ORPHAN DRUGS Alessandro Aiuti UNIVERSITA DI ROMA TOR VERGATA iget - - PowerPoint PPT Presentation

Alessandro Aiuti

GENE THERAPY-BASED ORPHAN DRUGS

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TELETHON INSTITUTE FOR GENE THERAPY

UNIVERSITA’ DI ROMA TOR VERGATA

Ex vivo approaches

Retrovirus Adenovirus Lentivirus

In vivo approaches

Adenovirus AAV DNA/Liposomes Lentivirus

Gene therapy strategies

Ex vivo

SCID-X1 ADA-SCID Wiskott-Aldrich Syndrome Beta-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

• rgans

Eye Skin Hematopoietic System 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 transfer vector 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 immunodeficiencies

Medicinal Product Starting material

Adenosine Deaminase-deficient SCID

Adenosine deaminase

dAdo, Ado dAXP

 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 recessive 1:375,000-1,660,000

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

• f 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

• n 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 GT 1 2 3 4 5 6 7 <0.01 0.1 1 10 100 % of vector of positive cells

CD34+ progenitors

Years after GT 1 2 3 4 5 <0.01 0.1 1 10 100 % of vector of positive cells

CD3+ T cells

Years after GT 1 2 3 4 5 6 7 <0.01 0.1 1 10 100 % of vector of positive cells

CD19+ B cells

Years after GT 1 2 3 4 5 6 7 <0.01 0.1 1 10 100 % of vector of positive cells

• A. Aiuti et al. NEJM, 2009

Long-term engraftment of gene corrected cells

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

Proportion
of
different
clones


No in vivo skewing

POLYCLONAL VECTOR INTEGRATIONS and REPERTOIRE

% TCRVbeta in CD3+ lymphocyte subset

TCR Vbeta Repertoire

2 4 6 8 10 12

Pt2 Pt3 Pt5 Pt6

2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12

Pt4

2 4 6 8 10 12

Pt7

2 4 6 8 10 12 2 4 6 8 10 12

Pt9 Pt10

2 4 6 8 10 12

Pt1 ND (n=46)

2 4 6 8 10 12

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 years

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 * Copies / 100 ng of DNA

Recent thymic emigrants

Recovery of thymic functions

anti-CD3 mAb

cpm

pre-GT 6 months 1 year 2 years 3 years Healthy controls

n=9 n=9 n=9 n=7 n=5 n=114 100 5000 50000 100000 200000 300000

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

5 10 15

Rate of severe infections

• N. events/10 person-months

Pre GT Post GT

20 infections/ 215.4 person-month 4 infections/ 394.5 person-month

Systemic detoxification and growth improvement

dAXP metabolites

Years after GT 1 2 3 4 5 6 7 nmoles/ml 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 PIP2 Plasma membrane GTP Cdc42 SH3 Nck Tec Grb2 Actin monomer Actin polymerization Arp2/3 Cytoplasm WIP

CELLULAR DEFECTS HSC migration T cells migration, immune syn. B cells migration, Ig production Platelets reduced size / number Dendritic cells migration Macrophages adhesion, antigen uptake NK 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 RRE cPPT PRE was 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

+

25 50 75 100

Spleen BM

* * * *

% WASP

+

cells

CD45

+ CD11b + B220 +

CD8

+

CD4

+

25 50 75 100

* * *

Spleen BM % WASP

+

cells

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

Ab TITER d=7

TP23 WT TP23 UT TP23 GT 50 100 150 200 250 300 350

TP23 WT TP23 GT TP23 UT

* *

T-cell functions

(ng/ml)

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 growth Colony assay (CFC,LTC-IC) Vector shedding Vector integrations Biodistribution Vector shedding Germline 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 <5 yrs)

Follow up: 3 years, then long-term safety protocol Study objective: Evaluate the safety, biological activity and efficacy of GT Financial sponsor: Fondazione Telethon PI: A Aiuti, MG Roncarolo, Co-PI: Fabio Ciceri Authorized by Ethical Committee (12/09) and National authority (ISS) (03/10)

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TELETHON INSTITUTE FOR GENE THERAPY

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

• f gene therapy-based ODD

Patients’

• rganization

San Raffaele Telethon Institute for Gene Therapy

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TELETHON INSTITUTE FOR 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