The Children’s Hospital of Philadelphia Integrative Genomics Discoveries and Development at The Center for Applied Genomics at CHOP

Novel Genome-based Therapeutic Approaches

Hakon Hakonarson, MD, PhD, Professor of Pediatrics CHOP’s Endowed Chair in Genetic Research Director, Center for Applied Genomics The Children’s Hospital of Philadelphia University of Pennsylvania, School of Medicine

Duke Center for Applied Genomics and Precision Medicine 2019 Genomic and Precision Medicine Forum

Nov 07, 2019 Genomics in the 21st Century Disclosures

Dr. Hakonarson and CHOP own stock in Aevi Genomic Medicine Inc. developing anti-LIGHT therapy for IBD. Dr. Hakonarson is an inventor of technology involving therapeutic development of ADHD, GLA and HCCAA Novel Therapeutic Stem Cell/ Editing Approaches

§ iPS and stem cell therapy shows early promise

§ Gene therapy for LCA (RPE65) at CHOP via AAV

§ Targeted T cell therapy for cancer (UPENN/CHOP)

§ CRISPR-cas9 gene editing

§ Single cell sequencing The Center for Applied Genomics (CAG) at CHOP

u Founded in June 2006

u Staff of 70

u Over 100 active disease projects with CHOP/Penn collaborators

u TARGET: Genotype 100,000 children

u ~450k GWAS samples >130k kids

u IC - participation in future studies >85%

u Database

u Electronic Health Records

u extensive information on each child

u >1.2 million visits per year to Population Genomics Research CHOP Recruitment of CHOP/PENN HealthCare Network Patients

u High-level of automation ADHD, Autism, Diabetes, IBD, Autoimmunity, Asthma/Atopy, Cancer, RDs - all high priority Children's Hospital of Philadelphia

Center for Center of Mitochondrial Applied and Center for Genomics Epigenomic Developmenta Medicine Center for l Biology and Biomedical Pediatric Informatics Disorders

Center for Center for Pediatric Cellular and Clinical Molecular Effectiveness Therapeutics

Center for Childhood PolicyLab Cancer Center for Research Center for Injury Autism Research and Research Prevention CAG Repository (Major Disease Areas)

Category # of § Major pediatric and samples adult diseases are represented Total CAG/CHOP (internal) 132,752 • Healthy Controls (0-21) 18,423 § EHR have unlimited potential regarding • Psychiatric 19,351 9 Longitudinal f/u • Autism/Developmental Delay 13,972 9 Medication use • Autoimmune/Inflammatory 31,643 9 Development • Cancer 9,585 9 AEs/SAEs/DDIs • Metabolic 13,760 § We have established • Malformations 8,954 over one hundred • Rare/Mendelian Diseases 22,436 collaborations world- wide for discovery Samples through collaboration 334,534 and replication Total number of samples @ CAG 467,286 purposes CAG Biobank – biomarker driven research

§ 450,000 unique patients in CAG biobank § Over 100,000 children from CHOP (>85% re-contact permission) § Blood samples from >95% § High quality DNA from with over 2/3 of samples GWAS genotyped § PBMCs - opportunity for EBV, iPS and tissue/organ differentiation § RNAseq, single cell sequencing § Cell based assays § Epigenetics § Plasma – for target driven biomarker measures § Thousands of different phenotypes in CAG § Clinical disease traits in the multi-hundred’s § Other: blood, chemistry, radiology, cardiology, sleep, u 2 Million Samples in tubes PFTs, meds etc. § Consortium networking u 10 Million Plated Samples collaborations § Multiple partnerships with biotech and pharma CAG CLIA certificate CAG Discovery and Development Pipeline

Objective: To identify biomarkers indicative of the genetic Family- underpinnings of a disease for based novel innovative therapies Analysis

u Three databases: 1. Family-based Samples 2. Genomic Database New drug 3. Health Records & Disease Phenotypes disovery and Genomic Database development u We integate these databases of biomarkers together in search for new target and diagnositc biomarkers and diagnostic products products

u High level of IT and Technology Structure Health Records u All cloud based and Disease u Highly integrative Phenotypes u Cost effective discovery platform Precision Medicine in the Context of “Big Data“

Creating and turning data assets into insights.

