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Terapia cellulare e genica per l’Emofilia Antonia Follenzi, MD, PhD Torino, 25 Novembre 2017 Dipartimento di Scienze della Salute Different degrees : mild (5-40% FVIII), moderate (1-5% FVIII) and severe (<1% FVIII) Treatment with plasma-derived or recombinant FVIII No definitive cure Op#mal target for cell and gene therapy approach

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  • Terapia cellulare e genica per lEmofilia

    Antonia Follenzi, MD, PhD Torino, 25 Novembre 2017

    Dipartimento di Scienze della Salute

    Different degrees : mild (5-40% FVIII), moderate (1-5% FVIII) and severe (

  • PureGeneTherapyApproach

    Successful HB gene therapy byt a r g e # n g F I X e x p r e s s i o n t ohepatocytesusingAAV

    FVIIIcanbetoolargeforAAVbutnewclinicaLtrialsarecoming

    Deve l opmen t o f an# - FV I I Ineutralizingan#bodies LV alterna#ve approaches bytarge#ng transgene expression tospecificcellsotherthanhepatocytes.

    Combininggeneandcelltherapycouldallowtodevelopaneffec#veandsafetreatmentforHA

    TereseWinslow2001

    JCI, 2008

    DAPI FVIII

    CD146 MERGE

    LSEC

    Haematologica 2015

    Intraperitonealinjec#on

    InNSG-HAmice107Hepatocites

    Cytodex3microcarriers

    107NPC

  • Tools to Optimize Cell-type Specific Transgene Expression

    Hepatocytes

    Space of Disse

    DC

    SC

    SC

    Hepatocytes

    Space of Disse

    miRNA complementary sequence (miRT):

    Promoter Transgene Promoter Transgene

    Tissue-specific promoter:

    ICAM2 Flk1 Tie2 VEC

    Alb TTR hAAT

    CD11b

    CD11c

    miR-T122

    miR-T126

    miR-T142-3p

    I

    I

    I

    II miR-T155

    CD105

    Endothelial-specific cell surface targeting

    HBVE

    Hepatocyte-specific envelope

    GP64

    KC LSEC

    KC

    van Golen R.F. et al 2012

    5

    Target Cells for HA Gene Therapy

    Blood, 2012

    Haematologica, 2015

    Liver is the main FVIII source of the body

    I n p a s t h e p a t o c y t e s w e r e cons idered the ma in FVI I I -producing cells

    Liver sinusoids unique anatomy can facilitate direct or indirect priming of lymphocytes and contr ibute to some of the immunological properties of the organ (e.g., induction of antigen-specific tolerance)

    Journ Thromb Haemost, 2013

    Blood, 2016

  • Aim

    To identify the best combination of cell-specific promoter and miRT for transgene expression in liver sinusoidal endothelial cells (LSECs)

    7

    8

    Targeting FVIII Expression in Endothelial Cells

    BDDhFVIIIVEC

    122 142

    BDDhFVIIIVEC

    Plasma dilution 1:2000

    Immuniza#on20ginIFA

    Immuniza#on20ginIFA

    109TU

    (n=5) (n=9)

    (n=5) (n=9)

  • 0

    0.1

    0.2

    0.3

    0.4

    0.5

    0

    2

    4

    6

    8

    10

    0 1 2 4 8 12 16 20 24 30 36 40 48 52 anti-

    FVIII

    IgG

    (OD

    450n

    m)

    % o

    f hFV

    III a

    ctiv

    ity

    Time after LV injection (weeks)

    LV.VEC-FVIII-122-142 aPTT Abs

    0

    0.1

    0.2

    0.3

    0.4

    0.5

    0

    2

    4

    6

    8

    10

    0 1 2 4 8 12 16 20 24 30 36 40 48 52 anti-

    FVIII

    IgG

    (OD

    450n

    m)

    % o

    f hFV

    III a

    ctiv

    ity

    Time after LV injection (weeks)

    LV.VEC-FVIII aPTT Abs

    hBDD-FVIII Activity of LV-VEC-FVIII 122-142 in Previously Immunized Mice

    20ghFVIII 109TU

    VEC-hFVIII 122-142 (n=3)

