2 (Figure

1). Of these patients, 921 (59·1%) were NAb positive and 284 (18·2%) were

considered ineligible for other reasons, leaving 353 (22·7%) with a qualifying

NAb titre (<1:2 or equivocal) who were eligible for further screening. Of these

patients, 103 (29·2%) were excluded for reasons summarized in Figure 1, and

250 patients were enrolled into the study and randomised. Two of 123

patients allocated to receive AAV1/SERCA2a and five of 127 patients

allocated to placebo did not receive study drug infusion (Figure 1). The

remaining 121 patients who received AAV1/SERCA2a and 122 patients who

received placebo constituted the mITT population that was the pre-specified

population for the primary efficacy analysis. Over the course of the study, 5

patients (3 in mITT) withdrew consent and 1 (in mITT) was lost to follow-up.

The participants were predominantly white and male with two-thirds from the

US (Table 1). A total of 135/250 (55·6%) patients had coronary artery

disease and HF was ascribed to an ischemic aetiology in 125/250 (51·4%)

patients. Patients had moderate to severe HF as evidenced by NYHA

Functional Class, ejection fraction, 6MWT distance, KCCQ score, and NT-

proBNP level. Baseline characteristics were balanced between groups.

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Median follow-up was 17·5 months since the study extended over 30 months

in order to allow all randomised patients to be followed for at least 12

months. At the time the last patient had been followed for 12 months, a total

of 232 recurrent and 65 terminal events had occurred in the mITT population.

Of the 232 recurrent events that qualified as primary endpoints, 128 were in

the placebo group and 104 were in the AAV1/SERCA2a group; most were HF

hospitalizations. Treatment with AAV1/SERCA2a failed to improve the rate of

recurrent events (HR, 0·93; 95% confidence interval [CI] 0·53 to 1·65;

p=0·81; Figure 2A and Table 2). Of the 65 terminal events that qualified as

secondary endpoints, 29 were in the placebo group and 36 were in the

AAV1/SERCA2a group; most were deaths (Table 2). AAV1/SERCA2a

administration failed to improve time to first terminal event (HR, 1·27; 95% CI

0·72 to 2·24; p=0·40; Figure 2B). AAV1/SERCA2a treatment also did not

improve time to all-cause death (Figure 2C).

No differences between treatment groups were detected in subgroup

analyses of the primary endpoint (Figure 3). In a pre-specified subgroup

analysis of the secondary endpoint, there was a significant interaction

between treatment and geography (Figure 3), with a higher HR in non-US

patients compared with US patients. However, the number of events in the

analysis of non-US patients was small (22 events in 85 patients), and baseline

disease characteristics suggest that non-US AAV1/SERCA2a patients may

have had more severe illness than non-US placebo patients (Appendix Web

Table 1). There was no such interaction for the primary endpoint. No other

significant interactions were detected for pre-specified subgroup analyses,

although a significant interaction was observed for the non-pre-specified

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subgroup of patients with diabetes. Post-hoc analyses of the primary and

secondary endpoints stratified by randomisation in the study “pre” or “post”

initiation of the protocol amendment designed to increase the risk for future

HF events showed that there was no meaningful difference in treatment

effect between these subgroups. For the primary endpoint, the HRs were

0·86 (95% CI 0·32 to 2·27) and 1·05 (95% CI 0·53 to 2·08) for “pre” and

“post” amendment patients, respectively, while for the secondary endpoint

the HRs were 1·14 (95% CI 0·53 to 2·44) and 1.38 (95% CI 0·59 to 3·25),

respectively.

There were no significant differences between treatment groups for any of

the exploratory efficacy analyses (change from baseline in NYHA class,

exercise ability as assessed by the 6MWT, quality of life as assessed by the

KCCQ, or levels of NT-proBNP) over 12 months of follow-up. No significant

treatment group differences were observed in the ITT analyses or in analyses

conducted in other pre-specified populations (Appendix p 5).