• CAG database has >450k patients GWAS genotyped and imputed to >30M variants • 15,000 whole genomes are sequenced and growing • 20,000 whole exomes are sequenced and growing • Thousands of phenotypes to mine for new targets Cells, RNA, Plasma available from these patients for biomarker development Representative CAG Milestones

Research is enabled by our unique, scalable biobank with sample collections that are highly enriched for rare disease causing variants

Reference, https://scholar.google.com/citations?user=nLerrWsAAAAJ&hl=en Personalized/Precision Medicine Paradigm

u There are 6 billion bases (3 million pairs) in the genome u SNPs occur every 100- 300 base pairs u The greatest number of DNA variations that are associated with diseases or traits are

u Missense mutations

u Nonsense mutations

u Deletions (structural variants) u Today we can run an exome for <$350 and a genome for <$1000 and identify all know PGx and disease causing variants Genetic Influence in Pediatric Diseases Impact of pediatric age on genetic risk

u Early Onset Disease:

u Distinct, genetically driven

u Severe burden of illness

u Aggressive progression

u Less responsive to standard of care

Pediatric studies generally produce much larger genomic signals than studies in adults. Neuroblastoma

u Embryonal cancer

u Misappropriation of normal sympathetic neurodevelopment pathways u Common pediatric cancer

u Median age diagnosis 17 months u 15% of childhood cancer mortality

u Approximately 2/3 of patients cured Familial Neuroblastoma 2p23-24

TELOMERE--NAG - DDX1 - MYCN - FAM49A - VSNL1 - KCNS3 - RDH14 - NT5C1B - OSR1 - TTC32 - WDR35 - MATN3 - LAPTM4A - SDC1 - PUM2 - RHOB - HS1BP3 - GDF7 - C2orf43 - APOB - ATAD2B - UBXD4 - LOC388931 - C2orf44 - FKBP1B - SF3B14 - TP53I3 - PFN4 - FLJ30851 - ITSN2 - NCOA1 - LOC391356 - CENPO - ADCY3 - RBJ - POMC - DNMT3A - DTNB - ASXL2 - KIF3C - RAB10 - HADHA - HADHB - GPR113 - SELI - C2orf39 - OTOF - LOC339778 - CIB4 - KCNK3 - C2orf18 - CENPA - DPYSL5 - MAPRE3 - FLJ20254 - AGBL5 - EMILIN1 - KHK - CGREF1 - ABHD1 - PREB - C2orf53 - TCF23 - SLC5A6 - C2orf28 - CAD - SLC30A3 - DNAJC5G - TRIM54 - UCN - MPV17 - GTF3C2 - EIF2B4 - SNX17 - ZNF513 - PPM1G - NRBP1 - KRTCAP3 - IFT172 - FNDC4 - GCKR - C2orf16 - ZNF512 - CCDC121 - XAB1 - SUPT7L - SLC4A1AP - MRPL33 - RBKS - BRE - FOSL2 - PLB1 - PPP1CB - SPDYA - FLJ20628 - WDR43 - LOC165186 - FLJ 34931 - CLIP4 - ALK - YPEL5 - LBH - LYCAT - CAPN13 - GALNT14--CENTROMERE Identification of Heritable Mutations in the ALK Tyrosine Kinase Domain

Unaffected grandfather

* Affected grandmother

Unaffected father 1

* Obligate carrier mother

Unaffected father 2

* Affected child

Affected child *

Affected child * Mosse, Nature 2008 ALK is the Major Familial Neuroblastoma Gene Familial NB

u A germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas u Resequencing in high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. u Nine of the ten mutations map to critical regions of the kinase domain (oncogenic drivers). u Several companies have ALK inhibitors in development (preclin to Phase I) u CHOP is pursuing clinical development and with first patient to be enrolled in April Neuroblastoma: Crizotinib Personalization and Repositioning Case Study

CHOP Target New Genomically Repositioned Identified Patient Enriched Mutations Compound Population Clinical Trials

• ALK • Lung cancer • Lymphoma • Phase I n=18

• Neuroblastoma • Phase I n=23

• mGluR • Dementia • ADHD • Phase I n=18 • Phase IIa n=60 u The tumors have responded in a robust way in those who are ALK mutation positive• Schizophrenia and receive• Future Xalkori trial • Autism • Future trial u Both germ-line and somatic mutation cases are responsive to Xalkori Discovery of Mutations (copy number variations/CNVs) in ADHD

u CNVs affecting glutamatergic neurotransmission observed to be over-represented in multiple ADHD cohorts (~10 fold)

u 3,500 ADHD cases and 12,000 controls

u Replication accomplished across multiple ADHD cohorts

GRM: Glutamate receptors metabotropic CNVs: Copy number variants Elia, Glessner et al. Nature Genetics, 2012 The Drug NFC1 Activates the mGluR Pathway

