levy cystic fibrosis - national jewish health

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Hara Levy, M.D., M.M.Sc.Associate Professor of Pediatrics

Division Head, Department of PediatricsPulmonary Medicine

National Jewish Hospital for Kids

February 5th-8th Keystone ConferenceKeystone, CO

Cystic Fibrosis-The Future is Bright

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• No financial interests to disclose

Disclosure

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1. Review current clinical guidelines for the diagnosis and treatment of patients with CF.

2. Evaluate current and emerging therapies and pharmacodynamics and their impact on patients with CF.

Learning Objectives

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Cystic Fibrosis: A Historical Timeline

Cystic fibrosis (CF) of the pancreas was described by Andersen.

The sweat defect was discovered by diSant'Agnese and colleagues when they noticed that many of the infants presenting with heat prostration during the “great summer heat wave” in New York City had CF.

CF was identified as an autosomal recessive disease.

The fundamental physiologic defects were clearly established by Knowles and colleagues and Quinton as the failure of cAMP regulation of chloride transport.

The genetic defect for CF was located on chromosome 7.

The gene encoding the CF transmembrane conductance regulator (CFTR) was identified by positional cloning.

CFTR was established to be a cAMP‐regulated chloride channel by complementation studies.

1938

1953

1983

1965

1985

19901989

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1950

1989‐2019 Prop

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Cystic Fibrosis: Median Survival Age, 1940-2010

pancreatic enzymes/nutritional support

antistaphylococcal antibiotics

lung Transplant

antipseudomonal antibiotics

DNase

Inhaled tobramycin

airway clearance

0

5

10

15

20

25

30

35

40

1930

1940

1950

1960

1970

1980

1990

2000

2010

Year

Projected Ag

e of Survival (years)

1st pathologicdescription

CF geneidentified

Discoveryof high saltin sweat

Sweat chloridetest developed

1st successfulpregnancy

CF proteinidentified

HT saline

azithro

aztrm

CFTR‐targeting2018:

‐Median predicted survival ~48 y‐53% of patients are ≥ 18 y

Slide courtesy of CFF

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Clinical GeneticsHigh

> 60

31-59

< 30

Low

Sw

eat C

hlor

ide

(mm

ol/L

)

CFTR-RDCRMS

PIMutation Class I, II, III

PSMutation Class IV, V

Clinical spectrum increasing disease severity

Classical CF

Levy et al., Clinical Genetics, 2016Prop

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Prospects for Correcting Cystic Fibrosis: Level of Correction

Chillon M, et al. N Engl J Med. 1996; 332:1475

unaffected

100% (wt, 9T/9T)

50% (wt, 9T, and mutant CFTR)

10% (wt protein, 5T/5T)

5% (wt protein, 5T, and severe mutant)

4% (R117H, 7T, and severe mutant)

1% (R117H, 7T, and severe mutant)

<1% (G551D, �F508)

vas deferens

sweat ductairway

pancreas

Tissue affected CFTR activity

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Cystic Fibrosis: Median Survival Age, 1940-2010

0

5

10

1520

25

30

1940 1950 1960 1970 1980 1990 2000

Med

ian survival age (years)

Year

3537.8

Cystic Fibrosis Foundation Registry

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Cystic Fibrosis: Epidemiology

Population

Caucasian (US)

Caucasian (Great Britain)

Hispanic

African American

Native American

Asian (US, England)

Israel

Southern Europe

Epidemiologic

1 in 1,900‐3,700

1 in 2,400‐3,000

1 in 8,000‐9,000

1 in 15,300

1 in 40,000

1 in 10,000

1 in 5,000

1 in 2,000‐4,000

Newborn Screening

1 in 3,400‐3,800

1 in 2,200‐3,200

‐‐

‐‐

‐‐

‐‐

‐‐

‐‐

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Classes of Cystic Fibrosis-Causing Mutations

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Cystic Fibrosis: Clinical Phenotype Associated with CFTR Mutations

Milder lung disease

Pancreatic sufficiency

Abnormal sweat chloride

Mild lung disease

Pancreatic sufficiency

Equivocal sweat chloride

R117H (5T)

3849 + 10kB C‐to‐T

2789 + 5 G‐to‐A

R334W

G85E

G91R

R347P

R347H

R347L

R117H (7T)

