UNIVERSITÀ DEGLI STUDI DI PERUGIADipartimento di Scienze Farmaceutiche Corso di Laurea Magistrale in Farmacia

UNIVERSITY OF VIENNADepartment of Medicinal Chemistry

Gene silencing of a receptor-targeted bioconjugate with enhanced siRNA loading capacity

Laureanda Relatori

Marta Marinucci Prof. Antimo Gioiello Dr. Johannes Winkler

Anno Accademico 2014-2015

RNAi is a fundamental pathway in eukaryotic cells by which a sequence-specific RNA molecule, named small interfering RNA (siRNA) is able to target and cleave complementary mRNA, thus causing selective gene expression inhibition [1]

RNA interference and gene silencing

RNAi is fundamental for: • Cell growth• Tissue differentiation• Heterochromatin formation• Cell proliferation

siRNAs are double-stranded molecules of RNA with 3’ overhangs at each end that can “interfere” with proteins translation [2]

1: Elbashir S.M. et al. (2001). Nature 411, 494-498 2: Dornseifer S. et al. (2015). Nucleic Acid Res. 43(22), 10623-10632

RNAi pathways and mechanisms

• Molecules of double-stranded RNA (dsRNA) are cleaved by the enzyme Dicer into siRNA

• siRNA is incorporated into RISC (RNA-induced silencing complex), that includes Argonaut 2

• Argonaut 2 unreels siRNA and the sense strand is cleaved

• Activated RISC, with the antisense strand of siRNA, selectively degrades mRNA [3]

3: Bernstein E. et al. Nature 409, 363-366

Hurdles of the therapy are associated with the need of the siRNAs to reach the cytoplasm, overcoming many obstacles:

• Absorbance barriers• Endothelial barriers• Cell membrane barriers• Degradation by exonucleases• Endosomal escape• Elimination by renal filtration

RNAi dysfunction is linked to cardiovascular diseases, neurological disorders and many types of cancer[4]

RNAi dysfunction and therapeutic potentiality of synthetic siRNA

4: Dorneseifer S. et al. (2015). Nucleic Acid Res 43(22), 10623-10632

Development of synthetic siRNA as chemical tools and therapeutic agents

EpCAM

• Polymeric or lipidic vectors to overcome lysosomal degradation• Membrane fusion events to alter confirmation in lysosomes• Chemical modifications of siRNAs to avoid nuclease degradation• Conjugation with small molecules to improve bio-distribution • Delivery systems to facilitate uptake into target tissues[5]

Strategies to increase siRNAs therapeutic efficacy

Cellular targetTarget cell Carrier Bioactive compound

DARPin siRNATumour cell

5: Kathryn A. et al. (2009). Nature Rev 8, 129-130

• Trans-membrane glycoprotein over-expressed in tumours cells

• Tumour suppressor or oncogene depending on the microenvironment[5]

Epithelial cell adhesion molecule (EpCAM): the cellular target

ONCOGENE- Inhibits molecular adhesion- Promotes cell mobility,

proliferation and metastasis formation

TUMOUR SUPPRESSOR- Cell adhesion molecule- Prevents the invasion

5: Van der Gun B.T.F et al. (2010). Carcinogenesis 31 1913-1921

Designed Ankyrin repeat proteins (DARPins) as carrier

• Class of non-immunoglobulin binding proteins • Designed via sequence alignments of natural Ankyrin repeat motifs [6]

- High affinity- High stability- Small size- High expression level- Rapidly selected from a library to bind desired targets

6: Winkler J. et al. (2009). Mol. Cancer Ther. 8(9)

Produce, analyse and evaluate gene silencing effect for multiplexed DARPin-conjugate of dT18 oligonucleotide in EpCAM positive (MDA-MB-468) and negative (HEK293) cell lines

AIMS OF THE WORK

PSO

O-HN S

O

S

N

NHOH

O

HS

PSO

O-HN S

O NHOH

O

S

dT18 3' NH2 dT18 3' disulfide

Ec4-Cys

HN

O

N

O

O

PSO

O-

HN

O

N

O

O

PSO

O-

NHOH

O

S

NHOH

O

HS

dT18 3' Sulfo-SMCC Ec4-C-dT18 3' Sulfo-SMCC

Ec4-Cys

Ec4-C-dT18 3’ Sulfo-SMCC Ec4-C-dT18 3’ SPDPdT18

spacer linker DARPin

dT18

spacer linker DARPin

Produce, analyse and evaluate gene silencing effect for multiplexed DARPin-conjugate of dT18 oligonucleotide in EpCAM positive (MDA-MB-468) and negative (HEK293) cell lines

