Antibody mediated in vivo

delivery of

small interfering RNAs

via cell-surface receptorsFrom “nature biotechnology”

Presented by

V.Esakki Muthu Lakshmi

Introduction

Protamine-antibody fusion(F105-P)

Fab fragment

siRNA delivery

Gene silencing in target cells

HIV env expressing cells

Cancer cells

siRNA function

19-25 nucleotides long

Antiviral defense mechanism

Transposon silencing

Gene regulation

Chromosomal modification

siRNA mechanism

Antibody structure

2 heavy chains

2 light chains

Disulfide bonds

Glycoproteins

Antigen specificity

Bivalent binding sites

Need for protamine-antibody fusion

Protamine

Cationic peptide

Binds to C terminus of Ab

Links Ab and siRNA

To avoid

Nonspecific gene silencing

Toxicity in bystander cells

Targeting of HIV env+ cells

COS cells

Fibroblast like cell line

Medium-RPMI1640 with 10% fetal bovine serum

Transfection with pCMV-F105-P

Collection of F105-P*

B16 cells

Melanoma cells

Transfection with expression vector for HIV env

FITC*-siRNA

FITC*-Flourescin isothio cyanate

F105-P* – protamine antibody against HIV env

F105-P binding assay

Addition of 200 pmol FITC-siRNA

Anti-protamine coated protein bound F105-P

overnight incubation at 4°C

Thorough washing to remove unbound FITC-siRNA

Determination of absorbance at 488nm

Construction of standard curve

1 F105-P binds to ~6 siRNA

molecules

RESULTS

siRNA delivery Mixing of F105-P & gag siRNA (6:1)

Incubation of mixture in PBS at 4°C

Addition of mixture to cells(B16)

Transfection of cells with oligofectamine*(control)

Analysis of gene expression 2days after of siRNA

delivery

gag siRNA-gene silencing in HIV env expressing cells

Oligofectamine*-tranfection reagent

RESULT(northern blot

analysis)

Analysis of target gene silencing

Transfection of HeLa* cells with plasmid encoding

EGFP

Tranfection of HeLa-GFP with HIV λHXB3

Treating of culture with F105-p EGFP siRNA

Analysis of HIV & GFP expression 2d after treatment

GFP expression by northern blot

HeLa*-immortal cell line

EGFP-enhanced green flourescent protein

RESULTAbsence of gene silencing

untransfected gag p24(-) cells

Irrelevant fas siRNA

Unmodified F105 in place of F105-P

Loading

control

Inhibition of HIV in infected T cells

Collection of CD4 T cells from normal donor

Stimulation with phytohemagglutinin*(4µg/ml) for 4d

Infection with HIV strain III B at a MOI of 0.1

Treating cells with F105-P -gag siRNA

Analysis of HIV replication

Phytohemagglutinin*-mitogen

RESULTS

Reduction in infected cells(85%-36%)

HIV gag staining-45%

ELIZA for HIV p24

Concentration of viral particle -170 ng/ml to

40 ng/ml(100 pmol of siRNA)

HIV gag staining

ELISA for p24 Ag

Tumor cell proliferation inhibition

BL6/C57 mice

Subcutaneous injection of gp160-B16 cells(5× 106 )

Intravenous injection of F105-P

1. c-myc siRNA

2. MDM2 siRNA

3. VEGF siRNA

F105-P:siRNA-1:6

Sectioning of tumor

from killed mice(9d)

Analysis of expression by

q RT-PCR or flow cytometry

RESULTS

F105-P-cmyc siRNA

Effect on gp160-B16 cells(si RNA conc>100nM)

No effect on B16-gp160(-) cells

F105-P-VEGF siRNA

Modest effect

Blocking of angiogenesis

F105-P -GFP siRNA(control)

Target tumor suppressor pp32

Contd…

F105-p-VEGF,MDM2&c-myc siRNA

The greatest effect on gp160-B16 cells

Determination of specificity

BL6/C57 mice

Subcutaneous injection of gp160-B16 cells(2× 106 )

Intravenous injection of 50µg F105-P-FITC

siRNA(day 9)

Transfection with oligofectamine

Cryosectioning of tumor from killed mice(16h)

OBSERVATION

Effect on gp160+ tumor cells

Flourescent signal in membrane & cytoplasm,not

in tumor cell nucleus

Is there any inflammatory responses?

Possibilities

Trigger an interferon responses

Binding of siRNA to TLR*

q RT-PCR

Interferon beta

2’,5’-oligoadenylate synthetase

Stat-1

Observation

No interferon responses

TLR*-Toll like receptor

RESULT

Single chain fragment variable

2 variable domains-VH&VL

High antigen specificity

Anti ErB2 ML39 scFV production

Transfection of SF9 cells with virus(ML39 scFV)

Extraction of ML39 scFV by 6M GuanHCl

Purification by Ni++ chromatography

Dialysis with PBS containing 5%glycerol,0.5M

arginine,1mM EGTA,1mM glutathione

Final dialysis with PBS containing 5%glycerol

FITC siRNA delivery by ML39 scFV-P

Targeting cells

ErB2 expressing cells

MCF7 breast cancer cells

SKBR3 cells

Observation

effect only in ErB2+ cells

Ku70 silencing in ErB2+ SKBR3 cells

Dose of ML39 scFV-P – 1000pmol

Conclusion

Rate limiting factor

Short in vivo half-life

Filtering of siRNA

Prone to Rnase activity

Gene silencing enhancement

Modified siRNA

Cocktails of siRNA

Chemokine analog