Gene Therapy Studies on the “Go”

Solving New Barriers

Roger J. Hajjar MD

Icahn School of Medicine at Mount Sinai,

New York

Injury / Damage

Coronary

Disease

Myocardial

Infarction

Familial/Genetic

Hypertension

Pregnancy

Valvular

Alcohol

Toxins

Mature

Dysfunctional

Dying

New

Fibroblasts

Extra-cellular Matrix

Blood vessel

Progenitors

Pathway to Heart Failure

PP2A

CaMKII

Ca2+ Ca2+

T-tubuleCa2+

NCX Na+/K+

Ca2+ Na+ K+

Na+ K+

LTCC

LTCC

RyR

SERCA2a

Calstabin2

Ca2+

Ca2+

Ca2+

PLB

Ca2+

PKA

βAR

NE

Ca2+TnI

Contractile apparatus

PLB

PKA

PP1

I1

PKA

SR Ca2+ content

SR Ca2+ uptake

MyofibrillarResponsiveness

SR Ca2+ leak

GRK2

Advanced Heart Failure

SERCA2aDeficiency

Advanced Heart Failure Leads to a SERCA2a Deficiency

Irrespective of the Underlying Cause of Heart Failure

Alcoholism and Drug Abuse

Pregnancy

Diabetes

Toxins, Infectious Agents,

Congenital Mutations

Hypertension

CoronaryHeart Disease

(Atherosclerosis, Leading to MI)

FamilialGenetic

Choice of Vectors

Modes of Delivery

Immune Response

Clinical Trials

Targeting by Gene Therapy

AAV Vectors Are Optimally Suited for

Delivery to the Myocardium in Heart Failure

Nonpathogenic

Low immune response

Contains no viral genes and is non-integrating, non-mutagenic

Glybera (AAV1 Genetic Enzyme Replacement Therapy) - received marketing approval from the EC in 11/2012

Able to be administered via coronary infusion (small size 20 nm)

Results in long-term expression

Broad distribution to the myocardium

Safety

Delivery

7

Calcium Up-Regulation by Percutaneous Administration of

Gene Therapy In Cardiac Disease (CUPID Trial)

Phase 1: Open-Label, Sequential Dose Escalation

1.4x1011, 6x1011, 3x1012, 1x1013 drp N=12 (3:3:3:3)

Phase 2: Double Blinded, Randomized, Placebo Controlled Trial

6x1011, 3x1012, 1x1013 drp, & Placebo N=39 (8:8:9:14)

AAV1.SERCA2a Improved in a Number

of Biological Parameters vs. Placebo

Efficacy Domain AAV1.SERCA2a Placebo / Optimized

Symptomatic

Quality Of Life Questionnaire

Functional

6 Minute Walk Test

VO2max

Biomarker

NT-proBNP

Remodeling

Ejection Fraction

End-Systolic Volume

* Circulation. 2011 Jul 19;124(3):304-313. Double arrows indicate that change from baseline at 6

months (primary endpoint) reached prespecified criteria for a clinically meaningful change.

Stable orImproved

Declined

= Improved

= Stable

= Worse

Time-to-Recurrent HF-Related Hospitalizations Adjusted for Competing Risk of Terminal Event (CV Death, Transplant, LVAD)

10

Hazard Ratio

(CI)0.12 (0.03, 0.49) 0.18 (0.03, 0.99)

Risk Reduction 88% 82%

p-Value p = 0.003 p = 0.048

Cum

ula

tive

Rate

of N

on

-Te

rmin

al

Eve

nts

Placebo

High Dose AAV1.SERCA2a

Through 1 Year Through 3 Years

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Jessup M, et al. Circulation. 2011;124(3):304-313; Zsebo et al, Circ Res. 2014; 114: 101-108

AAV1.SERCA2a 1x1013 DRP, N=125

Placebo, N=125

Sample Size/Power: N=125 per treatment group with 186 recurrent events provides:

83% power, 0.05 two-sided significance level, to detect at least a 45% risk

reduction (HR=0.55)

All Subjects Followed Quarterly for Clinical Events

Until:Last enrolled subject completes 12 months of observation AND

186 adjudicated HF-related hospitalizations have occurred

CUPID 2: A Phase 2b/3

International Study

* 60 centers in US, Western and Eastern Europe ; 50% from US or US-like countries;

on track for completion of enrollment 1H 2014

PRIMARY ENDPOINT

Time-to-recurrent HF-related hospitalizations in presence of terminal events

(all-cause death, heart transplant, and LVAD implantation)

SECONDARY ENDPOINT

Time-to-first terminal event (all-cause death, heart transplant, LVAD implantation)