In safety evaluations, there were 262 clinical events in placebo and 190 in

AAV1/SERCA2a patients (Table 3); most were hospitalizations. There were 20

deaths in placebo and 25 deaths in AAV1/SERCA2a patients, 18 and 22 of

which were adjudicated as being due to cardiovascular causes. Comparisons

of treatment-emergent serious adverse events occurring in ≥2% of either

treatment group identified only one significant difference between groups:

placebo patients had a higher rate of implantable defibrillator insertion than

AAV1/SERCA2a patients (6/122 [4·9%] versus 0/121 [0%]; p=0·03) (Appendix

Web Table 2).

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Since the results of CUPID 2 were divergent from those of CUPID 1, which

showed a beneficial effect of AAV1/SERCA2a on HF outcomes, post-hoc

analyses were performed to provide potential insights into the differences in

the efficacy of AAV1/SERCA2a therapy in CUPID 2 compared with CUPID 1.

There were no obvious important differences in study population

characteristics between these trials except for a higher use of cardiac

resynchronization therapy in CUPID 1 (Appendix Web Table 3), which

reflected the higher usage of this treatment modality in the exclusively US

population in CUPID 1 as compared with the international population enrolled

in CUPID 2. A review of manufacturing processes identified a difference in the

proportion of empty viral capsids (containing only the protein capsid and not

the single stranded DNA) between CUPID 1 (85%) and CUPID 2 (25%)

(Appendix Web Table 4), which may have affected transduction efficiency

(Appendix p 4 and Web Figure 1).

We assessed the presence of AAV1/SERCA2a in cardiac tissues from patients

whose condition deteriorated requiring either transplant, or MCSD

implantation and patients from whom cardiac tissue was obtained at autopsy.

A total of 23 heart tissue samples were obtained from 7 patients (Appendix

Web Table 5). The levels of vector DNA in these tissues (approximate median

of 43 copies/μg DNA; range <10 to 192) were at the lower end of the

threshold for dose response curve in pharmacology studies (<500 copies/μg

DNA). Although it is difficult to determine the number of cells that were

transduced due to the variable ploidy of cardiomyocytes in advanced HF

patients,15 these levels are most consistent with the likelihood that only a

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very small percentage of cardiomyocytes were expressing AAV1-delivered

SERCA2a in the myocardium of these patients.

Testing for the presence of AAV1 NAbs showed the expected high rate of

seroconversion in patients treated with AAV1/SERCA2a, but not in those who

were treated with placebo (Appendix p 5 and Web Table 6). These NAbs are

not expected to have influenced the level of SERCA2a expression, as an

antibody response occurs days to weeks after the cells take up the AAV

vector. Testing for the presence of an anti-AAV1 specific CD8 T cell response

was conducted and found to be mostly negative, so a cellular immune

response cannot explain the low level of transduced cells and lack of efficacy.

Discussion

CUPID 2 was the largest study of gene transfer performed in a HF population

to date and the first to look at clinical events as the primary endpoint. On the

basis of strong evidence demonstrating that a deficiency in SERCA2a

adversely affects cardiac function and favourable results with AAV1/SERCA2a

gene transfer in both experimental models and patients treated in pilot

studies,6,7,16-20 CUPID 2 was designed to assess whether AAV1/SERCA2a

administration improves the clinical course of moderate to severe HF patients

with reduced ejection fraction who were receiving contemporary guideline-

recommended therapy. The results showed that AAV1/SERCA2a at the dose

used did not reduce either recurrent HF events (primary efficacy endpoint) or

terminal events (secondary efficacy endpoint) in the overall study population

or in pre-specified subgroups. However, no evidence of worsening of the

clinical course of study patients emerged during the study.The negative

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results of CUPID 2 raise important questions. Although gene transfer is a

promising approach for treating human disease, there has been limited

success to date with this approach in treating patients with cardiovascular

disease. Previous experimental studies showed that reduced SERCA2a

activity was associated with abnormalities in calcium homeostasis and

cardiomyocyte function and that correction of these abnormalities by gene

transfer improved cardiac function and survival.2-5,16-19 In a pilot dose-finding