NFC-1 (fasoracetam):

üAn mGluR agonist

üWell tolerated in previous preclinical and clinical studies

üShown to ameliorate cognitive impairment and slow down locomotor activity in animal models

üStructure-similar compounds have good safety profiles Genotyping or resequencing methods identify the ADHD children at risk Prior Studies conducted on NFC-1

Preclinical (N=21)

Clinical (N=7) Clinical Trial Design ADHD (GREAT STUDY)

New IND filed to the FDA and approval obtained to treat 30 mGluR biomarker positive children for 5 weeks age 12-17 year old Week 1 Week 2 Week 3 Week 4 Week 5 Week 9 Day 7 (±2) Day 14 (±2) Day 21(±2) Day 28 (±2) Day 35 (±2) Day 75 (±2) Adverse event monitoring X X X X X phone Laboratory Safety Tests (blood and urine)A X X X X X Physical Examination X X X X X Vital Signs: BP, HR, RR X X X X X Body Weight (all points) & Height (week 1 X X X X X only) 12-lead ECG X X X X X Urine b-hCG test (menstruating females only) X X X X X Contraception verification (selected females) X X X X X Vanderbilt Parent Rating Scale X X X X X BREIF (Parent; Self) X X X X X QuotientâADHD test X X X X X PERMP-Math test X X X X X Actigraphy (continuous monitoring) X X X X X CGI-I & CGI-S X X X X X Dispense study drugB X X X X NFC-1 or placebo administration at homeC Placebo bid 50 mg bid 100 mg bid 200 mg bid 400 mg bid Retrieve pill bottle/pill count X X X X X A: Blood draws for hematology (RBC, WBC with differential, platelet count) and clinical chemistry (electrolytes, albumin, ALT, AST, alkaline phosphatase, bilirubin, BUN, creatinine, glucose, B: Study drug for Week 1 administered at end of PK study; study drug for next week dispensed at each clinic visit C: Dose escalations to be determined by CGI-S and CGI-I scores at end of each week of treatment; maximum doses indicated

Thirty mGluR pos ADHD children completed a 24 hr PK study and 5 wks on drug Placebo was given for 1 week – parents were blinded to placebo therapy ADHD Study: Repeated Measures Analysis increases confidence in demonstration of Treatment Effect

Average CGI - I score at the end of each week – all patients Week 1 Week 2 Week 3 Week 4 Week 5 P < 0.001 Mean 3.79 3.13 2.79 2.79 2.21

Average Vanderbilt score at each week – all patients

Week 1 Week 2 Week 3 Week 4 Week 5 P < 0.001 Mean 29.1 26.4 24.0 23.3 22.5 NFC-1 ADHD Study Results

u Seven kids had co-morbid autism demonstrating marked improvement in social behavior and language skills

u Two kids had 22q (one deletion/one duplication syndrome): Among the most robust responders

u Anxiety and mood symptoms improved across the board

u PERMP improved in Tier-1/Tier-2 mutation group

u Actigraphy measures of hyperactivity improved across all tiers (most robust in rapid repetitive movements, P<0.01) (Elia et al Nature Communication, 2018) mGluR enrichment in idiopathic ASD

u Enrichment of CNVs affecting glutamatergic neurotransmission genes also observed to be over-represented in multiple Autism

u Enrichment also observed in schizophrenia, anxiety, anorexia and mood disorders Pharmacogenomic Model in ADHD/ASDs

Neurological Genetic Stratify Intervention at New Drug Disease using Biochemical Disorder Screening Biomarkers Pathways Candidates ADHD SNPs/CNVs Gene network mGluR pathway mGluR agonist

ASD 22Q SCZ Fast-Track Clinical Trials Re-sequencing panel designed for diagnostic Market Test- and-Treat Product Precision Medicine First Pediatric IBD GWAS

u We identified and replicated significantly associated, previously unreported loci on 20q13 and 21q22 located close to the TNFRSF6B (DcR3) and PSMG1 genes, respectively

u Gene discovery studies in childhood-onset disease have unveiled genetic factors that are less likely to surface in adult studies

u We have identified multiple other novel IBD loci and replicated the vast majority of the pediatric-onset disease loci in the adult onset form of IBD Expression of DcR3 is altered in IBD

• The level of TNFRSF6B/DcR3 mRNA is elevated in intestinal biopsies from IBD

• Serum levels of DcR3 by ELISA are correlated with the haplotype DcR3 plays diverse roles in the immune system

Zhan C, Patskovsky Y, Yan Q, Li Z, Ramagopal U, Cheng H, et al. Decoy strategies: the structure of TL1A:DcR3 complex. Structure. 2011 Feb 9;19(2):162-71. Meta-analysis tags the DcR3 pathway

DcR3 TL-1A

Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, Ahmad T, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nat Genet. 2010 Nov 21.