3849 + 10kB C‐to‐T

G551S

D1152H

Severe lung disease

Pancreatic insufficiency

Abnormal sweat chloride

DF508

G542X

G551D

W1282X

N1303K

R553X

3120 + 1G‐to‐T

1078 del T

R75XProp

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Cystic Fibrosis

Organs affected by Cystic Fibrosis

SINUSES: sinusitis (infection)

LUNGS: thick, sticky mucus buildup, bacterial infection and widened airways

SKIN: sweat glands produce salty sweat

LIVER: blocked biliary ducts

PANCREAS: blocked pancreatic ducts

INTESTINES: cannot fully absorb nutrients

REPRODUCTIVE ORGANS: (male and female) complications

Normal airway (cross section)

Airway wall

Airway lined with a thin layer of mucus

Airway with CF (cross section)

Thick, sticky mucus blocks airway

Widened airway

Blood in mucusBacterial

infectionProp

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Pathophysiology of CF Lung Damage

Mutant CFTR Gene

Defective or Missing CFTR Protein/Impaired Ion TransportDehydrated Mucus

End Stage Lung Disease

Cycle of Destruction

Infection

Inflammation

Mucus Plugging

ScarringPropert

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WORK IN PROGRESS

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Wide Spectrum of Disease Severity in CF

Kerem et al NEJM 1990.

Lung function versus age in ∆F508 homozygotesFE

V1(%

pre

dict

ed)

Age (in years)

150

100

50

00 10 20 30 40

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Pathogenesis of Lung Disease in Cystic Fibrosis

Davis PB, et al. J Respir Crit Care Med. 1996;154:1229

Defective CF gene

Defective/deficient CFTR

Bronchial obstruction

Infection

Bronchiectasis

Inflammation

Abnormal airway surface milieu

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Treatment of Cystic Fibrosis Lung Disease

Defective CF gene



Infection

Bronchiectasis

Inflammation


Decrease mucus viscosityAugment clearance

Chest physiotherapyMucolytics (rhDNase)Hypertonic saline

Decrease bacterial load AntibioticsMacrolides

Reduce host responseCorticosteroids

Ibuprofen

Replace damaged lungs Transplantation

Current focus of modulator and

potentiator therapies

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Defective CF gene



Infection

Bronchiectasis

Inflammation





Reduce host response CorticosteroidsIbuprofen


Current focus of modulator and potentiator therapies

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Cystic Fibrosis: Bacterial Colonization

CFTR Antibacterial proteins

Impaired phagocytosisIncreased adherence

Impaired antimicrobial activity

asialoGM1

Impaired mucociliary clearance

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Effect of Pseudomonas aeruginosa Acquisition in Cystic Fibrosis

Demko CA, et al. J Clin Epidemiol. 1995;48:1041

0.9

0.8

0.7

0.6

Cumulative survival

1.0 Late acquisition (>6y)

Early acquisition (<6y)

malesfemales

6 8 10 12 14 16Age (y)

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Cystic Fibrosis: Organisms Isolated from the Lower Respiratory Tract

100

80

60

40

20

00‐1 2‐5 6‐10 11‐17 18‐24 25‐34 35‐44 >45

P. aeruginosa

S. aureus

H. influenzae

B. cepacia

Age (y)

Percen

tage positive

Data compiled from Cystic Fibrosis Foundation Patient RegistryProp

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Defective CF gene



Infection

Bronchiectasis

Inflammation







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Cystic Fibrosis: Airway Inflammation

respiratory epithelium

IL-8

pmn

Normal Cystic fibrosis

NE TNF-

mac

IL-1O2-

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Anti-Inflammatory Agents in Cystic Fibrosis: Corticosteroids

A four year, randomized double‐blind, placebo‐controlled trial that compared the efficacy of two doses (1 mg/kg/d and 2 mg/kg/d) of alternate‐day prednisone therapy with placebo in children with CF.

Eigen H, et al. J Pediatr. 1995;126:515.Lai HC, et al. N Engl J Med. 2000;342:851.

