Firenze, 15 aprile 2013

Letizia MazziniLetizia Mazzini

Centro Esperto SLA Centro Esperto SLA Clinica Neurologica Clinica Neurologica

Università del Piemonte OrientaleUniversità del Piemonte OrientaleAOU“Maggiore della Carità” AOU“Maggiore della Carità”

Novara Novara

Dal Laboratorio alla Clinica: lo stato dell’arte della ricerca sulla terapia cellulare nella Sclerosi Laterale

Amiotrofica

ALS is a rapid and progressive neurodegenerative disease that targets motor neurons in spinal cord, cortex and brain stem.

The selective degeneration of motor neurons manifests as a linear decline in muscular function eventually resulting in paralysis, speech deficits and dysphagia.

(Rowland LP et al., New Engl J Med, 2001; 344:1688-700).

Amyotrophic Lateral Sclerosis

Maladie de Charcot

Motor Neuron Disease

Lou Gehrig’s Disease

Multifactorial Disease

19 GENI

1. RNA processing 2. vesicle trafficking 3. oxidative stress 4. autophagy 5. Unknown function

Age Environment++ ++

Lost in Translation

Spatially diffuse death of MNs

Selective disruption of both short and long distance axonal connection

The chronic insidious but rapidneurodegenerative course

The lack of validated surrogate markers of disease.

Therapeutic Targets

Stem cells therapeutic strategy potentially target several Stem cells therapeutic strategy potentially target several of these putative mechanisms and can improve function of these putative mechanisms and can improve function

by replacing the lost neurons and glial cellsby replacing the lost neurons and glial cells

Cell types for transplantation

• Candidates to stem cell therapy in ALS must be able to survive and influence the pathological tissue environment, including inflammatory and immune reactions, and migrate into the sites of diffuse neurodegeneration.

• Moreover, it is fundamental for clinical application that stem cells are safe, and can be easily isolated and expanded.

Protection of neurons by adult stem cells

HuNi/DAPI

A B

hMSCs and hNSCs TRAPIANTO IN RATTI SOD1-G93A

Sopravvivenza

QUANTIFICATION OF LUMBAR MOTOR NEURONS

Transplanted Controls

Neuroscience Institute, University of Torino

CD11b

CD11b

GFAP

QUANTIFICATION OF MICROGLIA ACTIVATION

Transplanted Transplanted SOD1SOD1G93AG93A mice mice

Sham operated Sham operated SOD1SOD1G93AG93A mice mice

5458 5458 ++ 682 682 3549 3549 ++ 607 607 P<.005P<.005

T13T13 L2L2 L4L4

SHAM MICESHAM MICE 28.41128.411 28.48528.485 27.50027.500

TRANSPL MICETRANSPL MICE 21.64721.647 21.35721.357 20.35320.353

AND REACTIVE ASTROGLIOSIS

ANOVA p = 0,003286

IMMUNOMODULAZIONE – IBA1

Dati preliminari danno indicazioni di un effetto immunomodulatorio da parte delle hNSC trapiantate

SOD ventral horn SOD+NSC Wild type

Vescovi A. et al Department of Biotechnologies and Bioscience, Università Milano Bicocca

BEHAVIOURAL TESTS

PaGE

Score of motor

deficits

Rotarod test

MALES FEMALES

----- transplanted mice- - - control mice

Neuroscience Institute, University of Torino

macrophage/microglia infiltration

endogenous astrogliosis

neuronal phenotypeastrocytesoligodendrocytes

Clinical Translation

• Animal models do not accurately reflect the human disease and toxicological studies in animals are sometimes poor at predicting toxicity in humans.

• Transplantation studies where human cells are implanted in animals cannot provide full prediction of immune or other biologic responses to human cells in patients.

Clinical Translation

• Stem cells and their derivatives may act on several targets and exert both beneficial and detrimental effects, most notably, the risk of ectopic tissue and tumor formation.

• Cellular transplants may persist for many years in patients, or their actions may be irreversible, thus necessitating careful patient monitoring and extended follow-up.

http://www.isscr.org/clinical_transFrancese, tedesco, inglese

Stem cells Studyphase

Regulatory

Oversight

Route of delivery

Immunosuppressant

therapy

Number of cells

Nb of pts

tot 139

Patients (inclusion criteria)

Outcome Refs

Autologous BM MSCs

I National Institute of

HealthRegional and

Local Ethic Committees

Intraspinaltransplantation

(T4-T6) ( T7-T9)

no Mean:57×106 (Range: 7.0×106–152×106)

19 Age 20-75FVC>50%Different

Functional impairmentDuration of

the disease:8-80 m

Safe and well-tolerated even in

long-term (9 years)

Mazzini et al. 2003,2006;2008, 2010 2012

Autologous Bone marrow (BM)-derived hematopoietic progenitors

Open single arm

phase Itrial

ClinicalTrials Ethics

Committee of the University.

Agencia Española de Medicament

os.

