Prof. Giacomo P. Comi Dino Ferrari Center Department of Neurological Sciences University of Milan, Italy

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1 Prof. Giacomo P. Comi Dino Ferrari Center Department of Neurological Sciences University of Milan, Italy 2 Convegno AriSLA Nuove prospettive di ricerca. Per un futuro senza SLA!

2 ALS

3 Advances in ALS Pathogenesis: identification of new genes in familial ALS (FALS) C9ORF72: chromosome 9 open reading frame 72 UBQLN2 TDP-43: Transactive Response DNA- Binding Protein 43 FUS/TLS: Fused in Sarcoma/Translocated in Liposarcoma Optineurin Spatacsin DAAO D-Amino Acid Oxidase Gene CHMP2B: charged multivesicular body protein 2B gene PON: Paraoxonase Gene Cluster ATXN2: Ataxin-2

4 Therapeutic approaches Transfer of genes Culture Migration Survival Cell therapy Gene therapy Exon skipping Readthrough Ribozyme/ antisense Pharmacology Screening Mechanism of action Optimization Production Biodistribuition/Safety Targeting Immunology Routes of administration Functional evaluation

5 Stem cell therapy for ALS

6 We tested whether treatment of superoxide dismutase (SOD1)-G93A transgenic mouse, a model of ALS, with a neural stem cell subpopulation double positive for Lewis X and the chemokine receptor CXCR4 (LeX1+CXCR4+) can modify the disease s progression LeX RA+Shh+ Heparin+GFs HB9-GFP mice Neurospheres NSCs Cholinergic precursors Rotarod test Survival Engraftment into SOD1 spinal cord Corti et al., Brain 2007

7 Neural stem cell transplantation in ALS models 2006: human neural stem cells grafts ameliorate motor neuron disease in transgenic ALS rat (Xu et al.) 2007, 2009: human neural stem cells grafted in ALS rat differentiate and integrate into the segmental motor circuitry (Xu et al., Yan et al.) 2010: neural stem cells administered intravenously in a rat model of ALS differentiate and survive (Mitrecic et al.) 2011: Dual transplantation of human neural stem cells into cervical and lumbar cord of ALS rat ameliorates motor neuron disease (Xu et al.)

8 Phase I trial (safety trial) of spinal cord derived neural stem cells for patients with ALS Sponsor: Neuralstem, Inc. Center: Emory ALS center (Emory University in Atlanta), Prof. J. Glass FDA approved in September five-to-ten stem cell injections in the lumbar area Study duration: 24 months; 18 patients Treated up to now 12 patients: 3 unilateral (Group 1A), 3 bilateral (Group 2A): A= severe disease, 3 unilateral ambulatory (B), 3 bilateral ambulatory group (C) Next Group D: Eligibility Criteria Group D 1) Confirmed diagnosis of ALS by a neurologist 2)) Vital capacity greater than 60% of predicted value seated; greater than 50% measured flat on your back 3) Difficulty walking due to ALS 4) Lack of complicating medical conditions 5) Live in geographic proximity to Emory University Hospital 6) Ability to communicate vocally or with low-tech tools (writing or letter board) Updated 7 June 2011

9 Stem cell therapies ReN001 Nature Biotechnology, February 2011

10 Nat Neurosci, Lepore 2008 GRP transplantation extended survival, attenuated MNs loss and reduced microgliosis in SOD1G93A mice Human molecular genetics, 2011 C-Kit+ cell transplantation prolonged lifespan and promoted MNs survival of in SOD1 mice

11 NSCs MNs

12 Transplantation+cAMP GDNF and Rolipram treatment

13 Reprogrammed adult stem cells (ips) as a cell source of human NSCs and motor neurons

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16 16 human ipsc lines from 7 individuals of both sexes (5 healthy control and 2 ALS) ipsc and ESC lines (19/22) generated cells with a neuronal morphology and immunopositive for TUJ1, ISL, HB9 and ChAT ESC- and ipsc- derived neurons were physiologically active (electrophysiological studies)

17 ips derived neural stem cells for ALS (ipsals) Project aim We aim at exploring a possible therapeutic strategy for ALS that employs human NSC from ipscs, by testing the therapeutic efficacy and the molecular mechanisms linked to ipsc derived-nsc transplantation into ALS mice models (SOD1G93A and Prnp- TARDBP*A315T mice)

18 Specific aims 1) To determine the in vitro protocol for NSCs differentiation and isolation from human ipscs; 2) To establish cell transplantation protocols in the mice model of ALS; 3) To assess the engraftment and therapeutic efficacies of cell transplantation in the transgenic mouse models of ALS; 4) To characterize the mechanisms involved in the expected benefits of stem cell transplantation in these models; 5) To generate ipscs from ALS patients to create an in vitro experimental model of ALS for disease modeling, drug discovery, and eventually autologous cell replacement therapies.