�FVC

(% predicted

for a

ge

‐6‐5‐4‐3‐2‐101234

6 12 18 24 30 36 42 48 mos

1 mg/kg 2 mg/kg

placebo

p = 0.0001

high‐dose arm stopped

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Anti-Inflammatory Agents in Cystic Fibrosis: Azithromycin

An five‐month, randomized, double‐blind, placebo‐controlled trial that examined the efficacy of azithromycin in patients with CF (age > 6 years, N = 251), chronically colonized with P. aeruginosa, and lung disease (FEV1 > 30% predicted for age).

Saiman L, et al. JAMA. 2003;290:1749.

�FEV

1 (%

predicted

for a

ge)

‐6‐5‐4‐3‐2‐101234

28 84 168 196 days

azithromycin

placebo

5 p = 0.009

Drug stopped

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Defective CF gene



Infection

Bronchiectasis

Inflammation



Mucolytics (rhDNase)Chest physiotherapy




Block Na+ uptakeIncrease Cl‐ efflux

AmilorideUTP/ATP

Hypertonic saline

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Cystic Fibrosis: Alternative Therapies to Effect Bioelectric Properties of the Respiratory Epithelium

ClCa

CFTR

ENaC

Cl‐

Cl‐Na+ Na+Cl‐

CF Altering other channels

AmilorideUTP/ATPHypertonic

saline

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Aerosolized Hypertonic Saline for the Treatment of Cystic Fibrosis

Elkins MR, et al. N Engl J Med. 2006;354:229.

0

25

50

75

100

0 12 24 36 48

Period of observation (w)

Survival free

of sym

ptom

‐de

fined

exacerbations (%

)

hypertonic saline

control

9.2 w 36 w

An 48‐week, randomized, double‐blind, parallel‐group trial that examined the efficacy of inhaled hypertonic saline in patients with CF over 6 years of age.

p = 0.001

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Defective CF gene



Infection

Bronchiectasis

Inflammation



Mucolytics (rhDNase)Chest physiotherapy





( Ivacaftor/Potentiator)

(Lumacaftor/Corrector) (Tazacaftor/Corrector)

(Elexcaftor/Corrector)

PTC124

Increase CFTR proteinActivate mutant form

AmilorideUTP/ATP

Hypertonic saline

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Golgi

ER

Endosome

Nucleus

Cell membrane

apical trafficking

G551D CFTR

Golgi

ER

Endosome

Nucleus

Cell membrane

Proteasome

degradation

F508 CFTR

Low temperature

Glycerol

Cystic Fibrosis: Correcting CFTR Dysfunction

Zeitlin P. N Engl J Med. 2004;351:606

VX809translation

transcription

post‐translational folding

VX770

VX 770=Ivacaftor=PotentiatorVX 809=Lumacaftor =Corrector

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25

[Sweat chloride] (m

mol/L)

‐10

‐8

‐6

‐4

‐2

050 100 200

Cystic Fibrosis: Potentiating delF508 CFTR Dysfunction

A two‐week, randomized double‐blind, crossover trial that compared the effect of regular treatment with Lumacaftor with placebo in CF patients with delF508 mutation.

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Gentamicin-Induced Correction of CFTR Function in Patients with Cystic Fibrosis and CFTR Stop Mutations

Wilschanski M. N Engl J Med. 2003; 349:1433.

0

‐2

‐4

‐6

‐8

0 0.3 0.6 0.9 1.2

Gentamicin concentration (%)

Respon

se of n

asal PD to

chloride‐

free

isop

roterin

ol (m

V)

p = 0.03pre‐treatment

post‐treatment

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Analysis* Statistic Treatment Difference for TRIKAFTA (N=55) vs Tezacaftor/Ivacaftor# (N=52)

Primary

Absolute change in ppFEV1 from baseline at Week 4 (percentage points) Treatment difference (95% CI) P value 10.0 (7.4, 12.6) P<0.0001

Key Secondary

Absolute change in Sweat Chloride from baseline at Week 4 (mmol/L) Treatment difference (95% CI) P value -45.1 (-50.1, -40.1) P<0.0001

Absolute change in CFQ-R respiratory domain score from baseline at Week 4 (points)

Treatment difference (95% CI) P value 17.4 (11.8, 23.0) P<0.0001

ppFEV1: percent predicted forced expiratory volume in 1 second; CI: confidence interval; CFQ-R: Cystic Fibrosis Questionnaire-Revised. * Baseline for primary and key secondary endpoints is defined as the end of the 4-week tezacaftor/ivacaftor run-in period.# Regimen of tezacaftor 100 mg qd/ivacaftor 150 mg q12hr. USOU TRUJAFTA_21_October 2019