    Rechallenge4IUtailveinRechallenge4IUtailvein

    4w -FVIIIAbsproduc#on-FVIIIAbsproduc#on

    Rechallenge4IUtailvein

    Rechallenge4IUtailvein

    Rechallenge4IUtailvein Rechallenge4IUtailvein

    LSEC-induced Tregs Allow Tolerance to FVIII

    10

    Tregsdeple#onTregs

    deple#on

    109 TU (n=4-5)

    -CD25

    Tregsdeple#on

    Tregsdeple#on

    Tregsdeple#on

    Tregdeple#on(n=3)

    109TU5d

  • 0

    0.2

    0.4

    0.6

    0.8

    1

    2 4 8 12 16 20 24 28 36 40 48 52 Ctr+

    anti-

    FVIII

    IgG

    (OD

    450n

    m)

    Time after LV injection (weeks)

    VEC-hFVIII VEC-hFVIII-122-142 VEC-co-hFVIII-122-142 VEC-RH HA

    0 2 4 6 8

    10 12 14

    2 4 8 12 16 20 24 28 36 40 48 52

    % h

    FVIII

    act

    ivity

    Time after LV injection (weeks)

    VEC-hFVIII VEC-hFVIII-122-142 VEC-co-hFVIII-122-142 HA VEC-RH

    Engineered FVIII Forms

    Immuniza#on20ginIFA

    Immuniza#on20ginIFA

    BDDhFVIIIVEC

    122 142

    BDDhFVIIIVEC

    hFVIII.RHVEC

    122 142

    co-hFVIIIVEC

    109TU

    n=3-5

    24 weeks

    The presence of endothelial or myeloid specificpromoter with specific miRTs in LV were able to restrict transgene expression in cell-types capable of efficient and long term FVIII-expression without anti-FVIII antibodies formation Our data demonstrate a role for Tregs in establishing tolerance to FVIII during LV gene expression under the control of VEC promoter Endothelial-specific expression of FVIII-RH and codon-optimized FVIII results in higher FVIII activity without antibody formation

    12

    FVIII expression by liver sinusoidal cells may provide cellular models to acheive antigen-specific tolerance in

    gene transfer approaches reaching phenotipic correction in several hemophilia A mouse strains

    Conclusion I

    Merlin et al., Molecular Therapy 2017

  • LuciferaseassayshowedpF8ac#vityinhepa#ccelllinesStudyofTFthatbindpF8(inA-Eregions)iden#fiedCEBP/HNF3andHNF1byEMSA

    Role of Liver-Enriched Transcription factor HNF1 in transcriptional regilation of the FVIII gene

    McGlynn, Mueller, Begbie, Notley, and Lillicrap

    Mol Cell Biol, 16(5), 1936-1945 (1996)

    -Tocharacterizeinvitrotheac#vityoftwoFVIIIpromotersequences-TocharacterizeinvivoandexvivothecelltypesinwhichFVIIIpromoterisac#ve

    -Toinves#gatephenotypiccorrec#onofhemophiliaAmiceaeergenetherapyusingalen#viralvectorcarryingtheFVIIIunderthecontrolofFVIIIpromoter

  • h=p://alggen.lsi.upc.edu/recerca/menu_recerca.html

    Transcriptional Factor Expression and function

    TFII-D (7 nt) RNA Pol II

    TBP (10 nt) TATA binding protein

    HNF3-alfa (8 nt) Hepatocytes

    HNF1-alfa (8 nt) Hepatocytes

    C/EBP-alfa (7 nt) Hepatocytes, mieloyd differentiation

    c-Ets-1 (7 nt) Endothelial cells

    c-Ets-2 (9 nt) Endothelial cells

    PEA 3 (9 nt) c-Ets family

    STAT4 (6 nt) Mieloyd lineage

    GATA-1 (6 nt) Mieloyd lineage

    NF-Y (8 nt) Increasing during monocytes-macrophages differentiation

    IRF-2 (6 nt) Monocytes

    STAT1 (10 nt) Hematopoietic cells

    TCF-4E (10 nt) B cells

    Pax5 (7 nt) B cells

    NF-AT1 (10 nt) T cells

    Fox P3 (7 nt) T regulatory cells

    LEF-1 (8 nt) Pre B pre T cells

    Dissimilaritymarginlessthan5%

    Fominetal.,2014

  • INTRODUCTION to BOECs

    Blood outgrowth endothelial cells (BOECs) belong to the family of endothelial progenitors and they are generated from circulating endothelial progenitors found in adult peripheral blood