19 ips production We generated ipsc from wild-type fibroblasts with a non viral method We transfected cells (nucleofection) with non integrating vectors carrying 6 reprogramming factors We differentiated obtained ips through a motor neuronal differentiation protocol

20 ips characterization

21 ips: pluripotency in vitro and in vivo ectoderm Teratoma ips mesoderm endoderm

22 Gene expression analysis PEARSON CORRELATION ANALYSIS HEAT MAP ANALYSIS ipscs vs PARENTAL FIBROBLAST MULTISCALE BOOTSTRAP RESAMPLING

23 Motor neuronal differentiation Nestin SOX1 TuJ1 MAP2

24 AIM1: Establishing the in vitro protocol for NSCs isolation Using FACS, we will isolate a subpopulation of ALDHhiSSClo from ips cell lines differentiated in neuroectoderm. We will investigate the cells clonogenic and differentiative properties for the three neuroectodermal lineages. The hnscs from ipscs will be selected based on their high aldehyde dehydrogenase activity and low side-scatter in FACS analysis (ALDHhiSSClo) NSCs-iPScs

25 AIM1: Motor neuronal differentiation

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27 AIM2: Characterization of several aspects of cell transplantation protocols ips i) Define most effective cell type: unselected NSCs vs ALDHhiSSClo fraction ii) Dose/effect: optimal number of cells Intratechal injection+immunosuppression SOD1G93A mouse hnsc Prnp- TARDBP*A315T mouse

28 Mouse Models SOD1(G93A) Nervous system phenotype: decreased motor neuron number motor neuron degeneration, astrocytosis Behavior/neurological phenotype: abnormal motor capabilities/coordination/ movement decreased grip strength, paralysis Muscle phenotype: decreased muscle tetanic force, skeletal muscle atrophy hindlimb weakness from 4 months of age severe muscle weakness beyond 3 months of age TDP43A315T Mortality: survival curve showed an average survival of 153 days no mice have survived beyond 240 days Phenotype: mice appeared normal up to 3 months of age gait abnormality by approximately 3 4 months of age losing weight and developed a characteristic swimming gait by 4/5 months of age unable to hold their body off the ground

29 AIM3: Assessing the engraftment and therapeutic efficacy of transplanted cells Examine the acquired phenotype of donor cells in the host parenchyma Neuropathological analysis of host tissue Monitor the neuromuscular function and lifespan of treated and control mice

30 GFP NeuN Merge

31 AIM4: Characterization of mechanisms involved in stem cell transplantation outcomes Gene expression profiling using microarray and RT-PCR analysis of whole spinal cord and laser-dissected motor neurons GeneChip Mouse Exon 1.0 ST Array: gene expression and alternative splicing, without extra costs The data will be validated by proteomic (immunocytochemistry and Western blot) Laser capture microdissection (LCM) of motoneurons; Corti et al., 2008

32 AIM4: Characterization of mechanisms involved in stem cell transplantation outcomes Analysis of transplanted cells capacity to release neurotrophins and growth factors (Luminex and ELISA) Evaluation of axon lengths, axonal elongation and axon number Cell co-culture of NSC and murine and human ALS motor neuron (Non-autonomous cell death, organization, mitochondrial function, protein aggregates)

33 SOD1-G93A astrocytes are toxic for ips-derived motor neurons Corti et al., 2011

34 AIM5: Induction of pluripotent stem cells from ALS patient 1- Create patient and disease-specific stem cell lines (ALS ) 2- Generate NSC, neurons, motor neurons, and other relevant neuroectodermal cell populations 3- In vitro and in vivo use as described for WT

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36 Genetics and Biochemistry Lab, Dino Ferrari Center Department of Neurological Sciences, University of Milan Nereo Bresolin Neural Stem Lab Giacomo P Comi Stefania Corti F. Magri M. Nizzardo M. Ranieri S. Salani A. Govoni C. Donadoni F. Fortunato M. Nardini A. Bordoni C. Simone R. Del Bo M. Falcone S. Lucchiari G. Riboldi D. Ronchi F. Rizzo D. Saccomanno S. Pagliarani G. Ulzi D. Piga