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Transcript of Polymer Poster Symposium MCB Retreat Spring · PDF file 2014-02-18 · Polymer...

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    α-pPKC-θ (T538) Delivery via Cell Penetrating Peptide Mimics as a Novel Treatment of Aplastic Anemia E. Ilker Ozay1, Gabriela Gonzalez-Perez2, Joe Torres2, Gregory N. Tew1,2,3, and Lisa M. Minter1,2,3

    1Molecular and Cellular Biology Graduate Program, 2Department of Veterinary and Animal Sciences, 3Department of Polymer Science and Engineering, UMass Amherst, Amherst, MA, 01003

    Polymer Poster Symposium

    MCB Retreat Spring 2014

    Aplastic Anemia (AA)

    Factory for all blood cells

    Bone marrow

    In aplastic anemia (bone marrow failure),

    §  Bleeding

    §  Infection

    Autoimmune disorder

    “Aberrant T helper-1 (Th1)

    lymphocytes”

    Immune-mediated

    destruction

    Th1

    Th1

    Th1

    Current Treatments

    §  Bone marrow transplantation

    §  Anti-thymocyte globulin administration

    §  Blood transfusion

    Molecular Signaling in Aberrant Th1 Cells

    IFN-γ

    T-BET

    IL-2

    NOTCH1IC

    PKC-θ

    §  Protein kinase C-θ(PKC-θ) gets phosphorylated in

    activated Th1 cells (Threonine 538 residue for full activation)

    Roderick et al., JEM 210, 1311-1329, 2013; Young, N.S. & Maciejewski, J., N Engl J Med 336, 1365-1372, 1997 1

    Rationale behind targeting PKC-θ

    •  Intact PKC-θ signaling is necessary to facilitate disease progression in the mouse model of aplastic anemia.

    •  The disease can be completely rescued using Rottlerin by inhibiting PKC-θ function in the mouse model of aplastic anemia.

    •  The viral immunity can still be protected in PKC-θ knockout mice.

    •  Current treatment for aplastic anemia includes Anti-thymocyte globulin administration for Th1 cells targeting surface markers. However, 30-40% of the cohorts do not respond to this therapy.

    Inhibits the phosphorylation

    of PKC-θ, thereby its activation

    Matsumoto et al., Immunity 23, 2005; Springael et al., Biochem Pharma, 2007; Solomou et al. Blood, 2006; Scheinberg et al., The Journal of Pediatrics 153, 2008.

    2

    Targeting PKC-θ:  Using  An*bodies  to  Inhibit  Its  Func*on

    PKC-θ IS A POTENTIAL THERAPEUTIC TARGET FOR BONE MARROW FAILURE

    TREATMENT.

    INHIBITING PKC-θ FUNCTION IS BENEFICIAL TO TURN DOWN

    ABERRANT TH1 CELL ACTIVITY IN APLASTIC ANEMIA.

    STRATEGY

    PKC-θ

    T538

    α-pPKC-θ

    PKC-θ

    T538

    α- pP

    K C

    Interfering with the function

    Cell Penetrating Peptides (CPPs)

    §  Peptides which have the ability to internalize a cargo into cells.

    HIV-1 TAT

    RKKRRQRRR!49-

    -57

    §  Guanidinium-rich domain

    §  Covalently attached to cargo

    §  Responsible for cellular uptake

    §  More efficient than other cationic CPPs

    §  Preferable to lysine

    Polyarginine

    PEP-1

    §  Better uptake

    §  Both hydrophobic and lysine-rich

    §  Also non-covalent interactions

    Sgolastra,F., deRonde, B.M, Sarapas, J.M., Som, A., Tew, G.N.; Accounts Chem. Res. 2013. ASAP; Stanzl et al., Accounts Chem. Res., 2013, ASAP;

    Kurzawa et al., Biochimica et Biophysica Acta 1798, 2010, 2274-2285; Morris et al., Nature Biotech. 19, 2001, 1173-1176

    3

    T-cell Master Key

    CPPM

    Inspired by;

    HIV-1 TAT,

    Pep-1,

    and Polyarginine

    Design of Cell Penetrating Peptide Mimics (CPPMs)

    CPPMs are able to enter and deliver a bioactive

    cargo to T cells

    Research Aim and Questions

    Forming a complex of CPPMs and α-pPKC-θ (T538) and delivering them into T cells (hPBMCs) to neutralize PKC-θ activity both in

    vitro and in vivo with the eventual goal of treating AA

    §  Can we deliver α-pPKC-θ (T538) by using our CPPM?

    §  Can cell-penetrating α-pPKC-θ (T538) neutralize the actions of PKC-θ in vitro?

    §  Can in vivo delivery of α-pPKC-θ (T538) attenuate immune- mediated bone marrow failure in a “humanized” mouse model?