Intraspinal(T3-T4)

no 2 mL mononucleate

d cells

11 Age:33-61FVC>50%

Spinal onset

Safe and well tolerated (2yrs follow-up)

Blanquer Blanquer et al, 2012

Human spinal cord-derived stem cells (HSSC)

Phase I trial

University Institutional

review board

Intraspinal(lumbar

spinal cord)

Basiliximab Prednisolone

TacrolimusMycophenolate

5-10 injections

100,000 cells/ injection

12 Age >18 yrsALSFRS-R lower extremity subscore < 1 ( Group A)and >2 (Groups B,C)FVC > 60%

Safe and well tolerated.

Glass et al., 2012

Fetal olfactory ensheathing cells

Controlled pilot study

in accordance with

guidelinesissued by the

Chinese Ministry of

Health

Bilateral corona radiata

no 2x106 15 Age:Range:20-70

SafeThe mean ALSFRS

score remained stable

in the first 4 months

Huang et al.2008

Autologous blood purifyed CD133(+)

Single-center

pilot trial

Ethic and Research

committees of the hospital.

Frontal motor cortex

no 2,5-7,5x105 10 Age 38-62Duration of the disease Range:18-42months

Safe and well-tolerated (1 year

follow-up).Patients survival

significantly higher than

control group.ALS-FRS

improvement

Martinez et al., 2009

Stem cells Studyphase

RegulatoryOversight

Route of delivery

Immunosuppressant therapy

Number of cells

Nb of ptsTot 139

Patients (inclusion criteria)

Outcome Refs

Autologous Bone marrow (BM)-derived hematopoietic progenitors

Single-center

pilot trial

Regional Ethicsboard

Ministry of Health

Institutional

Intraspinal(C3-C4level)

CSFIV

no 4x106

15x106

5x106

13 Age 34-71Duration of the

disease Range:2-5yrs

Moderate-severe

No complications

Some degree of decline 1 year

after transplantation

Deda et al, 2009

AutologousHSCS

Single-center

pilot trial

institutional review board

Intravenous infusion following

total body irradiation;

immuno-suppression

Tacrolimusmethotrexate

= 6 Age:Range:35-69

FVC>60%Duration of the disease: Range:

5-30months

Tolerated.No clinical benefits.HSCTs enter the human CNS at sites of motoneuron and engraft as immunomodulatory cells.

Appel et al., 2008

AllogenicPBSCs

Pilot EthicsReview Board

of the University

Mobilization of autologous PBSC

with GCSF

no 3.3 2.0 (Range 1.5–

7.6)CD34 cells

(x10E6)/kg

826

Spinal onsetFVC>50%3m-4yrs

No adverse effects

Cashman et al.

2008

AllogenicPBSCs

Open-label,pilot study with a

phase I–II design

Ethics committees of

the participating

centers.

Mobilization of autologous PBSC

with GCSF

no WBC: (Range 40,290 - 43,425CD34 cells41 - 57.

Age:40–64Disease duration

<12 monthsModerate disabilityFVC>80%

Few and transitory adverse eventsNo significant reduction of decrease in ALSFRS-Rand FVC

Chiò et al,2011

Autologous BM MSCS

Phase 1/2 open-safety

clinical trial

Ethics committees of the hospitals.

Registeredin the

National Institutes of

Health database

Intrathecally and intravenously

no 54.7106 CSF24.5106 iv

19 Age 25-65 Feasible and safe .

Immediate immunomodulatory effects.

Karussis et al , 2010

How to get cells where they are needed?

The use of stem cells for therapy requires that they can easily access the target tissue to exert their therapeutic effect as the cells respond

to a particular pathological microenvironment. The proximity of grafted cells favours the diffusion

of trophic and immunomodulatory factors to MNs and surrounding glia.

Lumbar Transplantation

Lower Limbs

Cervical Transplantation

Upper LimbsDiaphram

Local propagation

Kanouchi T et al. J Neurol Neurosurg Psychiatry doi:10.1136/jnnp-2011-301826

Onset and regional spread mechanisms

Micropump injection system supported by a table-fixed arm

Floating cannula

Stem Cells Dev. 2013 Feb 13.

Allogenic vs autologous stem cells transplantation

Autologous transplantation may obviate the need for immunosuppression and also may facilitate the authorization of clinical studies. However, autologous cells might be more vulnerable to the disease

Immunosuppression

Although the brain remains an immunologically privileged site due to the blood–brain barrier, there is evidence that this barrier can be compromised in disease

Studies of cell graft survival demonstrate that immunosuppression increases the survival of graft tissue

Patient -Specific Stem Cell Therapy

What do we expect the stem cells to do in ALS patients, and what outcomes are predicted?

• Stem cells are new products

• Pilot trials cannot be performed on normal, volunteering subjects.

• Surgical trials is a new scenario in ALS

• High iatrogenic risk also in the long-term

Phase 1 trials

Phase 1 clinical trials

Study objectives

• Optimisation of intervention, feasibility, and preliminary safety and tolerability

Points to consider

• What is the primary outcome?