Triple combination therapy (elexacaftor/tezacaftor/ivacaftor; ivacaftor)

For patients (12 yrs and older) with one or two F508del mutations

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MODULATOR THERAPIES

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CFF Goals in CFTR Modulator Development

• Enable multiple CFTR modulator options - Patient Choice- Mitigate risk-

• Expand access to rare mutations leveraging in vitro models

• Lessons from highly effective modulator therapy –PROMISE study (TDN)Simplify study (TDN) Real World Research (CFF and NIH)

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Optimizing Treatment in the Era of Modulators

• Two significant variables‒ Level of CFTR activity provided by modulator ‒ Status of underlying lung disease

• Personalized medicine goes beyond genotype

‒ If there is more than one modulator, which one provides best benefit? ‒ What other therapies will be needed if modulators are started at birth? ‒ What therapies can be stopped if modulators are started later in life?

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Defective CF gene



Infection

Bronchiectasis

Inflammation



Mucolytics (rhDNase)Chest physiotherapyHypertonic saline





AmilorideUTP/ATP

VX809VX770PTC124

Increase CFTR proteinActivate mutant form

Gene therapyProvide normal gene

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Pretreatment In vivo transfection In vitro transfectionPretreatment In vivo transfection In vitro transfection

Administration of an Adenovirus Containing the Human CFTR cDNA to the Respiratory Tract of Individuals with Cystic Fibrosis

Crystal RG, et al. Nat Genet. 1994;8:42.Prop

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Prospects for Gene Therapy of Cystic Fibrosis: Submucosal Gland

http://www.medicine.mcgill.ca/dynhist/histoimages

epithelium (+)epithelium (+)

submucosal glands (++)submucosal glands (++)

duct (++++)duct (++++)

Sites of CFTR expression in the human airwaySites of CFTR expression in the human airway

Engelhardt JF, et al. J Clin Invest. 1994;93:737.Prop

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Prospects for Gene Therapy in CF: Obstacles

• Respiratory epithelial cells versus submucosalglands

• Target receptors?

• Difficulty to bypass physical and functional barriers in the airway

• Immunologic consequences

• Relevant outcome measures

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What is Our Near-Term Goal?

• 100% of people with CF have access to CFTR-based therapies

– Small molecules– Gene therapy approaches– mRNA therapy– Gene editing/stem cell therapy

• 100% of people with CF have access to the symptomatic therapies that they need but when? Right now one size fits all…

– Antimicrobials– Anti-inflammatories – Mucolytics– Nutritionals

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Relying on traditional

inputs is like flying blind

• Signs & symptoms

• Clinical exam

• Imaging

• Pulmonary function testingProp

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Global Analyses Needed

Aging

EN

VIR

ON

ME

NT

Gene Discovery ModifiersTGFβ1, IFRD1, APIP, EHF, DCTN4

Gene Expression Signature(time 1)

Gene Expression Signature(time n)

CF SeverityPulmonary function

Pulmonary sufficiencyCF disease spectrum

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Generation of Genomic and EpigenomicSignature

Next-generation sequencingMicroarray analysis

RNA seqCHIP seq

PERSONALIZED MEDICINE

AnalysisTechnical and

statistical validationData base LIMS

support

EpidemiologyDescriptive and

demographic analysis of cohort

Translational Molecular Biology: Cystic Fibrosis

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Approximately one-third of CF subjects are not responding well to treatment after early diagnosis through newborn screening. This observation confirms our previous observations with another cohort (Lai et al Pediatr 2009; 123:714-722).

These nonresponders are also evident in national data and are suspected to have detrimental genetic modifiers that can be detected by WGS (Corvol, Levy et al Nat Commun. 2015; 6:8382).

Although data from the CF Foundation (CFF) indicate that most children (<18 years) with CF have normal lung function, 20% of CF deaths occur before 20 years of age. This apparent discrepancy may result in part from the wide spectrum of clinical heterogeneity in CF.

Early life events such as bacterial infection, airway inflammation, and structural changes have long-term consequences in patients with CF, as do diet, exercise, treatment regimen, and infections, adding further complexity to disease progression and therapeutic outcome.

