De la génomique à la thérapeutique

Axel KAHN Directeur de l’Institut COCHIN et de l’IFR Alfred JOST

Genopole® : Genopole® La médecine de demainLa médecines’inventede demain aujourd’huis’inventeaujourd’hui THE DISEASES OF THE 21st CENTURY

Cancers Neurodegenerative disorders Cardiovascular diseases Nutrition diseases Infectious diseases

Monogenic diseases

Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 TheThe challengeschallenges forfor progressprogress inin therapeuticstherapeutics inin thethe XXIXXIst centurycentury

Variable targets

Complex physiopathogenic mechanisms

Chronic diseases with irreversible lesions

Genopole® LaAXEL médecine KAHNde demains’invente 3.06.2003aujourd’hui GENOMICS = LARGE SCALE STUDY OF THE GENOMES

Informational Genomics = maps, sequence

Functional Genomics = gene function (reverse ) expression maps - transcriptome - proteome Structural Genomics = from genetic information to protein structure

accumulation of a huge amount of more or less crude data in extensive databases How can we extract pertinent information from these data?

Biology, physiopathology hypotheses

bioinformatics ....for raising or studying the hypotheses Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 GENOMICS

genetic targets mechanisms r.proteins information

predictive drugs medicine

IMPROVED THERAPY Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 For improving pathophysiological understanding of common diseases and then identifying « validated targets »

Weak alleles involved in the common multigenic forms

Strong alleles involved in the rare (or exceptional) monogenic forms

: Atherosclerosis (LDL-R, ABC-A1, ABC-G5/ABC-G8,ARH)

: Alzheimer disease (PS1, PS2, APP)

: Obesity (leptin, leptin R, MCH and receptor, MSH and receptors) : Familial hypertrophic cardiomyopathy (AMPK) : Inflammatory anemias : Iron overload

Hepcidin Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 Although cancer usually :

- results from several associated molecular events - is associated with genetic instability

Is it possible to kill or to cure cancerous cells by acting on just one of the involved specific events ?

RA in PML

Gleevec in CML Kit (+) GIST

Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 databases

Drug design

Bioinformatics Candidate small targets Medicinal chemistry molecules the future?

combinatorial chemistry, HTS Physiopathology

THE BOTTLENECK Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 WORLWIDE SALES OF SMALL MOLECULES /vs RECOMBINANT PROTEINS (1997)

: 3 therapeutic proteins among the top ten selling drugs (EPO, insulin, G-CSF)

: 8.4 billions $ vs •• 25 billions total

already 1/3 of the sales (among the top ten...) .... while only •• 8.000 / 100.000 genes were exploited

Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 SOME PROMISES OF THERAPEUTIC PROTEINS

• cancer : antiangiogenic factors : antibodies (Herceptin) • diabetes type 2, lipodystrophies, obesity : leptin-like factors increasing insulin sensitivity ? : adiponectin • cardiovascular diseases : angiogenic factors • regenerative medicine : growth factors for specific cells and tissues - b cells (IDDM) - brain cells - nerves - spinal cord • autoimmune diseases, inflammations : b-interferon and multiple sclerosis : IL 13 antagonists and asthma

Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 PharmacogenomicsPharmacogenomics

Distinction of different groups of patients with respect to : drug toxicity drug efficacy

candidate gene polymorphism or alteration (e.g. P450 isoforms, susceptibility genes,disease genes)

tumor identity (gene markers, transcriptome, proteome) full genome screening with DNA microchips

Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 WhenWhen humanhuman genomicsgenomics failsfails toto leadlead toto diseasedisease etiologyetiology

Gastroduodenal ulcers - anti-histamine - anti-acids - hisR antagonists - proton pump antagonists

Helicobacter pylori

Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 3.06.2003 TREATMENT OF GENETIC DISEASES BY

: biological prosthesis / replacement - organ transplantation - administration of the lacking protein - gene transfer

: gene correction

Genopole® LaAxel médecine KAHNde demain 25.2.03s’inventeaujourd’hui TREATMENT OF GENETIC DISEASES BY SMALL CHEMICALS

Tyrosinemia type 1 NTBC

Familial ataxia (AVED), Vit E a-TPT deficiency Friedreich ataxia Quinones (Idebenone)

Lysosomal diseases Ligands of a-galactosidase Glucosyltransferase inhibitors (NB-DNJ and NB-DGJ) Congenital hypothyroidy T3,T4

Genopole® AxelLa Kahnmédecinede demain6.3.03s’inventeaujourd’hui PROSPECTS IN GENE THERAPY

• New mode of recombinant protein delivery; very active protein(EPO, angiogenic factors, clotting factors, etc…) • Corrected cells with a clear selective advantage(bone marrow,liver) • Correction of point mutations in the liver when partial correction is sufficient • …… but safety problems

• Cell autonomous effect of transferred or corrected genes • High % of correction required • Disseminated or poorly accessible target cells • High-level/prolonged transgene expression needed