AIMS OF THE WORK

1• Solid phase synthesis, analysis and purification of the dT18

oligonucleotide

2

• Oligonucleotide conjugation with Sulfo-SMCC or SPDP for the linkage with 4-arm spacer

3• Binding of the complex oligonucleotide-linker to DARPin

4• In vitro activity evaluation by Dual-Luciferase Reporter Assay

1

2

3

4

Solid phase synthesis of dT18 oligonucleotide

1

2

3

4

Analysis and purification of dT18 oligonucleotide

dT18 3’ NH2 purity and purification were assessed by:

1) Polyacrylamide gel electrophoresis

1: dT18 3’NH2; 2: dT18 3’NH2

2) RP-HPLC measurements

3) Size exclusion chromatography

dT18 3’NH2

• Conjugation with Sulfo-SMCC linker

PSO

O-

NH2

N

O

O

O

O

NO

OSO

O

O-Na+

HN

O

N

O

O

PSO

O-

dT18 3' NH2

Sulfo-SMCC

dT18 3' maleimide

• Purification of dT18 3’ maleimide

RP-HPLC Size Exclusion Chromatography

4

1

2

3

Oligonucleotide conjugation with Sulfo-SMCC or SPDP

25°, 1 h, DMSO

• Conjugation with SPDP linker

• Purification of dT18 3’ disulfide

RP-HPLC Size Exclusion Chromatography

4

1

2

3

Oligonucleotide conjugation with Sulfo-SMCC or SPDP

PSO

O-

NH2

dT18 3' NH2dT18 3' disulfide

N

O

O

O

O

SS

N

SPDP PSO

O-HN S

O

S

N

25°, 1 h, DMSO

HN

O

N

O

O

PSO

O-

HN

O

N

O

O

PSO

O-

NHOH

O

S

NHOH

O

HS

dT18 3' Sulfo-SMCC Ec4-C-dT18 3' Sulfo-SMCC

Ec4-Cys

Reaction monitoring by RP-HPLC

Michael-type addition

Binding of the complex oligonucleotide-linker to DARPin

4

1

2

3

25°, overnight

Binding of the complex oligonucleotide-linker to DARPin

4

1

2

3

• Removal of non reacted dT18 by nickel-NTA

HN

O

N

O

O

PSO

O-

HN

O

N

O

O

PSO

O-

NHOH

O

S

NHOH

O

HS

dT18 3' Sulfo-SMCC Ec4-C-dT18 3' Sulfo-SMCC

Ec4-Cys

Ni-NTA resin Ec4-C-dT18 3’Sulfo-SMCC

+

Ni-Ec4-C-dT18 3’Sulfo-SMCC

• Purification with complementary-biotinylated DNA• Purity monitoring with SDS-PAGE

3 2 1

1: Protein-Ladder2: Ec4-Cys3: Conjugate

PSO

O-HN S

O

S

N

NHOH

O

HS

PSO

O-HN S

O NHOH

O

S

dT18 3' NH2 dT18 3' disulfide

Ec4-Cys

• Removal of non reacted dT18 by nickel-NTA

• Purification with complementary-biotinylated DNA

• Final purification with SDS-PAGE

dT18 3’ SPDP Ec4-C-dT18 3’ SPDP

Nucleophilic substitution

Binding of the complex oligonucleotide-linker to DARPin

4

1

2

3

25°, overnight, GuHCl

EpCAM positive cell

EpCAM negative cell

+ pEGFP-Gluc (Firefly luciferase) pCMV-GLuc (Renilla luciferase) and Lipofectamine

+ anti-Luc siRNA and Lipofectamine

luminescence attenuation high luminescence

4

1

2

3

In vitro activity evaluation

high luminescence luminescence attenuation

DUAL-LUCIFERASE REPORTER ASSAY• Firefly luciferase is the experimental reporter

• Renilla luciferase is the internal control

In vitro activity evaluation

4

1

2

3

Luciferase assay in MDA-MB-468 cells

0.3 μl LF/pmol siRNA guarantees efficient downregulation at 10 and 1 pmol siRNA

The reduction of siRNA/well concentration is associated with a reduced down-regulation

Conditions: • siRNA dilutions: 10 pmol/well, 1 pmol/well, 0.1 pmol/well, 0.01 pmol/well• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA, 0.03 μl/pmol siRNA,

0.003 μl/pmol siRNA

LF

4-arm spacer

EpCAM +cells

siRNA

SulfoSMCC

SPDP

*

AIMS: establish the minimum amount of LF that guarantees efficient transfection and the siRNAs concentration to allow down-regulation.