    BOECs are self-renewing, clonogenic, able to form capillary-like structures and integrate into functional blood vessels both in vitro and in vivo

    They are a valuable source of cells to understand endothelial cell biology, to perform disease modeling and they can be a substrate for the generation of induced pluripotent stem cells (iPSCs)

    BOECs might represent a good target for gene delivery by lentiviral vector (LV) to cure HA

    AIMDevelopment of tools and technologies for a novel ex vivo cell-based therapy to treat HA

    Cellisola#on LVtransduc#on Cellexpansion

    Safetyandcellcharacteriza#onPre-clinicalstudies

    UKW-Germany UPO-Italy UNILO-UK

    UNILO-UKSERC-Canada

  • IN VIVO Non-transduced or transduced BOECs (107) were transplanted intraperitoneally in NSG-HA mice in association with microcarrier beads

    NSG HA HA BOECs: Only beads, n=2 Only HA BOECs, n=2 LV.VEC-GFP MOI 20, n=2 LV.VEC-FVIII MOI 20, n=3

    Weekly aPTT assay End point: bleeding assay

    BDDhFVIIIVEC

    HA BOECs secreted FVIII in vivo up to 7 weeks after transplantation

    Only beads (n=2)

    LV.VEC-FVIII MOI 20 (n=3)

    aPTT assay on plasma obtained from HA BOECs-injected mice

    0

    2

    4

    6

    8

    10

    12

    14

    1w 2w 3w 5w 7w 1w 2w 3w 5w 7w 1w 2w 3w 5w 7w 1w 2w 3w 5w 7w 10w 13w

    FVIII

    act

    ivity

    (%)

    Only HA BOECs (n=2)

    LV.VEC-GFP MOI 20 (n=2)

  • NSG-HA mice ip injected with FVIII-corrected HA BOECs showed a blood loss similar to NSG control mice

    Bleeding assay performed on NSG-HA mice ip injected with LV.VEC-FVIII BOECs

    0

    0,5

    1

    1,5

    2

    2,5

    3

    A.U

    . (O

    D57

    0nm)

    NSG NSG-HA 7w

    HA BOECs LV.VEC-FVIII MOI 20

    13w

    BOECs were isolated and expanded by both healthy and hemophilic donors

    BOECs were efficiently transduced by LV carrying FVIII under the VEC promoter

    LV.VEC-FVIII transduced BOECs survived and secreted FVIIII up to 10 weeks in NSG-HA mice

    NEXT:

    Long term evaluation of BOEC tumorigenesis

    Transplantation of FVIII-corrected BOECs in a small implantable device (Cell PouchTM)

    CONCLUSIONS AND FUTURE PLAN

  • Thanks to UPO Simone Merlin, PhD Diego Zanolini, PhD Stefania E Cannizzo, PhD Rosella Fam, PhD Ester Borroni, PhD Chiara Borsotti, PhD Cristina Olgasi, PhD Kevin Bellofatto, PhD stud Valentina Bruscaggin, BSc Prof. Maria Prat, PhD Prof. Guido Valente, MD FREE UNIVERSITY OF BRUSSELS (VUB) Thierry VandenDriessche, PhD Marinee K. Chuah, PhD CHILDREN'S HOSPITAL OF PHILADELPHIA Valder R. Arruda, MD, PhD

    AKNOWLEDGMENT

    UniversityofPiemonteOrientaleHistologylab:Valen#naBruscagginSimoneMerlinCris#naOlgasiChiaraBorsok

    UniversityHospitalofWrzburgDepartmentofTissueEngineeringandRegenera#veMedicine:SarahFroschPatrickBinorfThorstenBergmannJorisBraspenning

    SernovaCorpKelceyPanersonDelfinaMazzucaPhilipToleikis

    LoughboroughUniversityCentreforBiologicalEngineering:NicholasWraggAndreaIeimia-ManderAdeoluAdewoyeAlexandraStolzing

    IntegrierteManagementSystemee.K.SamimAziziMar#nZierau

    ARTTICPatriziaTorremanteKathrinSunner