This complexity underscores the need for more precise predictors of infection, inflammation, and the structural and functional impacts thereof in CF.

UNKNOWN UNKNOWNS

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Unique Molecular Signatures Correlate with Disease Severity

PI:HCn=1043

PS:HCn=870

n=224

n=819

n=51

Unionn=1094

A B

PS-HC PI- PI+

-2 +2Fold‐Change

HC CFPS+C

Levy, et al. Physiologic Genomics , Jan 2019

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4.0

6.0

8.0

9.5

10.5

6.5

7.4

8.4

10.5

14.6

15.2

16.0

16.6

16.9

18.8

1.5

2.5

3.5

5.9

6.4

7.2

9.0

10.0

11.0

Longitudinal Differential Gene Expression

Fold-Change- 4 +4

IL6MMP1TFPI2F3TNFIL1F9SOD2TNFAIP6SERPINA1PTX3CCL4CCL20CXCL2PTGS2IL1ACXCL3PLAURIL8IL1BTNFSF15ADAMDEC1RSAD2THBDCLEC5ACCL2CXCL5IL24IRAK3MGST1CCR1IL1RNDUSP6TLR2IL1R1TNFSF14THBS1LYZCD9TGFBIIL13RA1IFI30CXCL16TNFRSF21CXCL6CD14

Subject

Age

CF 1 CF 2 CF 3

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Microarrays have the potential

to predict CF course and therapeutic

response

• Airway clearance• Antibiotics• Hypertonic saline• Mucolytics• Potentiators and

correctors

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Future DirectionsComparative analyses with RNA-seq to analyze spliced isoforms and non coding RNAs

Application in other cohorts such as COPD looking at CFTR in chronic bronchitis

Comparison of signature in probands of differing CFTR mutation status, parents and to determine impact of CFTR on expression array; can parents and siblings inform relevant genes

Investigate epigenomic underpinnings of expression signature such as miRNA specific profile impacting expression and lncRNA’s

Single cell sequencing comparing CF PBMCs, CF sputum macrophages and epithelial cells over course of infection (Chan Zuckerberg Grant)

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Acknowledgments

Levy Laboratory, National Jewish Health• Xi Zhang, M.D., Ph.D.• Stephanie Jump, B.A.

Collaborators Northwestern Feinberg School of Medicine• Susanna McColley, M.D.• Scott Budinger, M.D.• Alexadra Mischarin, Ph.D.

Collaborators National Jewish Health and Univ Colorado

Russ Bowler, M.D., Ph.D.Camille Moore, Ph.D. Pam Zeitlin, M.D., Ph.D.Katie Hierst, M.D., Ph.D.Eszter Vladar, Ph.D.

Medical College of Wisconsin and University of Wisconsin, Madison Collaborators• Martin Hessner, Ph.D., McGee Center,

Children’s Hospital of Wisconsin

• Shuang Jia M.S., McGee Center, Children’s Hospital of Wisconsin

• Philip Farrell M.D., Ph.D. UW School of Medicine and Public Health

• HuiChuan Lai, Ph.D. UW School of Nutritional Sciences

• Pippa Simpson, Ph.D, Quantitative Health Services Center, Children’s Hospital of Wisconisn

• Amy Pan Quantitative Health Services Center, Children’s Hospital of Wisconsin

Funding Sources• NIH-NHLBI DP2 HL074202

• NIH-NHLBI R21 HL102523-01

• National Jewish Heath

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Methodology: GeneChip® Scanning and Microarray Analysis

After an overnight hybridization with the labeled mRNA the chips are scanned. A laser scans each DNA spot on the microarray.

A spot with a large amount of labeled mRNA will have a high intensity of light, and a spot with little or no labeled mRNA with have a low light intensity.

The intensity information is sent to a computer for data analysis.

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Methodology: GeneChip® Scanning and Microarray Analysis

Once the image intensity data has been analyzed, statistically significant changes can be identified.

Differential expression performed using ANOVA and principle components analysis as part of Parteck Genomics Suite 6.5.

From that information a heat map, a visual representation of the expression data, is generated by Genesis. Down-regulated genes are shown in green and up-regulated genes are shown in red.

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levy cystic fibrosis - national jewish health

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