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CELL-PENETRATING PEPTIDES FOR PERSONALIZED MEDICINE Diaspora în cercetarea științifică și învățământul superior din România – Diaspora și prietenii 2016” - Workshop: "Perspective in medicina personalizata - de la concept la aplicatii clinice". Timisoara, 26-27 April, 2016 “Aurel Vlaicu” University, Elena Dragoi Str., Nr. 2, 310330 Arad, Romania Institute of Technology, Tartu University, Nooruse Str., Nr. 1, 504 11 Tartu, Estonia E-mail: [email protected] Dana Maria Copolovici

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CELL-PENETRATING PEPTIDES FOR

PERSONALIZED MEDICINE

”Diaspora în cercetarea științifică și învățământul superior din România – Diaspora și prietenii

2016” - Workshop: "Perspective in medicina personalizata - de la concept la aplicatii clinice".Timisoara, 26-27 April, 2016

“Aurel Vlaicu” University, Elena Dragoi Str., Nr. 2, 310330 Arad, Romania

Institute of Technology, Tartu University, Nooruse Str., Nr. 1, 504 11 Tartu, Estonia

E-mail: [email protected]

Dana Maria Copolovici

Summary

IntroductionAim of the StudyMaterials and MethodsDevelopment of PepFect peptidesDevelopment of NickFect peptidesDevelopment of gHoPe2 vector into the gliomaConclusionsAcknowledgments

Examples of nanoparticle therapeutics

A. Ediriwickrema and W. M, Saltzman, Nanotherapy for Cancer: Targeting and Multifunctionality in the Future of Cancer Therapies, ACS Biomater. Sci. Eng. 2015, 1, 64−78

- gene therapy- antisense technology- splice correction- RNA interference

Regulation of gene expression

Figure. A schematic overview of gene therapy and gene expression modulation approaches.

Introduction: Cell-Penetrating Peptides (CPPs)

► CPP-s: - 30 amino acid-long peptides– capable of transporting various cargo molecules – high cell internalization efficacy– easy to produce and to modify

Protein-derived Chimeric Synthetic

PenetratinTAT

pVECVP22

MPG transportan

TP10M918Pep-1

OligoarginineMAP

CADY

Cationic Amphipathic

PenetratinTAT

Oligoarginine

TransportanTP10MPGPep-1CADY

►Clasification of CPPs

Intracellular delivery of CPP-cargo complexes.

To obtain highly effective and not harmful cell-penetrating peptides

to deliver therapeutic agents (e.g. nucleic acids, chemical drugs, etc.) in vitro and in vivo.

Aim of the Study

Copolovici et al, ACSNano, 2014.

Uptake mechanisms of CPP or CPP-cargo complexes.

Aim of the Study

Copolovici et al, ACSNano, 2014.

► N-Fmoc solid peptide synthesis strategy, RP-HPLC purification, analytical-HPLC and MALDI-TOF mass spectrometry identification of the products

► non-covalent co-incubation approach to form stable peptide:nucleic acid nanocomplexes

►to obtain potential therapeutic agents by forming compounds with covalent bond between CPP and cargo

► splice correction assay on HeLa pLuc 705 cells

► pDNA delivery in vitro and in vivo

► to silence genes in vitro by using siRNA

► in vitro toxicity: cell proliferation assays

►animal experiments

Materials and Methods

Protocol of formation of non-covalent CPP-nucleic acid nanocomplexes

Stearyl: (CH3–(CH2)16–CO–)

Development of PepFect Peptides

Pooga et al., FASEB J. 1998, Soomets et al., 2000

Mäe et al., J. Control. Release., 2009

Oskolkov et al., Int. J. Pept. Res. Ther., 2011

AGYLLGKINLKALAALAKKIL-NH2galanin sequence mastoparan (wasp venom sequence)

Stearyl-AGYLLGKINLKALAALAKKIL-NH2

Transportan 10 (TP 10):

PepFect 3 = Stearyl-TP 10:

PF6-mediated siRNA delivery into reporter cells. (a) RNAi response in luc-HEK cells. (b) Dose-response in HepG2 cells. (c) Impact of cell confluence. Luc-U2OS cells seeded at indicated densities 1 day prior experiment were treated and analyzed using 50 nM siRNA with PF6, LF2000 or RNAiMAX. (d) Flow cytometry histogram analysis of EGFP RNAi response in EGFP-CHO cells at 48 h post treatment with 100 nM free EGFP siRNA (mock) or complexed with LF2000 or PF6. (e) Flow cytometry analysis of EGFP knockdown decay kinetics following single treatment with siRNA. Treatments were performed by using 50 nM siRNA.