study of AAV1/SERCA2a (CUPID 1) in patients with HF, administration of

1x1013 DRP was associated with stabilization or improvement in several

independent measures of patient wellbeing and cardiac function. There was

also a reduction in the recurrent event rate compared with patients who were

treated with placebo.6,7, These results provided the rationale for and informed

the design (including dose) for CUPID 2. The reasons for the failure of

AAV1/SERCA2a to improve the clinical course of HF patients and the

differences between the results of CUPID 1 and CUPID 2 are unclear. The

entry criteria and treatment algorithms were similar between the studies, and

although CUPID 2 added the requirement for elevated natriuretic peptide

levels or a recent hospitalization during the course of the study to enrich for

recurrent HF events, comparison of the profile of the patients included in the

studies reveals no striking differences. Moreover, post-hoc analyses indicated

that the amendment did not meaningfully affect the response to treatment

for either the primary or secondary efficacy endpoints. However, the crude

recurrent event rate in placebo patients was higher in CUPID 1 compared

with CUPID 2 (1·27 per patient/yr vs. 0·7 per patient/yr, respectively), and

CUPID 1 was a small study, with only 14 patients receiving placebo and nine

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receiving 1x1013 DRP AAV1/SERCA2a. These factors raise the possibility that

the positive results observed in CUPID 1 were due to chance and/or to

greater severity of illness in patients randomised to placebo. The negative

results of CUPID 2, however, do not appear to be related to the high

percentage of coronary artery disease (56%) in the population enrolled in the

study, as there were no differences in outcomes by HF aetiology.

Another possibility is inadequate delivery and uptake of the vector in the

hearts of patients enrolled in CUPID 2. It is possible that other approaches for

introducing AAV1/SERCA2a to the heart might have enhanced uptake into

cardiomyocytes.21,22 Intracoronary delivery of AAV1/SERCA2a, however, is

simpler and more practical than other modes of delivery, and this technique

was associated with significant increases in SERCA2a gene expression both in

a large animal model using the same vector as in CUPID 218 and in pilot

studies in which HF patients were treated with intracoronary delivery of

AAV1/SERCA2a.6,7,20 However, in the intervening period between CUPID 1 and

CUPID 2, the work of Mingozzi et al. showed that not only is the quantity of

full AAV viral capsid particles (containing the single stranded DNA and used

for dose determination) important for in vivo activity, but also the total viral

particle dose, including the proportion of empty capsid particles contained in

the preparation.23 Though perhaps counterintuitive, the possibility that a

higher proportion of empty capsids improves gene transfer is supported by

results presented in Appendix p 4 and Web Figure 1. These findings differ

from results of earlier studies showing improved gene delivery with fewer

empty capsids,24 likely related to the fact that previous work did not address

the neutralization that might occur with vascular delivery of AAVs in vivo.

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Thus, empty capsids may serve as “decoy” proteins that block the inhibitory

activity of antibodies and possibly of other serum-based interfering

substances.23 The presence of even low titres of NAb (<1:2) or other

interfering substances in vivo can shift the dose response curve; with a lower

percentage of empty capsids in the preparation, higher doses are required in

order to achieve the same level of gene transfer. The difference in the

proportion of empty capsids in preparations used in CUPID 1 and CUPID 2,

with a lower total particle dose infused in this study, may have contributed to

a reduction in gene transfer efficiency in CUPID 2. Although the low level of

vector DNA in the limited number of CUPID 2 patients from whom tissue was

available is consistent with this possibility, these patients may not be

representative of the overall study population since their condition had

deteriorated to the point where they required advanced therapies. A

fundamental question is whether SERCA2a was an appropriate target for

therapy. Although deficiencies in SERCA2a activity in the failing heart and

their correction by gene transfer have been demonstrated in experimental

models,16-19 it is possible that these findings are not applicable in human HF

and that, regardless of the level to which SERCA2a activity is raised, the

impact would be insufficient to alter the trajectory of the disease. It is also

possible that post-transcriptional or post-translational regulatory factors in

patients may have negated enhanced transgene expression or enzyme

activity in treated patients and that the earlier findings in animal studies

showing significant improvement using this same vector do not translate to

humans with HF.