    4

    Delivery of CPPM/α-pPKC-θ into T Cells

    Blank

    Agent1/FITC-IgG

    CPPM/FITC-IgG

    Comparison between commercial agent and CPPM

    5

    Population shifted to higher fluorescence with CPPM

    α-­‐pPKC-­‐θ  

    WITHOUT CPPM

    α-­‐pPKC-­‐θ  

    WITH CPPM

    Acknowledgments & Funding

    Lisa M. Minter

    Gregory N. Tew

    Barbara A. Osborne

    Tew Lab

    Federica Sgolastra

    Brittany M. deRonde

    Bob Fu

    Michael Lis

    Katie Gibney

    Cathy Walker

    Joel Sarapas

    Madhura Pawar

    Coralie Backlund

    Minter Lab

    Gabriela Gonzalez-Perez

    Christina Arieta Kuksin

    Joe Torres

    Karthik Chandiran

    Wesley Rossiter

    Victoria Mello

    Jessica Jarmolowicz

    Osborne Lab

    Rebecca Lawlor

    Anushka Dongre

    Furkan Ayaz

    Joseph Homsi

    Manit Munshi

    Charles H. Hood Foundation for Child Health Research

    12

    §  We are able to successfully deliver α-pPKC-θ (T538) via our CPPM into hPBMCs

    §  Our novel CPPM design has much better uptake of the antibody with compared to commercially available antibody delivery reagent

    §  We are able to interfere with the actions of PKC-θ by observing reduction of T cell immune response marker expressions

    §  AA model of mice treated with CPPM/α-pPKC-θ complex had longer survival compared to control mice

    §  In vitro and in vivo studies of α-pPKC-θ (T538) delivery into hPBMCs have promising results for the treatment of aplastic anemia for clinical studies

    Conclusions

    11

    In Vivo Humanized Mouse Model of Aplastic Anemia

    10

    9

    6

    7

    8

    In Vivo Humanized Mouse Model of Aplastic Anemia

    NSG (NODscidIL2Rγcnull)

    female mice

    CPPM treated human PBMCs

    Rest mice for 4 hours

    Human PBMCs

    CPPM/

    α-pPKC-θ

    Wait for

    17 days

    γIR

    CPPM treated mice lived LONGER (up to 36 days)

    Gabriela Gonzalez-Perez & Joe Torres

    41%  

    Mouse  CD45  

    Hu m an  C D4

    5  

    86%   92%  

    56%   88%   94%  

    Bone  Marrow   Spleen   Peripheral  Blood   DMSO  treated  hPBMCs:  

    Bone  Marrow   Spleen   Peripheral  Blood   CPPM/α-­‐pPKC-­‐θ  treated  hPBMCs:  

    11%  

    Human  CD4  

    Hu m an  C D8

     

    9%   20%  

    18%   15%   25%  

    Bone  Marrow   Spleen   Peripheral  Blood   DMSO  treated  hPBMCs:  

    Bone  Marrow   Spleen   Peripheral  Blood   CPPM/α-­‐pPKC-­‐θ  treated  hPBMCs:  

    54%   27%   70%  

    53%   28%   63%  

    Engraftment of Human CD45 Cells in Peripheral Tissues

    Infiltration of Human CD4 and CD8 T Cells in Peripheral Tissues

    0 10 20 30 40 0

    20

    40

    60

    80

    100 Blank Rottlerin P13D5/α-pPKC-θ

    Days

    Su rv

    iv al

    (% )

    DMSO

    Rottlerin

    CPPM/α-pPKC-θ

    CPPM treated Humanized

    Mouse Model of AA

    SURVIVAL  STUDY  

    Gabriela Gonzalez-Perez & Joe Torres

    T-cell Activation and Disease Markers

    In Vitro Experimental Design

    Cytokine Expression

    CELLULAR VIABILITY

    AFTER 24 H OF TREATMENT

    CD25-MFI

    24 h

    48 h

    72 h

    0

    2000

    4000

    6000

    8000

    10000

    12000

    **

    *

    *

    Hours after Treatment

    C D

    25 (M

    FI )

    ns ns ns

    NOTCH1IC-MFI

    24 h

    48 h

    72 h

    0

    1000

    2000

    3000

    4000

    *

    * *

    Hours after Treatment

    N O

    TC H

    1I C

    (M FI

    )

    ns nsns

    CD69-MFI (24 h)

    Un sti mu lat ed

    DM SO

    Ro ttle rin

    CP PM /α- pP KC -θ Tre atm en t

    0

    200

    400

    600

    800

    1000 *

    C D

    69 (M

    FI )

    **

    T-BET-MFI

    24 h

    48 h

    72 h

    0

    500

    1000

    1500

    2000

    ***

    * *

    Hours after Treatment

    T- B

    ET (M

    FI )

    ns ns ns

    IFN-γ

    24 h

    48 h

    72 h

    0

    10000

    20000

    30000

    40000

    *

    ns

    ***

    Hours after Treatment

    IF N

    -γ c

    on ce

    nt ra

    tio n

    (p g/

    m L)

    nsns ns

    IL-2

    24 h

    48 h

    72 h

    0

    1000

    2000

    3000

    4000

    5000

    **

    ns *

    Hours after Treatment

    IL -2

    C on

    ce nt