ZebrafishFishing for New Therapies for DBA

Camp Sunshine 2017 Elizabeth Macari, PhD

Lab of Leonard Zon, MD Boston Children’s Hospital

Transparent embryo

Circulating blood in 23 hours

Transgenic fish

Genetic screens Gene knockdowns

Chemical screens

Conserved blood lineages

Adult

100s of progeny / week

Strengths of studying zebrafish

Zebrafish embryoHuman embryo(28 days) (19 hours)

Wild Type (Healthy Fish)

Mutant that lacks

hemoglobin

Visualizing red blood cells in zebrafish

Hemoglobin Stain

Twomethodstomodifyzebrafish

X

Chemical treatment to induce random mutations

+/+ +/ m

Inject genetic material into 1 cell stage

+/+ +/+

Casper

Transparent,adultzebrafish

CasperlackspigmentofWTfish

WT

Casper

sis (grx5)

cia (transferrin receptor ) cdy (dmt1) drc(fch) frs (mitoferrin) cha (protein 4.1)

Lymphoid progenitor T cell

B cell

Myeloid

Erythroid

assam camomile darjeeling earl grey (sart 3) jasmine

Hematopoietic Stem Cell

bls

vld (gata1) mon (tifγ)

gata2

Erythroid -myeloid progenitor

DBA (rps29) ret (band 3) ris (b-spectrin) sau (alas2) weh(fpn1) yqe (urod) zin (globin)

Zebrafishbloodmutantgenes

Wild Type (Healthy Fish)

DBA model Mutation in

rps29

DBA zebrafish model

Hemoglobin Stain

Twomethodstomodifyzebrafish

X

Chemical treatment to induce random mutations

+/+ +/ m

Inject genetic material into 1 cell stage

+/+ +/+

Thismethodisusefultoaddbackagenethatismissing

Human RPS29 RNA can rescue hemoglobin in rps29 mutant DBA model

MirabelloL,MacariEetal.Blood2014

rps29-/-

InjectHealthyhuman(WT)RPS29RNA

OrRNAfromDBApaMent 48h

StainforHemoglobin

+ WT RNA

+ I31F RNA uninjected + I50T RNA

Chemical screen using DBA zebrafish model

DBA zebrafish model

Chemicallibraries600compounds

Chemical“hits”

Screenforrescueof

hemoglobin

Chemical screen identifies calmodulin inhibitors increase hemoglobin

StainforHemoglobin

rps29+/- rps29-/-

VehicleCaMinhibitors

Trifluoperazine(TFP)

TFP improves anemia in DBA mouse model

Kavitha Siva and Johan Flygare

TFP (5 mg/kg IP)

Blood/BM Collection

2 wks

**p<0.01

RPS19mutantDBA

Mouse

HealthyMice

Healthy mouse

DBA mouse

DBA mouse + TFP

0

50

100

150

200

Hem

oglo

bin

(g/L

) **

DBAMice

DBAMice+TFP

Hemoglobin

RPS19R94XSteroidresponsive10yearoldpaMent

TFPincreasesredbloodcellsinDBApaMentbonemarrowsamples

RBC Differentiation BM cells

TFP5days4days

SergeiDoulatovandLindaVo

Analysisforredbloodcellmarkers

CD71

TFP5µm

GLYA

Healthybonemarrow DBApaMentbonemarrow

89 20 50

PaMentderivedinducedpluripotentstem(iPS)cells

Patient

Patient-derived iPS cells

Too few patient blood cells

Blood cells

Disease modeling and drug screens

Skin cells Respecify

Patient fibroblasts: RPS19 RPL5

DBAiPScellsshowimpairedabilitytomakeredbloodcells

RPS19+/-Control RPL5+/- Controlheme RPL5+/-heme

GLYA

CD71

33 11 10

RBC Differentiation iPS cells Analysisforredbloodcell

markersandhemoglobinDBApaMentskincells

Chemical screen using DBA iPS cells

DBA patient derived iPS cells

Chemical“hits”

Screenforchemicalsthatgivemoreredbloodcells

Chemicallibraries1200compounds

SMER28

SMER28increasesnumberofredbloodcells

RPL5+/- DBA Cells RPL5+/- DBA Cells

+SMER28

Healthy DBA RPS19 mutant cells

CD71

GLY

A

1.0 µM SMER28

89 54 76.5

Control

RBC Differentiation iPS cells

SMER28

Analysisforredbloodcellmarkers

SMER28increasesredbloodcellsinDBApaMentsamples

GLY

A

CD71

DBA Patient 1 RPS19

DBA Patient 2 RPS29

untreated SMER28 1.0 µM

20 55

15 41

RBC Differentiation BM cells

SMER284days

Analysisforredbloodcellmarkers

SMER28improveshemoglobininDBAzebrafishmodel

Healthy (WT)

DBA DBA

1µM SMER28

Chemical screens to discover potential therapies to treat DBA

DBA patient derived iPS cells

DBA zebrafish model

TFP SMER28

ClinicaltrialstarMnginadults

FurthertesMngandbeferderivaMvesneeded

Funding NHLBI F32 and NHBLI U01

BCH FACS Core Ronald Mathieu Mahnaz Paktinat Lund University Johan Flygare Kavitha Siva Northwell Health Jeff Lipton Adrianna Vlachos

Acknowledgements

Zon Lab

Len Zon Alison Taylor Emma Stillman Julia Huber Kate McGrath Serine Avagyan Julien Ablain Julie Perlin Christie Ciarlo Jess Humphries Alessia Giannozzi Entire Zon Lab!

Kara Maloney Chris Lawrence Bruce Barut Hannah diCicco

Daley Lab Sergei Doulatov Linda Vo

Ebert Lab Dave Raiser Anu Narla

Taplin Mass Spec Ross Tamaino

NCI Blanche Alter Lisa Mirabello Sharon Savage

DBA patients and their families