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During the course of the study no signals regarding safety emerged. While it

is reassuring that the intracoronary delivery of the drug can be safely

performed in patients with moderate to severe HF due to reduced ejection

fraction, concerns about the efficiency of AAV1/SERCA2a delivery raise the

point that conclusive data on the safety of AAV1/SERCA2a will require

demonstration of greater uptake and expression of the transgene in

cardiomyocytes.

Although we did note a significant interaction between treatment group and

geography, suggesting that the risk of terminal events might be greater in

the AAV1/SERCA2a group than in placebo in non-US patients, the number of

patients and terminal events in this sub-group was small and the non-US

AAV1/SERCA2a patients appear to have been somewhat sicker at baseline

than non-US placebo patients. Thus, this finding was likely due to chance,

sicker patients being randomized to AAV1/SERCA2a, or both. The lack of an

increased HR for recurrent events, which should have been influenced in the

same direction, suggests chance as the most likely explanation.

While the results of CUPID 2 show that antegrade coronary delivery of 1x1013

DRP of AAV1/SERCA2a does not alter the clinical course of HF patients with

reduced ejection fraction, they raise a number of questions that will need to

be addressed if future studies in this area are to be successful. For the

development of AAV1/SERCA2a, evidence that efficiency might have been

compromised by the lower number of empty capsids raises the possibility

that the latter was responsible for the negative results of CUPID 2 and it

provides the rational for further studies using drug with higher numbers of

total capsid proteins, which is best achieved by increasing the dose of

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AAV1/SERCA2a. In addition, the issues raised by the negative results of

CUPID 2 need to be considered in designing trials with other constructs

meant to enhance gene expression in the failing heart in the future,25 and

they suggest that it will be important to characterize serum effects from the

target patient population for their potential impact on the biological potency

of drugs used for gene transfer.

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ContributorsAll authors contributed to the interpretation of the results and writing of the manuscript and all authors approved the decision to submit the manuscript for publication. BG, JB, GMF, PP, AAV, ASD, DB, AB, BJ, and ARL were investigators in this study. JMP was the study statistician. JJR, KMZ, and JMP were involved in study design. BG wrote and prepared the first draft of the manuscript, with input from the other authors.

Declaration of interestsBG, JB, GMF, PP, AV, AD, DB, AB, BEJ, and ARL received financial support from Celladon Corporation, the sponsor of this trial, in the form of grants, personal fees, and other financial support. JMP, JJR, and KMZ were employees of Celladon during the CUPID 2 trial.

AcknowledgmentsThis trial was funded by Celladon Corporation. Celladon also provided funding for

manuscript and graphics support. We would like to thank all of the investigators

(Appendix p 3) and patients involved in this study. We wish to thank Roger Hajjar,

M.D. (Mt. Sinai, New York, NY), for his guidance on AAV1/SERCA2a development. ARL

wishes to acknowledge support from the National Institute for Health Research

Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, and the British

Heart Foundation. We wish to thank Sharon L. Cross, Ph.D. for providing manuscript

support and Julia Andres for providing graphics support on behalf of Celladon.

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Table 1: Characteristics at baseline in the modified intention-to-treat population.