Development of PepFect Peptides (PF 6)

El Andaloussi, Nucleic Acids Res., 2011.

Structures of NickFect peptides: NF1, NF2, NF3, NF11, NF51, NF53, NF61 (stearyl moiety is CH3–(CH2)16–CO–).

Development of NickFect peptides

Oskolkov et al., Int. J. Pept. Res. Ther., 2011. Ülo Langel, et al. EPO Priority number: EP11155275; Priority date: 22.02.2011, WO 2012113846.

Arukuusk et al, Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013.

Abbrev Sequence

NF1 Stearyl-AGY(PO3)LLGKTNLKALAALAKKIL-NH2

NF2Stearyl-AGYLLGKT(PO3)NLKALAALAKKIL-NH2

NF3 Stearyl-AGY(PO3)LLGKT(PO3)NLKALAALAKKIL-NH2

NF11 Stearyl-AGYLLGKTNLKALAALAKKIL-NH2

NickFects are chemically modified Stearyl-TP-10 peptides

Plasmid delivery (pGL3) mediated by NF1 (A) and NF2 (B) in CHO cells, and

NF1 (C) and NF2 (D) in MEF cells.

U

CR

1

CR

2

CR

3

CR

4LF20

00

10 3

10 4

10 5

10 6

10 7

10 8- serum+ serum

A

RL

U/m

g

U

CR

1

CR

2

CR

3

CR

4LF20

00

103

104

105

106

107

108- serum+ serum

B

RL

U/m

g

U

CR

1

CR

2

CR

3

CR

4LF20

00

103

104

105

106

107

108- serum+ serum

D

RLU

/mg

U

CR

1

CR

2

CR

3

CR

4LF20

00103

104

105

106

107

108- serum+ serum

C

RL

U/m

g

Development of NickFect peptidesNF 1: Stearyl-AGY(PO3)LLGKTNLKALAALAKKIL-NH2

NF 2: Stearyl-AGYLLGKT(PO3)NLKALAALAKKIL-NH2

Electron micrographs of the translocation and intracellular localization of ON–NG–NF complexes in HeLa pLuc 705 cells. Association of ON–NG–PF3 complexes as dense particles with cell surface (a) localization in endolysosomal structures (b). Interaction and translocation of ON–NG–NF1 particle into cell (c), localization diffusely in cytosol close to the cell nucleus (n) (d), and inside nucleus (arrows in e).Spherical particles of ON–NG–NF2 complexes associating with cell surface (f), localizing in vesicular structure (arrow in F), and freely in cytosol (g).

Oskolkov et al., Int. J. Pept. Res. Ther., 2011.

Development of NickFect peptides

Plasmid transfection efficacy of NickFects. a) NickFects and stearyl-TP10 mediated pDNA delivery in CHO cells in serum free medium. b–d) Delivery efficacy of NF51/pDNA nanoparticles in hard to transfect cell lines: MEF, Jurkat, A20. Concentration of peptides: 1.5 µM for CR2, 2.25 µM for CR3, 3 µM for CR4. Untreated cells (U) were used as negative control and Lipofectamin™ 2000 (LF2000) as a positive control.

Arukuusk et al, Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013.

Development of NickFect peptides

Arukuusk et al, Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013.

Development of NickFect peptides

Gene knockdown by NickFects/siRNA nanoparticles. RNAi response in EGFP-CHO cells measured by FACS analysis 48 h after single treatment with a) NickFects and 100 nM siRNA, molar ratio 10, controls Lipofectamine™ 2000and Lipofectamine™ RNAiMAX were used according to manufacturer's protocol and b) NF51/siRNA complexes, molar ratio 10, at different siRNA concentrations (12.5–100 nM).

drugCPP

homing peptidereceptor

cell

- fluorescent dye or

chemotherapeutic drugCargo

Tumor-targeting strategies

Names Sequence

pVEC LLIILRRRIRKQAHAHSK-NH2

gHo NHQQQNPHQPPM-NH2

FAM-pVEC aLLIILRRRIRKQAHAHSK-NH2

FAM-gHo aNHQQQNPHQPPM-NH2

FAM-gHoPe2 aLLIILRRRIRKQAHAHSKNHQQQNPHQPPM-NH2

FAM-gHoPe3 aNHQQQNPHQPPMLLIILRRRIRKQAHAHSK-NH2

Dox-gHoPe2 bXCLLIILRRRIRKQAHAHSKNHQQQNPHQPPM-NH2

a5(6)-carboxyfluoresceinyl modification at N-terminus; bdoxorubicin; X - succinimidyl-4-(Nmaleimidomethyl) cyclohexane-1-carboxylate (SMCC)

Development of gHoPe2 vector into the glioma

Eriste et al, Bioconj. Chem., 2013.

gHoPe2

gHo

pVEC

doxorubicin

FAM

FAM-gHoPe2

Dox-gHoPe2

Development of gHoPe2 vector into the glioma

Eriste et al, Bioconj. Chem., 2013.