• What are the secondary outcomes? Serious measures to collect data regarding several kinds

of side effects and data for general, unexpected adverse events have to be properly acquired in an effective manner

Phase 1 Clinical Trial (Inclusion Criteria)

The principal aim is to test the safety and feasibility Therefore, all the criteria adopted in the design of the studies are intended to ensure the greatest safety to the patient and minimize the possible iatrogenic damage.

• High Functional Impairment• Good Respiratory Function• Good Psychological Acceptance • Good Understanding of the Informed Consent

Informed consentInformed consent

How to measure safety

Cell transplants may survive for several years in patients, or their effects may be irreversible: therefore, stem cell therapy requires extended follow-up.

• Adverse events records (WHO severity scale )• Pre and post treatment course of the disease• Neuroradiological records• Post-mortem studies

No correlation was found with the number of the injected cells or the injection’site

Adverse Events (WHO Grade I-II)

2012 Mar 13.

Trapianto intramidollare di cellule staminali neuronali umane come terapia putativa per la SLA: proposta di un trial clinico di fase I

EudraCT: 2009-014484-39

Eventi avversi minori

Adverse event Nb of patients Mean duration (days)

OMS Grade

Pain 4/6 (70%) 4 (Range 2-6) II

Pneumonia 1 7 II

How to measure safety

Cell transplants may survive for several years in patients, or their effects may be irreversible: therefore, stem cell therapy requires extended follow-up.

• Adverse events records (WHO severity scale )• Pre and post treatment course of the disease• Neuroradiological records• Post-mortem studies

Neurotrophic Bone Marrow Cellular Nests Prevent Spinal Motoneuron Degeneration in Amyotrophic Lateral Sclerosis Patients: A Pilot Safety Study

Miguel Blanquer Blanquer , Jose M. Moraleda Jiménez , Francisca Iniesta Martínez et al

2012 Mar 13. [Epub ahead of print]

How to measure safety

Cell transplants may survive for several years in patients, or their effects may be irreversible: therefore, stem cell therapy requires extended follow-up.

• Adverse events records (WHO severity scale )• Pre and post treatment course of the disease• Neuroradiological records• Post-mortem studies

MRI follow-upbeforebefore 15 days15 days 3 m3 m 6 m6 m 9 m9 m 12 m12 m 18 m18 m

MRI (9 years after surgery)

T2-weighted MRIT1-weighted MRIT2-weighted MRI

DTI Tractography

How to measure safety

Cell transplants may survive for several years in patients, or their effects may be irreversible: therefore, stem cell therapy requires extended follow-up.

• Adverse events records (WHO severity scale )• Pre and post treatment course of the disease• Neuroradiological records• Post-mortem studies

T1 T2-T3 T3-T4-T5

2012 Mar 13. [Epub ahead of print]

Neurotrophic Bone Marrow Cellular Nests Prevent Spinal Motoneuron Degeneration in Amyotrophic Lateral Sclerosis Patients: A Pilot Safety Study

Miguel Blanquer Blanquer , Jose M. Moraleda Jiménez , Francisca Iniesta Martínez et al

Post-mortem studies

Are there sufficient data regarding the following to justify phase II/III clinical trials: safety, feasibility, dose, delivery, and

participant numbers?

• Stem cells research and application is opening great opportunities in ALS treatment. The scientific community and patients urgently need safety and efficacy to be addressed properly in the framework of rigorous controlled clinical trials.

• Numerous clinics around the world are exploiting patients’ hopes by offering expensive, new and effective stem cell therapies for ALS patients, without credible scientific rationale, transparency, oversight, or patient protection. Recruitment and selection of appropriate patients for larger trials will be a challenge and will require national and/or international multi-center collaboration with multidisciplinary groups.

• Translation, by which we mean advancing scientific discoveries from the laboratory into practical applications for patient benefit, i.e., ‘‘bench to bedside,’’ requires a comprehensive collaborative team approach: research scientists and clinicians must work closely with regulatory agencies, patient advocacy groups, ethic bodies, cell manufacturing facilities, and industry to achieve the quality of studies and necessary funding to ensure success.

FONDAZIONE CAVALIERI OTTOLENGHI

University of TorinoUniversity of TorinoNeuroscience InstituteNeuroscience Institute

Alessandro VercelliAlessandro Vercelli

Dpt of Biotechnologies and Bioscience

Università Milano Bicocca “Casa Sollievo della Sofferenza”Hospital

San Giovanni RotondoAngelo Vescovi

Dpt of Experimental Dpt of Experimental NeurosurgeryNeurosurgery

““E. Geuna”, NovaraE. Geuna”, NovaraGabriele PanzarasaGabriele Panzarasa

Dpt of NeurosurgeryDpt of NeurosurgeryS Giovanni Bosco S Giovanni Bosco

H,TorinoH,TorinoGiuseppe OliveriGiuseppe Oliveri

Stem Cell Transplantation and Cellular Stem Cell Transplantation and Cellular Therapy Unit; Therapy Unit;

Pediatric Onco-Hematology DivisionPediatric Onco-Hematology Division Regina Margherita Children’s

HospitalTorinoFranca Fagioli

ALS Center “Maggiore della Carità” Hospital

University of NovaraLetizia Mazzini

Thank you for your attention