CharacteristicPlacebo

(n = 122)

AAV1/

SERCA2a

(n = 121)

All patients

(N = 243)

Age (years) 58·4±12·26 60·3±9·77 59·3±11·11

Female sex 24 (19·7%) 21 (17·4%) 45 (18·5%)

Race

White 98 (81·1%) 99 (81·8%) 198 (81·5%)

Black/African American 22 (18·0%) 18 (14·9%) 40 (16·5%)

American Indian/Alaskan Native 0 (0·0%) 1 (0·8%) 1 (0·4%)

Native Hawaiian/Pacific Islander 0 (0·0%) 1 (0·8%) 1(0·4%)

Other 1 (0·8%) 2 (1·7%) 3 (1·2%)

Ethnicity

Not Hispanic 118 (96·7%) 114 (94·2%) 232 (95·5%)

Hispanic 4 (3·3%) 7 (5·8%) 11 (4·5%)

Country

United States 79 (64·8%) 79 (65·3%) 158 (65·0%)

Non-United States* 43 (35·2%) 42 (34·7%) 85 (35·0%)

Coronary artery disease 67 (54·9%) 68 (56·2%) 135 (55·6%)

Six-minute walk test (meters) 336·6 (71·29) 319·9 (91·47) 328·2 (82·23)

Left ventricular ejection fraction (%) 24·0 (6·26) 23·0 (6·48) 23·5 (6·37)

NYHA functional class

II 21 (17·2%) 22 (18·2%) 43 (17·7%)

III 100 (82·0%) 96 (79·3%) 196 (80·7%)

IV 1 (0·8%) 3 (2·5%) 4 (1·6%)

KCCQ (overall score) 59·2 (22·7) 58·4 (19·76) 58·8 (21·02)

NT-proBNP (pg/mL) 1504 1754 1679

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CharacteristicPlacebo

(n = 122)

AAV1/

SERCA2a

(n = 121)

All patients

(N = 243)

(849-3031)(843-3785)

(843-3561)

Heart failure aetiology

Ischemic 63 (51·6%) 62 (51·2%) 125 (51·4%)

Idiopathic 50 (41·0%) 48 (39·7%) 98 (40·3%)

Hypertensive 5 (4·1%) 5 (4·1%) 10 (4·1%)

Familial 1 (0·8%) 2 (1·7%) 3 (1·2%)

Peripartum 2 (1·6%) 0 (0·0%) 2 (0·8%)

Other 1 (0·8%) 7 (5·8%) 8 (3·3%)

Heart failure regimen

ACE inhibitor/ARB 110 (90·2%) 111 (91·7%) 221 (90·9%)

Aldosterone antagonist 74 (60·7%) 83 (68·6%) 157 (64·6%)

Beta blocker 117 (95·9%) 117 (96·7%) 234 (96·3%)

Diuretic 109 (89·3%) 111 (91·7%) 220 (90·5%)

Digoxin 48 (39·3%) 45 (37·2%) 93 (38·3%)

OAC/NOAC 81 (66·4%) 76 (62·8%) 157 (64·6%)

Cardiac resynchronization therapy 39 (32·0%) 53 (43·8%) 92 (37·9%)

Implantable cardioverter-

defibrillator89 (73·0%) 98 (81·0%) 187 (77·0%)

Other medical history

Chronic renal insufficiency 37 (30·3%) 36 (30·0%) 73 (30·2%)

Type 2 diabetes 49 (40·2%) 59 (48·8%) 108 (44·4%)

Atrial fibrillation 49 (40·2%) 44 (36·4%) 93 (38·3%)

COPD 18 (14·8%) 15 (12·5%) 33 (13·6%)

25

Data are mean (standard deviation) or n (%) except for NT-proBNP, which is median (IQR). There were no significant differences between the two groups in baseline demographic or disease characteristics.*Sweden (8/7/15), Great Britain (6/8/14), Denmark (5/6/11), Poland (6/5/11), Germany (5/5/10), Hungary (5/3/82), Israel (5/3/8), Belgium (3/4/7), and the Netherlands (0/1/1) for Placebo, AAV1/SERCA2, and All Patients, respectively . ACE inhibitor=angiotensin-converting enzyme inhibitor. ARB=angiotensin-receptor blocker. COPD=chronic obstructive pulmonary disease. IQR=interquartile range. KCCQ=Kansas City Cardiomyopathy Questionnaire. NT-proBNP=N-terminal pro-B-type natriuretic peptide. NYHA=New York Heart Association. OAC/NOAC=oral anti-coagulant/novel oral anti-coagulant.