Genopole® Axel KAHN 25.2.03 La médecinede demains’inventeaujourd’hui Genopole® AXEL KAHN 3.06.2003 La médecinede demains’inventeaujourd’hui Genopole® AXEL KAHN 3.06.2003 La médecinede demains’inventeaujourd’hui DNA and gene….. as a vaccine

Instead of developing antibodies against microorganisms and proteins... Injection of DNA will direct cellular synthesis of antigens safe easy stable cheap cellular immunity May be a breakthrough in preventive or curative vaccinology Genopole® AxelLa médecine KAHNde demain 25.2.03s’inventeaujourd’hui REGENERATIVE MEDICINE

A new concept for the future

Its goal : to replace aged, diseased cells (or tissues) by young genetically similar cells (or compatible tissues) assuring a restored function

Could revolutionize medicine in the new century

Genopole® La médecineAxel deKAHN demains’invente 25.2.03aujourd’hui REGENERATIVE MEDICINE

Neurodegenerative diseases

Eye, ear degenerative diseases

Spinal cord damages

Bone marrow diseases cells Engineered Diabetes cells Heart failure Joint diseases Skin diseases

Tissue engineering (bladder, vessel, skin..) Genopole® AXEL LaKAHN médecine de25.2.2003 demains’inventeaujourd’hui HSC

MSC Mesangioblasts Tissue-Specific

3 2

Pluripotent Egg ESC (MAPC)

Totipotent Multipotent 1

1 : « therapeutic » 2 : multipotent adult stem cells. 3 : reprogrammed adult cells Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 25/02/03 Differentiated ES Cells : therapy in vivo

Rats with PD models 1. Undifferentiated cells • Teratomas (5/25) • In situ dif. into dopaminergic neurons 2. Ex vivo differentiation into dopaminergic neurons • transformed with a nurr-1 transgene • normal function in vivo, some improvement Ex vivo differentiation into b cells • 2-5% of insulin content • May be, no insulin synthesis at all • Transient effet Ex vivo differentiation into HSC •<2% of immunocompetent cells

Genopole® La médecinede demains’inventeAXELaujourd’hui KAHN 12.06.2002 Cell therapy with somatic (stem) cells already a reality in Usually ….. • HSC and hematological diseases • Keratinocytes and skin grafts (burnt patients) Experimentally…… • Fœtal neural cells in neurodegenerative diseases • Islets b-cells in diabetes mellitus • Hepatocytes in liver failure and metabolic diseases • Muscle cells and bone marrow cells in myocardium infarction

Genopole® AXELLa médecine KAHNde demain 25.2.2003s’inventeaujourd’hui Genopole® 3.06.2003 La médecinede demains’inventeaujourd’hui Genopole® 3..06.2003 La médecinede demains’inventeaujourd’hui Genopole® 3.06.2003 La médecinede demains’inventeaujourd’hui ADULT CELL REPROGRAMMATION

« Therapeutic » cloning

Nucleus exchange with ESC

In vivo/ex vivo spontaneous reprogrammation ? (e.g,MAPCs)

In vivo/ex vivo transfer of differentiation master gene(s) (e.g, Pdx1-Vp16, Neurod, betacellulin)

In vivo/ex vivo cell fusion

In vitro reprogrammation with cell free extracts (permeabilized cells + nuclear extracts of SC or differentiated cells)

Genopole® La médecinede demains’inventeaujourd’hui AXEL KAHN 25/02/03 LIVER OVAL CELL High glucose

Insulin- secreting cells Neurod + btc Pdx 1/VP16 « hit and run » system

HEPATOCYTES ® AXEL KAHNGenopole La médecinede demains’invente3..06.2003aujourd’hui Multipotent Somatic Stem Cells

Bone Marrow MSC

Mesoangioblasts Multipotent Adult Stem Cells § bone §Cartilage §Fat Vessels Endothelial progenitors §Vessels Cord §Cardiocytes Cord blood §Myocytes §Hepatocytes Somatic epidermal SC §Glial cells §Neurons §…….etc

Skin Skin-derived precursors Genopole® La médecinede demainAXELs’invente aujourd’huiKAHN 12.06.2002 Pluripotent and tissue-specific stem cells

• Brain • MSC • Heart • Skeletal muscle • Blood (HSC) • Skin • Pancreas • etc….

Genopole® La médecinede demains’inventeaujourd’hui Axel Kahn 20/1/2004 Major hurdles with -based regenerative medicine

° To obtain enough starting stem cells (SC) (from adults) ° To control SC in vitro amplification and differentiation ° To eliminate non-differentiated, potentially tumorigenic cells before transplantation (cell sorting, differentiation- specific positive or negative selection markers)

Persisting uncertainties ° Even if enough differentiated cells can be obtained, what will be their long-term - function - viability - tumorigenic potential Genopole® LaAXEL médecine KAHNde demains’invente3.06.03aujourd’hui GENOME PROGRAMME

Differentiation and Growth factors

STEM CELLS expansion (embryonic / DIFFERENTIATED CELLS adult) differentiation

Genopole® AXELLa médecine KAHNde demains’invente 3.06.03aujourd’hui