Fas ligand Sequencing of TNFRSF6B in pediatric IBD cases and controls

Case/Ctrl status Homozygous Missense WT carriers IBD 521 11 Controls 555 3 Odds ratio = 3.9, p < 0.025 by Χ2

Case/Ctrl status Homozygous Missense WT carriers Crohn’s 521 11 Exome seq 4200 24 project Odds ratio = 3.7, p < 1.37×10–4 Secretion of DcR3 from IBD cases (293T cells) Comparison of non-secretors

Normal secretor Non-secretor Crohn’s cases 388 5 Controls 554 1

Odds ratio = 7.1, p < 0.037 by Χ2

Cardinale et al, Genes and Immunity, 2013 Decoy Receptor 3 (DCR3) is a Natural “Off-Switch” for Immune and Inflammatory Response

Normal Patient Normal Patient LIGHT Activates Immune Response DCR3 Turns Off Immune Response

DCR3 LIGHT

Normal Patient

NoInflammation Inflammation No Inflammation CertainOur Approach:IBD Patients Replace Have Mutations Deficiency that withResult anti in -LOFLight of Ab DcR3 Therapeutic Antibody that “Mimics” DCR3

DCR3- IBD Patient DCR3- IBD Patient No “Off” Switch Replacement Tx with anti-LIGHT mAb

Anti-LIGHT LIGHT Antibody

Chronic Inflammation No Inflammation Anti-LIGHT mAb Program u First-in-Class Biologic from Japan

Immune u Initial development in Cell Severe Pediatric Onset IBD Decoy Receptor 3 DcR3 LIGHT u Phase 2 ready human monoclonal antibody

u Program Update HVEM LTbR u Successfully transferred and approved IND

u Requalified clinical trial material

u Cleared by FDA to start trial Therapeutic antibody which mimics DcR3 regulation by binding LIGHT u Study began at CHOP and 3 other sites

u Initial data expected Q4 Heritability and Shared Genetic Architecture in Pediatric Autoimmune Diseases (pAIDs) Ten pAID case cohorts and top pAID association loci

u A: Details of the 10 pAID cohorts and common population-based controls

u B: Top pAID association signals identified by performing a GWAS merging cases from the 10 pAIDs (Left) or by performing an inverse chi-square meta-analysis (Right)

u C: Novel and established pAID association loci (n=47) u IL2RA, IL12B, CD40LG, SMAD3

Li et al, Nat Med, 2015 46 Loci Are Highly Associated (p < 1x10-6) with 27 of These Reaching Genome-Wide Significance (p < 5x10-8) Pharmacogenomic Model in IBD/pAID

Inflammatory Genetic Stratify Intervention at New Drug Disease using Biochemical Disorder Screening Biomarkers Pathways Candidates IBD SNPs Gene network DcR3 pathway aLIGHT-Ab pAIDs

Fast-Track Clinical Trials

Market Test- and-Treat Product Precision Medicine CAG RARE DISEASE PROGRAM

Mendelian Disease Project at CAG % • Number of WE sequenced samples: Disease Area Breakdown 2019 2012 Change 6,740 (>1500 families) Musculosketal 4,920 2,459 +50% • Hundreds of additional families in Nervous System 4,855 2,652 +45% Cardiovascular 3,289 2,225 +32% pipeline Endocrine/Immune 2,846 1,337 +53% Blood Diseases 1,649 816 +51% Circulatory 1,418 795 +44% Genitourinary 1,027 645 +37% Eye/Face/Neck 909 571 +37% Chromosomal Abnormality 662 347 +48% Other 316 125 +60% Mitochondrial 165 76 +54% Skin 121 76 +37% Digestive 112 64 +43% Perinatal 92 15 +84% Respiratory 55 42 +24% Total 22,436 12,245 +45% Generalized Lymphatic Anomalies (GLAs)

u Orphan disease(s) with pleiomorphic presentation: some malignant/fatal

u Defined defects in mTOR/PI3KA pathway

u Somatic mutations been identified in a subset of patients

u Sirolimus been used anecdotally as therapy (subset of patients) Generalized Lymphatic Anomaly (GLA) Gorham-Stout Disease (GSD) u Central Collecting Lymphatic Anomalies (CCLA) Molecular diagnosis missing in Klippel-Trenaunay Syndrome (KTS/CLVM) most cases Kaposiform Lymphangiomatosis (KLA) Kaposiform Hemangioendothelioma (KHE) Family 1 enrolled