Cellular internalization (a) and toxicity assay (b) on U87 glioma cells.

a)

b)

Development of gHoPe2 vector into the glioma

Eriste et al, Bioconj. Chem., 2013.

Localization of FAM-labeled gHo and gHoPe2 in subcutaneous U87 tumors. Labeled peptides were administered i.v. 3 h before tissue collection. Cryosections from the subcutaneous tumors, as well as from brain, kidney, and liver were counterstained with DAPI and visualized by fluorescent microscopy. gHoPe2 (green) localized in tumors. FAM and DAPI are merged in the images. Scalebar 100 µm.

Development of gHoPe2 vector into the glioma

U87 subcutaneous tumor growth dynamics after treatment with either free doxorubicin (i.v. 1 mg/kg twice a week) or with an equivalent molar dose of dox-gHoPe2 (treatment days indicated with arrows). Star represents Tukey posthoc comparison of treatment groups after significant repeated-measures ANOVA.

Development of gHoPe2 vector into the glioma

Eriste et al, Bioconj. Chem., 2013.

Coronal section of a mouse brain with U87 tumor in the right striatum. (A) A Nissl-stained reference section (Allen Institute, http://mouse.brain-map.org) of a normal mouse brain at the same level and size where tumor sections B and C are viewed. The respective tumor area is circled with a dotted line. The inlet shows the whole coronal section. (B) H&E stained hemisphere of brain and glioma, reconstructed from single microscopic images. The animals received an i.v. injection of FAM-labeled gHoPe2 3 h before tissue collection. (C) Fluorescence image from the same location (reconstructed from single fluorescence microscopic images), indicating global FAM localization in the tumor area.

Development of gHoPe2 vector into the glioma

Eriste et al, Bioconj. Chem., 2013.

Conclusions

New CPPs have been designed and synthesized and then used as delivery vectors of therapeutic agents (nucleic acids, chemical drugs).

► PepFects (PF3, PF6) are stearylated-peptides derived from TP10 which are able to: (a) deliver splice correction oligonucleotides in in vitro model and in vivo; (b) increase gene expression after efficient delivery of pDNA in several cell lines; (c) induce gene silencing.

► NickFects (NF1, NF2 and NF51) are chemically modified peptides derived from TP10 which are able to: (a) deliver splice correction oligonucleotides in in vitro model; (b) increase gene expression after efficient delivery of pDNA in several cell lines; (c) induce gene silencing by targeting endogenous mRNA.

►Dox-gHoPe2 showed effective targeted localization to the glioma demonstrating cytotoxic activity in vitro and anti-tumor activity in vivo. The efficacy of a chemotherapeutic drug conjugated to a CPP vector (Dox-gHoPe2) was demonstrated.

► PepFects and NickFects did not exhibited long-term toxicity.

► PepFect peptides and their CPP-cargo complexes did not showed toxicity and immunological response in vitro and in vivo.

► more studies have to be undertaken, especially in vivo experiments, in order to reveal the potential of the CPPs in pre-clinical and clinical studies in the future gene therapy, cancer applications, and personalized medicine.

Conclusions

Laboratory of microscopy(TEM/STEM, JEOL 2100HR), SEM-FIB-EDX (LYRA3,TESCAN), AFM-Raman (NT-MDT, Renishaw)

Institute of Technical and Natural Sciences Research-Development-Innovation of “Aurel Vlaicu” University, Aradcestn.uav.ro

Laboratory of chromatography (U-HPLC-MS-MS, GC-GC-MS with thermodesorbter, Shimadzu), X-Ray Diffractometer (Miniflex 600, Rigaku)

D.M. Copolovici was supported by a post-doctoral fellowship of the Estonian Science Foundation (MJD64 ).This study was supported by the EU through the European Regional Development Fund: Center of Excellence in Chemical Biology, Estonia; by the targeted financing SF0180027s08 and ETF 9438 grantsfrom the Estonian Ministry of Education and Research, and by Cepep Eesti OÜ.

Acknowledgments

Ülo Langel, Professor

Nikita Oskolkov, PhD

Elo Eriste, PhD

Kaido Kurrikoff, PhD

Piret Arukuusk, PhD

Julia Suhorutšenko, PhD

Kent Langel, PhD

Thank You for attention!