The gene silencing effect is smaller than in MDA-MB-468 cells

Even if the higher amount of LF/pmol siRNA is used, the extent of down regulation is lower

Luciferase assay in HeLa cells LF

4-arm spacer

EpCAM +cells

siRNA

SulfoSMCC

SPDP

Conditions: • siRNA dilutions: 10 pmol/well, 1 pmol/well, 0.1 pmol/well, 0.01 pmol/well• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA, 0.03 μl/pmol siRNA,

0.003 μl/pmol siRNA

*

siRNA

siRNA

Luciferase assay

siRNA

4-arm spacer

DARPinEpCAM

siRNA

In order to increment siRNA cellular uptake a 4-arm spacer was prepared and used:

AIM: produce a delivery system in order to increment cellular uptake and endosomal release using the minimum amount of LF and increasing the siRNAs quantities.

Luciferase assay in MDA-MB-468 cells siRNA

SulfoSMCC

SPDP

4-arm spacer increases the down-regulation of luciferase gene when compared with the siRNA monomer

The trend of the down-regulation is likely to be concentration dependent The higher amount of LF/pmol siRNA is associated with a better transfection

LF

4-arm spacer

EpCAM +cells

siRNA

SulfoSMCC

SPDP

Conditions: • siRNA dilutions: 40 pmol/well, 20 pmol/well, 4 pmol/well• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA

*

0.075 μl/pmol siRNA 0.3 μl/pmol siRNA

*

Higher is the concentration of 4-arm spacer, greater is the down regulationThe higher amount of LF/pmol siRNA is associated with better transfection

Luciferase assay in HeLa cells siRNA

SPDP

LF

4-arm spacer

EpCAM +cells

siRNA

SulfoSMCC

SPDP

Conditions: • siRNA dilutions: 40 pmol/well, 20 pmol/well, 4 pmol/well• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA

0.3 μl/pmol siRNA 0.075 μl/pmol siRNA

1: 4-arm spacer+3x siRNA+ conjugate+LF2: 4-arm spacer+3x siRNA+ LF3: 4-arm spacer+3x siRNA+conjugate4: 4-arm spacer+3x siRNA5: siRNA (positive control) + LF

4-arm spacer seems to be more effective in gene silencing than siRNA as monomer

The full assembly resulted in a slight target down regulation

4-arm spacer with conjugate and without LF did not produce significant down regulation confirming the role of LF in transfection

Luciferase assay in MDA-MB-468 cells

Conditions: • 4-arm spacer dilutions: 10 pmol/well, 1 pmol/well• LF amounts: 0.001 μl/pmol siRNA• 4-arm spacer was used alone or with Sulfo-SMCC with or without LF

LF

4-arm spacer

EpCAM +cells

siRNA

SulfoSMCC

SPDP

MDA-MB-468

• dT18 oligonucleotide was efficaciously synthesized • DNA oligonucleotides dT sequences were successfully attached to an EpCAM-specific

DARPin• Analytical data clearly showed successful removal of unreacted components and high

substance purity• The use of conjugate attached to a 4-arm spacer seems to increase the cargo-uptake per

receptor internalization though no significant gene silencing effect was found• The oligonucleotide complexation can be a useful strategy to produce stronger effects

than standard siRNA-transfection

CONCLUSIONS…

• More efficient methods for endosomal escape need to be investigated• Use of different modified oligonucleotides will be explored

…and FUTURE PROSPECTIVES

Thank you!!!

The use of conjugates is not associated with higher down regulation if compared with 4-arm spacer without conjugate

LF is essential for transfection

Luciferase assay in MDA-MB-468 cells

Conditions: • 4-arm spacer dilutions: 333 pmol/well, 167 pmol/well,33 pmol/well• No LF amounts was used• 4-arm spacer was used alone or with Sulfo-SMCC or with SPDP

MDA-MB-468

LF

4-arm spac

er

EpCAM

+cells

siRNA

SulfoSMCC

SPDP

The use of conjugate is not associated with higher down regulation if compared with 4-arm spacer alone

LF is necessary for knock-down

Luciferase assay in MDA-MB-468 cells

Conditions: • 4-arm spacer dilutions: 10 pmol/well, 1 pmol/well • LF amounts: 0.001 μl/pmol siRNA• 4-arm spacer was used alone or with Sulfo-SMCC with or without LF

MDA-MB-468

LF

4-arm spac

er

EpCAM

+cells

siRNA

SulfoSMCC

SPDP

The use of conjugate is not associated with higher down regulation if compared with 4-arm spacer alone

LF is necessary for knock-down

Luciferase assay in MD-MB-468 cells

Conditions: • 4-arm spacer dilutions: 10 pmol/well, 1 pmol/well • LF amounts: 0.005 μl/pmol siRNA• 4-arm spacer was used alone or with Sulfo-SMCC with or without LF

MDA-MB-468

LF

4-arm spac

er

EpCAM +cel

ls

siRNA

SulfoSMCC

SPDP