I u Lead proband with history of complex 1 2 lymphovascular disease since birth, II manifesting primarily as a right-sided 1 2 3 4 5 6 7 8 chylous effusion III u At birth - near complete opacifications of 1 2 3 4 5 6 7 8 9 his lungs IV u 4 months- pulmonary 6 lymphangiomatosis u 11 years - chylous effusion was drained and the fluid re-accumulated within 12 hours u Family history – venous stasis was a consistent feature and variability in abdominal distension u A stillbirth baby was affected by hydrops

Li, D. et al. Hum Mol Genet 438, 946 (2018) Proband 1

A A'

A'' A'''

Central Conducting Lymphatic Anomaly (CCLA)

Li, D. et al. Hum Mol Genet 438, 946 (2018) WES revealed splice-altering mutation in EPHB4

I 1 2

II 1 2 3 4 5 6 7 8 * **

III 1 2 3 4 5 6 7 8 9 ** ** * ** * * ** *

IV 1 2 3 4 5 6 ** ** * ** **

Tested positive EPHB4: * Tested negative EPHB4: **

c.2334+1G>C

Li, D. et al. Hum Mol Genet 438, 946 (2018) EphB4 signaling in ECs controls various cellular responses

Füller et al. Journal of Cell Science (2003) vol. 116 (Pt 12) pp. 2461-70 Knockdown study - lymphatic vessel misbranching phenotype

A B

Control 54 hpf ephb4a MO 54 hpf C D

Control 4 dpf ephb4a MO 4 dpf

Li, D. et al. Hum Mol Genet 438, 946 (2018) Lymphatic misbranching can be rescued by mTORC1 inhibitors

p-mTOR (S2448) u Perturbation of EPHB4 in p-p70S6K (T389) developing zebrafish p-ERKs (T202/Y204) activates mTORC1 b-actin signaling

Morpholino: EPHB4 EPHB4 EPHB4 Control Control Control u Activation of mTORC1 can be inhibited by Treatment: Control

BEZ235 treatment Rapamycin

Li, D. et al. Hum Mol Genet 438, 946 (2018) Summary I u Splice-altering mutation in EPHB4 leads to nonframeshift 4 amino acids insertion in the highly conserved catalytic loop of kinase domain u The insertion decreases the state of EPHB4 protein u Modeling the splicing-altering mutations in zebrafish results in vessel misbranching and deformities in the lymphatic vessel development u Drugs that inhibit mTOR or PIK3A kinase signaling effectively rescued the misbranching phenotype in a comparable manner Patient 2: Central Conducting Lymphatic anomaly

a) b) c)

a) T2 weighted MRI demonstrating large pericardial effusion (arrow). b) Dilated and tortuous thoracic duct (TD; arrow) retrograde perfusion of the periportal lymphatic networks (arrowhead) and abnormal mediastinal and pulmonary perfusion (boxed region). C) Contrast lymphangiogram of the boxed region in (b) demonstrates dilated and tortuous terminal TD with multiple dilated ducts perfusing both lung fields (arrows). Recurrent ARAF mutation

Patient P1 Patient P2

T C C A C G T C C A C T C T C C A C G T C C A C T C T C C A C G T C C A C T C

Mother of patient P2

T C C A C G Y C C A C T C T C C A C G Y C C A C T C T C C A C G T C C A C T C

ARAF c.640T>C ARAF c.640T>C Daughter of patient P2 (p.S214P) (p.S214P) in patient P2

ARAF c.640T>C (p.S214P) CR1 CR2 CR3 *

Human PLQRIRST S TPNVHMV Rhesus PLQRIRST S TPNVHMV Mouse PLQRIRST S TPNVHMV PLQRIRST S TPNVHMV Elephant PLQRIRST S TPNVHMV X_tropicalis SLQRHRST S TPNVHID Zebrafish --QRLRST S TPNVTML B-RAF LSQRDRSS S APNVH-I C-RAF FGQRQRST S TPNVHMV ARAF signaling cascade

wt mt

Wellbrock, C., Karasarides, M. & Marais, R. Nat Rev Mol Cell Biol 5, 875 (2004)

