Alessandro Aiuti

GENE THERAPY-BASED ORPHAN DRUGS

igetTELETHON INSTITUTEFOR GENE THERAPY

UNIVERSITA’ DI ROMA TOR VERGATA

Ex vivo approaches

RetrovirusAdenovirusLentivirus

In vivo approaches

AdenovirusAAV

DNA/LiposomesLentivirus

Gene therapy strategies

Ex vivo

SCID-X1ADA-SCID

Wiskott-Aldrich SyndromeBeta-thalassemia

Metachromatic Leukodystrophy

In vivo

Duchenne Muscular Distrophy Alpha-Sarcoglycanopathy

Gamma-Sarcoglycanopathy

Glycogen storage dis. type II (Pompe's disease)

Retinitis pigmentosa Leber’s amaurosis Stargadt’s disease

Epidermolysis bullosa

Gene therapy-based EU designated Orphan Drugs

Muscle

Multiple organs

Eye

Skin

HematopoieticSystem

CNS &PNS

Primary immunodeficiencies

•  Alterations in development and/or functions of adaptive/innate immunity•  Higher susceptibility to infections•  Failure to thrive•  Increase risk of autoimmunity and cancer

ADA-SCID

INNATE IMMUNITY ADAPTIVE IMMUNITY

WISKOTT-ALDRICH SYNDROME

ADA-SCID

Gene transfervector Autologous gene modifed

HSC

-Autologous procedure (No rejection/ GVHD)

- Reduced toxicity

- Selective advantage for gene corrected cells

- Data on safety and efficacy from preclincial studies and pilot studies

HSC gene therapy for primary immunodeficienciesMedicinal Product

Starting material

Adenosine Deaminase-deficient SCID

Adenosinedeaminase

dAdo, AdodAXP

  Bone Marrow Transplantation  Enzyme Replacement Therapy (PEG-ADA)  HSC Gene Therapy

TREATMENT OPTIONS

•  T, B, NK, lymphopenia•  Severe recurrent infections•  Autoimmunity

•  Bone and growth abnormalities•  Organ toxicity (lung, liver)•  Neurological and behavioral alterations

Autosomal recessive1:375,000-1,660,000

RATIONALE FOR GENE THERAPY

Unmet medical need •  90% of children lack an histocompatible donor in the family •  High risk of bone marrow transplant from alternative donors (30-65%

survival)

•  Treatment with bovine enzyme (PEG-ADA) (80% survival) not a definitive cure, not always effective, very expensive

Scientific rationale •  The ADA gene is constitutively and ubiquitously expressed •  Gene-corrected lymphocytes have an advantage over ADA-deficient

cells.

•  10% of normal ADA expression may be sufficient

Day -4: Purification of BM CD34+ cells

Gene transfer protocol into autologous bone marrow CD34+ cells

Day-4: Prestimulation (TPO, FLT3-ligand, SCF, IL-3)

Days -3 to -1: 3 cycles of transduction on retronectin + cytokines

Busulfan 2 mg/Kg/day x 2 (days -3, -2)

Day 0: Infusion

ADA SV Neo

No PEG-ADA

A. Aiuti, MG Roncarolo, C. Bordignon, 2002

MLV LTR

BM Harvest

CD15+ granulocytes

Years after GT0 1 2 3 4 5 6 7

0,0

5,0

10,0

15,0

20,0

0
 20
 40
 60
 80
 100
 120
 140


4 yrs after infusion

Diversity of integrations in T lymphocytes

Prop

ortion


of
d

ifferen

t
clone

s


No in vivo skewing

POLYCLONAL VECTOR INTEGRATIONS and REPERTOIRE %

TCR

Vbet

a in

CD3

+ ly

mph

ocyt

e su

bset

TCR Vbeta Repertoire

0246810

12

Pt2

Pt3

Pt5

Pt6

02468

1012

02468

1012

02468

1012

Pt4

02468

1012 Pt7

02468

1012

02468

1012

Pt9

Pt10

0246810

12

Pt1 ND (n=46)

02468

1012

Aiuti et al., JCI 2007 and unpublished results

Immune reconstitution after GT

Thymus 3 yrs post-GT

Median (n=9)

CD3+CD4+CD8+

1500

+3 years (n = 8) (n = 6)

+1 year +2 years00

500

1000

Cells

/µl

+3 years

T- cell reconstitution after gene therapy

PRE-GT 1y FU 2y FU >3y FU BMT HC

10

100

1000

10000 *

Cop

ies /

100

ng

of D

NA

Recent thymic emigrants

Recovery of thymic functions

anti-CD3 mAb

cpm

pre-GT 6 months 1 year 2 years 3 years Healthy controlsn=9 n=9 n=9 n=7 n=5 n=114

100

5000

50000100000200000300000

T-cell functions (anti-CD3)

Aiuti et al., NEJM, 2009

•  IVIg discontinued in 6 pts with proven antibody response

(TT, DT, Pertussis, Haemophilus, Pneumo)

