Genes - Where Do We Go From Here?

Camp SunshineMonday, July 13, 2015

Dr. Dave Bodine, Ph.D.Chief, Genetics and Molecular Biology BranchNational Institute for Human Genome Research49 Convent Drive, MSC-4442, Room 4A04Bethesda, MD 20892Phone: (301) 402-0902email: tedyaz@mail.nih.gov

MACROPHAGE

STEMCELLS

ERYTHROCYTES

PLATELETS

T CELLS

B CELLS

BASOPHILS

EOSINOPHILS

NEUTROPHILS

COMMITTEDPROGENITOR CELL

ADA Deficiency: A Combined Immunodeficiency

ADA

Courtesy of Fabio Candotti

ADA

MACROPHAGE

STEMCELLS

ERYTHROCYTES

PLATELETS

T CELLS

B CELLS

BASOPHILS

EOSINOPHILS

NEUTROPHILS

COMMITTEDPROGENITOR CELL

Gene Therapy for ADA Deficiency

Virus containing a normal ADA gene

Courtesy of Fabio Candotti

ADA

CollectMarrow

IsolateCD34+Cells SCF

MGDFFL

CombineAliquots

Backup Marrow

MND-ADA

GCsapM-ADA

Transduce x 3 Days

Gene Therapy of ADA deficiency

Busulfan75 mg/m2

ADA-

DiscontinuePEG-ADA

Reinfuse

ADA?

PROSPECTS FOR GENE THERAPY FOR DIAMOND BLACKFAN ANEMIA: MIXED

RPS19RPL5RPL11RPS26RPS24

RPL35aRPS10RPS17

RPS7Other – inc. GATA1, MCM2

deletions

unknown

deletions

genotype unknown

No need to have the gene regulated.Modest expression levels should have positive results.

Need to improve gene transfer frequency by 2-3 fold at a minimum.Need to develop multiple gene transfer vectors.

+

-

SOME NEW FINDINGS ABOUT DBA

COST OF SEQUENCING A GENOME

SEQUENCING PIPELINE

SEQUENCING PIPELINE

DECIPHERING THE DATA

RPS19 DELETIONS

COST OF SEQUENCING A GENOME

SEQUENCING PIPELINE

New genes that cause DBA

Small deletions that cause DBA

HUMAN CHROMOSOMES

HUMAN CHROMOSOMES

THE DBA DEFECT IS AT THE BFU-E STAGELT-HSC

CLPCMP

ST-HSC

MEP

GMP

ERY

Plt

Neu Eo

NKMono

Bas

Mac

TB

MEG

MPP

LMPP

HSC

CFU-MkCFU-E

Lipton JM, Kudisch M, Gross R, Nathan DG (1986). “Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia”. Blood. 67: 962-8. Iskander D, et al (2015). “Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs”. Blood. 125 :2553-7.

THE DBA DEFECT IS AT THE BFU-E STAGE

Question 1: Where can we get DBA erythroid progenitor cells?

Question 2: Can enough DBA erythroid cells be cultured from peripheral blood CD34+ cells for analysis?

Lipton JM, Kudisch M, Gross R, Nathan DG (1986). “Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia”. Blood. 67: 962-8. Iskander D, et al (2015). “Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs”. Blood. 125 :2553-7.

LT-HSC

CLPCMP

ST-HSC

MEP

GMP

ERY

Plt

Neu Eo

NKMono

Bas

Mac

TB

MEG

MPP

LMPP

HSC

CFU-MkCFU-E

CULTURE OF CIRCULATING CD34+ CELLS

1. Measure growth and differentiation of patient and control cells in vitro

2. Analyze the transcriptional profiles of phenotypically similar patient and control cells in vitro

LT-HSC

CLPCMP

ST-HSC

MEP

GMP

ERY

Plt

Neu Eo

NKMono

Bas

Mac

TB

MEG

MPP

LMPP

HSC

CFU-MkCFU-E

Miyake K et al. (2008). “Ribosomal protein S19 deficiency leads to reduced proliferation and increased apoptosis but does not affect terminal erythroid differentiation in a cell line model of Diamond-Blackfan anemia.” Stem Cells. 26: 323-9. Narla A et al. (2011). “Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis.” Blood. 118: 2296-304. Moniz H, et al. (2012). “Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro.”Cell Death Dis. 3:e356.

Kelly O’Brien

D

REDUCED GENERATION OF ERYTHROID CELLS FROM DBA PATIENTS

Control

DBA

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

Days in Culture

Nu

mb

er

of

Ce

lls

7x106

6x106

5x106

4x106

3x106

2x106

1x106

0

Kelly O’Brien

MICROARRAY ANALYSIS OF DBAERYTHROID CELLS (RP MUTATIONS)

Top Upstream Regulator: GATA1Top Functions: Hematological System Development and Function;

Increased HematocritIncreased RBC

DBA – R

PS17

DBA – R

PS24

DBA - Unkn

own

DBA – R

PL5

DBA - Unkn

own

DBA - Unkn

own

DBA – R

PS17

Controls

DBA Patients and Families: YOU ARE NOT ALONE!

Each of the investigators in this photo represent teams of dedicated people who are trying to understand, treat and

cure DBA.

FINAL THOUGHTS

FINAL THOUGHTS

THANK YOU

Thanks to all of you for being here. You are providing the most valuable contribution of all:

Hope, Support and a Sense of Community.

No DBA patient or family could feel alone in the presence of people like you.

No researcher could not be inspired by people like you.