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Table 2: Primary and secondary endpoints at the primary analysis data cutoff in the modified intention-to-treat population

OutcomePlacebo(N=122)

AAV1/SERCA2a(N=121)

Hazard ratio(CI)

p value

Primary endpointRecurrent events 128 (73·9) 104 (62·8) 0·93

(0·53-1·65)0·81

HF- related hospitalizations

121 (69·8) 96 (57·9)

Ambulatory treatment for worsening HF

7 (4·0) 8 (4·8)

Secondary endpointFirst terminal event 29 (16·7) 36 (21·7) 1·27

(0·72–2·24)0·41

Death 19 (11·0) 24 (14·5)Heart transplant 2 (1·2) 5 (3·0)Durable MCSD implant

8 (4·6) 7 (4·2)

Data are n (rate per 100 patient-years). CI=confidence interval. HF=heart failure. MCSD=mechanical circulatory support device.

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Table 3. Rates of adjudicated clinical events in the safety population*

OutcomePlacebo(N=122)

AAV1/SERCA2a(N=121)

All clinical events† 262 (147) 190 (111)All-cause hospitalizations 240 (135) 172 (100)

HF- related hospitalizations 121 (67·9) 99 (57·7)Ambulatory treatment for worsening HF 7 (4·0) 8 (4·8)Non-fatal myocardial infarctions 5 (2·8) 3 (1·7)Non-fatal strokes 3 (1·7) 5 (2·9)Heart transplant 4 (2·2) 7 (4·1)Durable MCSD implant 8 (4·5) 7 (4·1)Deaths 20 (11·2) 25 (14·6)

Non-cardiovascular 2 (1·1) 3 (1·7)Cardiovascular 18 (10·1) 22 (12·8)

Pump failure 11 (6·2) 14 (8·2)Sudden death 3 (1.7) 7 (4·1)Presumed sudden death 1 (0·6) 0 (0)Arrhythmia 2 (1·1) 0 (0)Fatal stroke 0 (0) 1 (0·6)Non-traumatic subdural hematoma

1 (0·6) 0 (0)

Data are n (rate per 100 patient-years)* The numbers of recurrent and terminal events differ slightly from the numbers in the efficacy analysis shown in Table 2 due to specific definitions used for primary and secondary endpoints. For example, in the primary efficacy analysis, only first terminal events were counted, and recurrent events that occurred after terminal events were not counted.†Excluding all heart failure (HF) hospitalizations and any other clinical event already counted as a hospitalization. MCSD=mechanical circulatory support device.

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Figure legendsFigure 1. Trial profileFx=failure. HF=heart failure. I/E=inclusion/exclusion. ITT=intention-to-treat. mITT=modified intention-to-treat. NAb=neutralizing antibodies. URI=upper respiratory infection.

Figure 2. Kaplan-Meier curves for cumulative number of recurrent events per patient at the primary analysis data cutoff (A), the probability of being terminal-event free at the primary analysis data cutoff (B), and the probability of death from any cause (C) in patients assigned to AAV1/SERCA2a (blue) or placebo (yellow) in the modified intention-to-treat population CI=confidence interval.

Figure 3. Subgroup analyses Hazard ratios (HR) for recurrent events (primary endpoint) and first terminal event (secondary endpoint) in the listed subgroups. The size of the square corresponds to the number of patients in each subgroup. Pre-specified analyses consisted of overall patient population, geography, heart failure (HF) aetiology, New York Heart Association (NYHA) class, and years of age. CI=confidence interval. ICD=implantable cardioverter-defibrillator. ITT=intent-to-treat. LVEF=left ventricular ejection fraction. mITT=modified ITT. NT-proBNP=N-terminal pro-B-type natriuretic peptide. US=United States

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