ADDITIONAL ENDPOINTS

Symptoms, Exercise Capacity and Quality of Life

Study Population

• 18-80 years of age

• Systolic HF

• Ischemic or non-ischemic

• EF ≤35%

• NYHA Class II to IV

• Maximal, optimized HF regimen & high risk for HF-related hospitalizations

• AAV NAb titer negative

Enrollment Conduct (12 Months on Study) Analysis3 Months

PRIMARY ENDPOINTCumulative Number of Recurrent Events per Patient

Secondary Endpoint: Probability of Being Terminal Event Free

Subgroup Analyses

Potential Reasons for the Neutral

Results of CUPID 2

• The target: SERCA2a

• Different lots & formulation

• Myocardial uptake insufficient

– AAV concentration too low

– Large animal studies with AAV serotypes not

predictive of human uptake

• T cell response

• Methods of gene transfer inadequate

SERCA2a

In all preclinical studies, there has been a linear relationship between dose, % transduction of cardiomyocytes, and efficacy. Preclinical data from laboratories around the world support SERCA2a as an important target for the treatment of Heart Failure

Cardiac:

• Increase in Contractility

• Decrease in Ventricular Arrhythmias

• Improvement in Energetic Profile

• Decrease in Cardiac Hypertrophy

Vascular:

• Increase in Blood Flow - eNOSactivation

• Decrease in Smooth Muscle Cell Proliferation

• Improvement in Pulmonary Hypertension

ADP + Pi

Ca2+

ATP

ADP + Pi

Ca2+

SERCA2a

Study Subject Time Post-Infusion

Heart Tissue AAV1/SERCA2a (ss copies/µg DNA)

CUPID-1 091006 Month 18 Posterolateral wall <20

CUPID-1 091007 Month 11 Left ventricular apical core (LVAC) >20 to <200

Month 23 Anterior Septum Posterior Septum

LVAC Right ventricular apical core (RVAC)

Anterior Wall Posterolateral Wall

561 365

230 250

>20 to <200 >20 to <200

CUPID-1 011005 Month 31 LVAC >20 to <200

CUPID-1 011010 Month 22 Posterolateral Wall

Anterior Septum Posterior Septum

Anterior Wall

223

>20 to <200 >20 to <200

>20 to <200

CUPID-2 021020 Month 12 LVAC 14-62

CUPID-2 161039 Month 13 Anterior septum Posterior septum

Anterior Wall Posterolateral Wall

80 71

36 77

CUPID-2 501024 Month 1.5 Anterior Septum

Posterior Septum Anterior Wall

Posterolateral Wall LVAC

RVAC

26

37 40

134 <10

<10

CUPID-2 231028 Month 8 Anterior Wall Heart [1]

115 72-123

CUPID-2 251008 Month 14 Anterior Septum

Posterior Septum Anterior Wall

Postolateral Wall

27

84 62

102

CUPID-2 501002 Month 10 Anterior Septum Posterior Septum

Anterior Wall Posterolateral Wall

LVAC

43 33

53 37

36

CUPID-2 021009 Month 20 Heart [2] 192

CUPID-2 111033 Month 16 LVAC <10

Viral Uptake in Animal Models of Heart Failure where

SERCA2a Gene Transfer was found to be Beneficial

Animals Virus Delivery ss copies of viral DNA/mg DNA

% Infected Cardiomyocytes

Mice AAV9.SERCA2a Intravenous 42,000 ~75%

Rats AAV9.SERCA2a Intravenous 30,000 ~70%

Pigs AAV1.SERCA2a Intracoronary 8,000 ~30%

Sheep AAV6.SERCA2a

AAV1.SERCA2a

Intracoronary

Surgical, MCARD

9,000

13,000

~33%

~42%

Humans AAV1.SERCA2a CUPID 1

Intracoronary

CUPID 2

Intracoronary

561

192

3%

<1%

T Cell ReponseSafety data from CUPID 1 and CUPID 2

has not indicated any evidence of a T

cell response against transduced cells

(specific for AAV1 capsid proteins

analyzed by ELISPOT), nor any other

evidence of toxicity. Review of all safety

data has provided a clean bill of health.

Modes of Gene Delivery

Potential Modes of Delivery

Surgical Technique:

• Antegrade Infusion during

Bypass

• Retrograde Infusion

during Bypass (MCARDTM)

Catheter Based Techniques:

• Extracoporeal Re-

Circulating System

(Osprey Medical)

• Retrograde Perfusion

• Antegrade Epicardial

Coronary Artery Infusion

(AECAI)