2 Pts ongoing IVIg discontinuation

•  MMR vaccine in 1 pt led to protective antibodies

•  Four patients experienced varicella

without complications

Immune response and protection from infections

05

1015

Rate of severe infections

N. e

vent

s/10

per

son-

mon

ths

Pre GT Post GT20 infections/ 215.4 person-month 4 infections/ 394.5 person-month

Systemic detoxification and growth improvement

dAXP metabolites

Years after GT0 1 2 3 4 5 6 7

nmol

es/m

l

0

100

200

300

400

500

*

T cell

B cell

NK cell

Monocyte

Erythrocyte

Granulocyte Platelet

Outcome of ADA-SCID GT

Clinical translation in ADA-SCID

First attempts Pilot studies Phase I/II

EMEA ODD 2005 1991 2000 2002

Towards registration

Enrolment closed

2008

Nonclinical

FDA ODD 2009

EMEA protocol assistance

Wiskott-Aldrich Syndrome (WAS)

Autoinhibited WASP

Active WASP

PH-EVH1 GBD

Pro

A C V V

PIP2Plasmamembrane GTP

Cdc42

SH3NckTecGrb2

Actin monomer

Actin polymerization

Arp2/3Cytoplasm

WIP

CELLULAR DEFECTSHSC migrationT cells migration, immune syn.B cells migration, Ig productionPlatelets reduced size / numberDendritic cells migrationMacrophages adhesion, antigen uptakeNK cells cytotoxic activity

  Eczema   Bleeding   Infections   Autoimmunity   Tumors

X-linked, 1,250.000 newborn male

Life expectancy: 15 years in severe forms (WAS-negative)

Current Treatment

Filipovich, Blood 2001; Kobayashi, 2006; Ozsahin, Blood 2008

GA RREcPPT PREwas WASP CMV

LENTIVIRAL VECTORS

•  HIV derived, self-inactivating system

•  Safer integration profile

•  Physiological promoter

•  Improved GT efficiency into HSC

WAS‐/‐
donor
(male/CD45.2)


BM
harvest


Transplantation
i.v.
2.5x105
‐
106
lin‐
BM
cells


700
rads
(sublethal)


WAS‐/‐
recipient
(female/CD45.1)

LV
transduction


12
hrs


w1.6W_WPREmut


GA RRE cPPT WPRE
h1,6WASP
 hWASP
RSV

SAFETY AND EFFICACY OF WAS GT IN WAS KO MICE

Low MOI High MOI

CD45+ CD11b+ B220+ CD8+ CD4+0

25

50

75

100

SpleenBM

* ***

% W

ASP+

c

ells

CD45+ CD11b+ B220+ CD8+ CD4+0

25

50

75

100 * **

SpleenBM

% W

ASP+

c

ells

No long-term toxicity

No vector derived tumors

No increase in tumor incidence

98 mice followed for 4-16 months

Engraftment and selective advantage

FUNCTIONAL CORRECTION OF T-CELLS AND B CELLS

anti-CD3 2 µg/ml

Pneumo23 vaccine challenge TP23 - P23 immunization

Ab TITER d=7

TP23 WT TP23 UT TP23 GT0

50

100

150

200

250

300

350 TP23 WT

TP23 GTTP23 UT*

*

T-cell functions(n

g/m

l)

1

10

100

1000

10000 ** ****IL-2

SI

1

10

100

1000

10000 ** ****PROLIFERATION

wt

Was-/-w1.6W high MOI

w1.6W low MOI

Lin- Was-/-F. Marangoni, A. Villa, M. Bosticardo

300+250
rads


Rag2‐/‐
γc‐/‐
(neonates)


SUMMARY OF TOXICITY AND SAFETY STUDIES (CD34+ cells)

In vitro growthColony assay (CFC,LTC-IC)Vector shedding

Vector integrations

BiodistributionVector sheddingGermline transmission

The path to clinical trial in WAS

Vector & Proof of concept

Preclinical studies

Validation

2002 2004 2006 2008

Manufacturing and quality

Phase I/II trial

2010

•  Lack of toxicity

•  Safety and efficacy in the animal model

•  Selective advantage for gene corrected cells

•  Efficient gene transfer in human CD34+ stem cells

Design: non-randomized, open label, single center

Population: 6 patients -Severe WAS mutation or WAS clinical phenotype

-No HLA-identical sibling -No HLA-matched UR BM or UCB donor (Pts

Very rare population!

“Personalised” therapy

Single curative injection

“Old” therapy approaches Gene/cell therapy for rare diseases

•  Often very rare populations•  Long-term safety•  Mainly academic-driven, high costs•  Limited interest for pharma company’s investment•  Manufacturing and standardization •  Rapidly evolving scientific field and regulation

Funding agencies

Regulatory Agencies (National level)

Manufacturing (Biotech or Academic)

Investigators (PRECLINICAL AND CLINICAL)

EC Regulatory Agencies

Manufacturing (Biotech)

Industrial partner

National Health system

European Community

Partners required for clinical development of gene therapy-based ODD

Patients’ organization

San Raffaele Telethon Institute for Gene Therapy iget

TELETHON INSTITUTEFOR GENE THERAPY

S. Marktel B. Cappelli L. Callegaro M . Casiraghi V. Bergamante F. Ferrua

I Brigida B. Cassani S. Scaramuzza A. Ripamonti L. Biasco A. Sauer

M. Bosticardo A. Villa

L. Naldini M.G. Roncarolo

Pediatric Clinical Research Unit

MolMed S.p.A M. Salomoni M. Dieci

Main European collaborators (WAS) A. Thrasher (London) A. Galy (Paris)

All participating physicians worldwide

HSR BMT Unit F. Ciceri

U. Bicocca MG Valsecchi S. Galimberti

Univ. of Siena A.  Tabucchi F. Carlucci

Univ. Wien M. Eibl