Molzan, M. et al. Mol Cell Biol 30, 4698–4711 (2010) ARAF mutations induce ERK activation

HEK293T

Whole Cell a-FLAG-IP lysate

FLAG (ARAF) HEK293T

14-3-3 (Pan) p-mTOR (S2448)

s.e. E V WT p-ERKs - FLAG (ARAF) S214P l.e. - ARAF

ARAF p-p70S6K (T389) b-actin -

E V p-AKT (S473) WT S214P ARAF

- l.e. p-ERKs (T202/Y204) ARAF s.e.

p-p38 (T180/Y182)

b-actin EV WT

Transfection: - S214P - ARAF ARAF

The mutated serine is part of the motif that interacts with 14-3-3. The S214P mutation disrupts the interaction between ARAF and 14-3-3, resulting in dramatic ERK activation in 293T cells. ARAF mutations induce ERK activation

p-ERKs (T202/Y204)

FLAG (ARAF)

ERK

[Trametinib] nM nM nM nM nM nM nM nM nM nM 30 30 DMSO 100 300 DMSO 100 300 1000 3000 1000 3000 ARAF-WT ARAF-S214P

When expressed in primary human dermal lymphatic endothelial cells (HDLECs), ARAF S214P induces potent ERK phosphorylation, which is inhibited by the MEK inhibitor trametinib. ARAF mutations induce sprouting formation

ARAF S214P induces spontaneous sprouting in spheroids formed from HDLECs

ARAF-WT

ARAF-S214P

Trametinib 0 nM 30 nM 100 nM 300 nM ARAF mutations result in internalization of VE-cadherins reversed by MEK inhibitors

ARAF-WT + DMSO ARAF S214P induces elongation of HDLECs in monolayers and internalization of VE-cadherin. Changes are reversed by Trametinib.

ARAF-S214P + DMSO ARAF-S214P + 100 nM Trametinib

VE-cadherin staining ARAF mutations result in actin filament derailment reversed by MEK inhibitors ARAF-WT + DMSO

Morphology changes induced by ARAF S214P are reflected in actin arrangement as well. Multiple, roughly parallel actin filaments are DMSO observed in the interior cell bodies of ARAF-WT expressing cells, while ARAF-S214P expressing cells lack clear filaments except along the cell perimeter (cortical actin). Changes are reversed by Trametinib.

ARAF-S214P + DMSO ARAF S214P + 100 nM Trametinib Phalloidin staining Overexpression of human ARAF mutant in zebrafish results in dilated lymphatics - v2a - Mrc1a:Araf S214P mcherry Disrupted normal vasculature can be reversed by MEK inhibitor

TD PCV mrc1a:ARAFwt

mrc1a:ARAFS214P No treatment

TD PCV mrc1a:ARAFS214P Cobimetinib Clinical Response of ARAF Proband to a MEK Inhibitor

Improvements in ARAF S214P patient following trametinib treatments

Baseline Post Therapy 03/17/2017 04/04/2017 05/04/2017 10/23/2017 03/08/2018 Parameter Unit Pre % Ref Pre % Ref Pre % Ref Pre % Ref Pre % Ref Weight kg 38 40 42 40 39 Height cm 142 142 143 145 148 FVC L 0.58 23 0.88 35 0.80 31 0.95 35 1.18 40 FEV1 L 0.52 23 0.77 34 0.72 31 0.85 35 1.09 42 FEV1/FVC % 89.8 105 87 102 89.3 105 89.0 105 92.0 108 FEF25-75 L/s 0.8 29 1.12 41 1.10 39 1.46 50 1.79 58 TLC L 0.93 29 1.23 38 1.28 39 1.51 45 1.98 56 RV L 0.27 31 0.35 40 0.48 55 0.56 62 0.80 86 RV/TLC % 28.94 107 28 104 37.67 140 37 137 40 154 DLCO [Hb] mL/min/mmHg ------9.99 54 9.9 52

DLCO/VA mL/min/mmHg/L ------9.08 135 7.67 116

MIP cmH2O 51.6 71 - - 62.2 82 70.0 95 85.0 115 MEP cmH2O 69.4 66 - - 77.6 74 89.0 82 83.0 75 O2 Sat % 92 - 97 100 97

