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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.

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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

KC

-θ

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

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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?

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

Blank Agent1/FITC-IgG CPPM/FITC-IgG

Comparison between commercial agent and CPPM

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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

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§  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

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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  

Human  CD4

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  

Human  CD8

 

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 30 400

20

40

60

80

100BlankRottlerinP13D5/α-pPKC-θ

Days

Surv

ival

(%)

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

24h

48h

72h

0

2000

4000

6000

8000

10000

12000

**

*

*

Hours after Treatment

CD

25 (M

FI)

ns nsns

NOTCH1IC-MFI

24h

48h

72h

0

1000

2000

3000

4000

*

**

Hours after Treatment

NO

TCH

1IC (M

FI)

ns nsns

CD69-MFI (24 h)

Unstimulated

DMSO

Rottlerin

CPPM/α-pPKC-θ Treatment

0

200

400

600

800

1000*

CD

69 (M

FI)

**

T-BET-MFI

24h

48h

72h

0

500

1000

1500

2000

***

**

Hours after Treatment

T-B

ET (M

FI)

ns ns ns

IFN-γ

24h

48h

72h

0

10000

20000

30000

40000

*

ns

***

Hours after Treatment

IFN

-γ c

once

ntra

tion

(pg/

mL)

nsns ns

IL-2

24h

48h

72h

0

1000

2000

3000

4000

5000

**

ns *

Hours after Treatment

IL-2

Con

cent

ratio

n (p

g/m

l)

n/a ns ns

0

20

40

60

80

100ns

*

Cel

lula

r via

bilit

y (%

)

CD25%

24h

48h

72h

0

20

40

60

80

100

*

***

nsns ns

UnstimulatedDMSORottlerinCPPM/α-pPKC-θ Treatment

Hours after Treatment

CD

25+ %

Cel

ls

CD25%

24h

48h

72h

0

20

40

60

80

100

*

***

nsns ns

UnstimulatedDMSORottlerinCPPM/α-pPKC-θ Treatment

Hours after Treatment

CD

25+ %

Cel

ls