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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