55 121 ) lbs

45 99 Weight ( Weight (kg) 35 77

25 55

22.5 8.9 10 12 14 Age (years) GLA patient with end-stage disease unresponsive to Rapamycin

• We discovered a new mutation guiding us to a new therapy in a 12 year old child who was dying from the disease • Zebrafish studies expressing the human gene mutation developed lymphatic abnormalities, rescued with a drug blocking the abnormal gene function • Endothelial cell studies showing uninhibited growth that was fully rescued with inhibitor therapy • Patient had rapid improvement, fluid resolved, lung function reversed in 4 MRI lymphangiogram before therapy MRI after 4 months therapy months; Off supplemental oxygen, walking to school • Total remodeling of his lymphatic system

Li et al, Nature Medicine, 2019 Patient Gene Mutation Origin Main Phenotypic Features

1 ARAF c.640T>C:p.S214P Somatic Central Conducting Lymphatic Anomaly

2 ARAF c.640T>C:p.S214P Somatic Lymphangiomatosis Lymphatic malformation with cardiofaciocutaneous syndrome and 3 KRAS c.35G>A:p.(G12D) Somatic chylothorax Lymphatic malformation with cardiofaciocutaneous syndrome and 4 BRAF .1403T>C:p.(F468S) Germline chylothorax c.475_476del:p.(L159Gfs*20 Lymphatic disorder with chylous pericardial effusion and non-immune 5 RASA1 Germline de novo ) hydrops 6 SOS1 c.2536G>A:p.(E846K) Germline de novo Noonan syndrome with lymphatic maformation and left pleural effusion

7 PTPN11 c.1504T>G:p.(S502A) Germline de novo Noonan syndrome with lymphatic disorder and chronic severe chylothorax Noonan syndrome with lymphatic disorder and acute onset of chylous 8 PTPN11 c.1510A>G:p.(M504V) Germline ascites 9 PTPN11 c.1530G>C:p.(Q510H) Germline de novo LEOPARD syndrome with lymphangiectasia and respiratory distress

10 PTPN11 c.1507G>C:p.(G503R) Germline Plastic bronchitis and chylothorax with heart defects c.1236+4A>G and 11 ITGA9 Recessive Lymphatic anomaly, chylothorax, lymphedema c.289T>G:p.C97G Noonan; Chylothorax, Lymphatic disorder, Valvular pulmonary 12 RAF1 c.433A>C:p.Thr145Pro (n) De novo stenosis 13 RASA1 c.2246G>C p.Arg749Pro (n) inherited Baby Boy is a 2 month old male with congential lymphatic disorder Noonan syndrome. In infancy her course was complicated by 14 RIT1 c.270G>T:p.M90I unknown pulmonary lymphangiectasis and plastic bronchitis. 15 PIEZO1 c.7289C>T:p.P2430L Recessive Lymphedema and lymphatic conduction disorder

16 EPHB4 c.2288G>A:p.R763Q M inherited Central Conducting Lymphatic Anomaly

17 EPHB4 c.2654A>G:p.K885R M inherited prenatal onset nonimmune hydrops, ascites, and subcutaneous edema Summary II

u A recurrent mutation in ARAF was identified in the conserved phosphorylation site in two unrelated patients u ARAF mutant leads to enhanced MAPK activity u Transgenic expression of human ARAF mutation in zebrafish leads to dilation of thoracic duct u This lymphatic defect is treatable with a MEK inhibitor u Submitted for a Frontier program this year and was awarded Hereditary Cystatin-C Amyloid Angiopathy

Cystatin C amyloid deposition in samples from the Background: cerebrum, cerebellum, midbrain and thalamus in HCCAA. Patients suffering from HCCAA have amyloid fibrils precipitate in the cerebral arteries, resulting in cerebral hemorrhage, dementia and death in early adulthood (Wahlbom et al 2007)

The molecular pathophysiological process involves the transformation of a soluble functional monomeric Cystatin C/Amyloid complex into potentially toxic aggregates (Ostner et al 2013).

L68Q cystatin C, the mutated form causing HCCAA, has an increased propensity to form dimers compared with that of wt cystatin C.