Next Steps for SERCA2a

Program

• Increasing AAV1.SERCA2a

concentration

• Other disease states

• Novel Re-engineered AAV vectors

• Different Targets

• Small molecule program

Ovine model of Ischemia Induced HF

ITR ITR

TnT+Enhancer

Cardiac-specific

Promoter

Intron PolyA

AAVRe-eng/SERCA2a-CCN5

huSERCA2a-2A-CCN5

HIGH DOSE AAV1.SERCA2a Phase 2 Randomized Double-Blind, Placebo-Controlled

10 study centers

Startup 3mP1 Enrollment 6m Conduct 12m Follow-Up 12m

Years 1 2 3

Close-OutP2 Enrollment 6mStartup 3m

Individual Subject Timeline

MCSD = Mechanical Circulatory Support Device

AAV1.SERCA2a 1x1014 DRP, N=20Patient Population

• 18-80 years of age, inclusive

• Chronic systolic HF

• Ischemic /non-ischemic CM

• EF ≤35%

• NYHA Class III or IV

• Optimized drug/device therapy

• AAV NAb titer negative

• ICD

• Recent HF hosp or NT-proBNP

• All subjects followed quarterly by

telephonic interview for a total of 24

months of observation and follow-up

• Clinical events collected and

adjudicated through Month 24 for each

subject

• Subjects undergoing MCSD or

transplant on-study will continue to be

followed

Long-Term Follow-Up

Placebo, N=10Statistical Methodology

Analyze treatment effect using composite outcomes in 5 domains:• Symptomatic: NYHA class, KCCQ• Functional: 6MWT• Biomarker: NT-proBNP• LV function/remodeling: LVESV• Clinical outcome: recurrent & terminal events

Safety Endpoints:

• Proportion of subjects who complete the study

• AE incidence, severity & relationship to IMP or procedure (M6 &12)

• Conmed use and changes in HF-related medications (M6 &12)

• Incidence and event rates of clinical events

• Changes from baseline in laboratory tests, ECG, VS & PE (M6 &12)

• Changes from baseline in ICD interrogation parameters (M6 &12)

Exploratory Efficacy Endpoints:

• Rate of recurrent events (HF hosp, ambulatory WHF)

• Rate of terminal events (MCSD, transplant, death)

Exploratory Activity Endpoints: (M6 & 12)

• Change from baseline in: LVESV, 6MWT, NT-proBNP, NYHA classification &

KCCQ

• Presence of AAV1/SERCA2a transgene and SERCA2a expression in available

tissue samples

Data Analysis Cuts

Screening 12-Month Active Observation Phase Long-Term Follow-Up

0 M1 M3 M6 M9 M12//

-60d

Prescreen AAV1 NAb

& NT-proBNP

W W1 2 M21M15 M24M18-30d

Other Trials with AAV1.SERCA2a

• Patients with pulmonary arterial hypertension

Distribution of Neutralizing Antibodies

Against AAV1 in Europe & the US

Designer AAV Vectors

• Highly Tropic for Cardiac Muscle

• Highly Efficient in Primate & Human Cardiac Myocytes

• Has very little/no tropism to liver, lungs, spleen, kidneys

• Resistance to Antecedent Neutralizing Antibodies

The Heart Specific BNP is a chimeric of several rAAV capids. It represents a heart

targeted vector which is more “resistant” to neutralizing antibodies.

HEART SPECIFIC AAV Mutant

Courtesy of R. Jude Samulski, UNC

Differential NAb Sensitivity of AAV2i8, AAV2 and AAV8

Serum AAV2i8 AAV2 AAV8

1 <1/2 1/64 1/16

2 <1/2 1/16 1/2

3 1/2 1/64 1/16

4 1/2 1/4 1/2

5 1/2 1/2 1/8

6 <1/2 1/4 1/8

7 <1/2 <1/2 1/8

8 1/2 1/4 1/128

9 1/2 1/4 <1/2

10 <1/2 1/4 1/8

11 <1/2 1/4 1/4

“Average” ≤1/2 ~1/15 ~1/18

AAV2i8.I-1c Program in Heart Failure

• Planned Start of

Enrollment for Phase 1 in

2016

• Phase 1: 12 patients

– 3x1012 vg (n=3)

– 1x1013 vg (n=3)

– 3x1013 vg (n=3)

– 1x1014 vg (n=3)

• Phase 2: 45 patients

– 3x1013 vg (n=15)

– 1x1014 vg (n=15)

– Placebo (n=15)

• 8 US medical centers

Post Translational Modification of SERCA2a in

Heart Failure

We identified a small molecule

that activates SERCA2a

through SUMOylation and

induces beneficial effects in

vitro and in vivo. This First-in-

Class small molecule activator

may provide a novel therapeutic

platform for the treatment of

heart failure.

Cardiomyopathy Associated with Phospholamban R14del Mutation

• Familial dilated cardiomyopathy first

identified in a Greek family

• R14del mutation associated with

dilated and arrhythmogenic right

ventricular cardiomyopathies

• Arrhythmogenic cardiomyopathy and

DCM in the Netherlands –15%

prevalence).

[Ca2+]i transients and Stress Markers in

Corrected IPS-CMs

Karakikes et al. Nature Comm. 2015

• AAV has proven to be a safe vector in CV Diseases

• Effective uptake of vector is necessary

• Antecedent neutralizing antibodies: Major obstacle to effective gene transfer

• Improved gene delivery systems need to be developed

• Modified AAV vectors with engineered capsids are new generation vectors

• Familial cardiomyopathies associated with specific mutations can now be targeted.

Future of Gene Therapy in

Cardiovascular Diseases

Thank you for your attention