Wahlbom et al 2007

Objective: To identify therapeutics agents capable of stopping the production of oligomers and fibrils of L68Q Cystatin C/Amyloid, thereby preventing vascular events and dementia. N-acetylcysteine blocks oligomerization of Cyst-C L68Q

• We found a drug, Cystatin C L68Q GSH, that % Oligomer in supernatant completely GSH NAC blocked the (mM) (mM) amyloid 100 aggregation 1 3 10 1 3 10 HMW DTT *

50 • The drug is well 250 * * * tolerated and a * 150 HMW * * precursor version Oligo 0 100 of it (NAC) can be DTT 75 given as a tablet Glu 1mMGlu 3mM untreated Glu 10mMNAC 1 mMNAC 3 mM a-Cyst-C NAC 10 mM % Monomer in supernatant • The oligomers 50 essentially Trimer + 100 + disappeared so 37 + + there is no new dimer Mono precipitation of 25 50 amyloid in the brain and brain 20 Mono vessels. 0 Incubation for 24 h at 37C with indicated concentrations DTT Glu 1mMGlu 3mM untreated Glu 10mMNAC 1 mMNAC 3 mM • A skin biomarker Drug can be given orally NAC 10 mM confirmed response to therapy in patients Cystatin C/amyloid protein aggregate in the skin in HCCAA following NAC therapy

~70% reduction in proband’s amyloid precipitation in skin biopsy Proband in 15 months of NAC therapy

~50% reduction in the father’s skin biopsy

Father following 6 months of therapy

u Clinical trial in Iceland with 50 patients with HCCAA to be treated for 12 months with NAC (FPI July 01, 2019) u Dementia Rating Scale (DRS) administered every 3 months u Biomarker measures – skin and blood u Results will inform on therapy of vascular dementia and Alzheimer disease (next trial) Genome-Based Diagnosis And Development at CAG

Appropriately consented biobanking efforts are a pre-requisite for these opportunities

CAG Phenotyping Genotyping Variant Targeted Biobank Identification therapies • EPIC • GWAS POC clinical • Common • ANNOVAR • ADHD • CHOP • Sequencing trials Diseases Clinicians • WES/WGS • PennCNV • Autism • Rare • Recontact • SNVer • IBD, pAIDs Test&Treat Diseases • RNAseq • ChIPseq • GLAs • HCCAA

Highly feasible and cost-effective to perform POC studies (Rx/Dx) within academic infrastructure with focus on genetic/genomic biomarker-stratified patient populations Acknowledgements

CAG staff: Patrick Sleiman, Dong Li Collaborators: Avni Santani Clinical CAG staff: Collaborators: Yoav Dori Charlly Kao Mcckenzie Behr Russell Buono Raquel Gur Tom Farraro Lifeng Tian Alexandria Thomas Renata Pellegrino Bob Baldassano Michael Sperling Nora Robinson Sarah Shepard John Maris Maja Bucan Shanell Harrison Jo Elia Joseph Glessner Hongzhe Li Jean Belasco Elizabeth Bhoj Carlita Gibbons Mingyao Li Christoph Seidler John Connolly Kisha Harden Jason Christie Tara Wenger Allan Pack Huiqi Qu Iris Ortiz-Rivera Xiang Wang Jonathan Perkins Dan Rader Salley Freda Athena Zuppa Alvaro Gutierrez Muredach Reily Betsy Goldmuntz Michael March Tyeisha Rex Garret Fitzgerald Bob Schultz Berta Castillo Daniel Senicola Kate Nathanson Kenny Nguyen Debra Abrams Michael Grunstein Gholson Lyon Margaret Harr Jonathan Spergel Steven Albelda Julian Allen Mike Gonzalez Genetics: Hans Bisgaard Nancy Spinner Struan Grant Donna McDonald-McGinn Kathleen Barnes Dennis Dlugos Frank Mentch Elaine Zackai Bill Cookson Haijun Qiu Marni Falk Eric Rappaport Adnan Custovic Xiao Chang Matt Deardorf Susan Levy Eric Topol Bob Berkowitz Jonathan Bradfield Beverly Emanuel John Trojanovski Dimitri Monos Yiran Guo Donna McDonald-McGinn Virginia Lee Babette Zemel Christopher Cardianle Marcella Devoto John Asplin Marina Bakay Charlie Stanley David Golfarb Rahul Pandey Charles Bailey ….and many others Garrett Brodeur Fernanda Marfa Larus Hermannson Edward Attyeh Zhi Wei Asbjorn Kristbjornsson Management: Yael Moss George Otieno Janine Larr Madeline Bell Bill Gaynor Amber bender Jennifer Lebold Bryan Wolf Sandra Deliard Scott Cook-Sather … and many others Maria Garris Cuiping Hou Adam Wenocur