Antibody mediated in vivo delivery of siRNA through cell surface receptors.i presented this paper during asso hour in my final year UG

1. Antibody mediated in vivo
delivery of
small interfering RNAs
via cell-surface receptors
From “nature biotechnology”
Presented by
V.Esakki Muthu Lakshmi

2. Introduction
Protamine-antibody fusion(F105-P)
 Fab fragment
siRNA delivery
Gene silencing in target cells
 HIV env expressing cells
 Cancer cells

3. siRNA function
 19-25 nucleotides long
 Antiviral defense mechanism
 Transposon silencing
 Gene regulation
 Chromosomal modification

4. siRNA mechanism

5. Antibody structure
 2 heavy chains
 2 light chains
 Disulfide bonds
 Glycoproteins
 Antigen specificity
 Bivalent binding sites

6. 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

8. 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

9. 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

10. RESULTS
1 F105-P binds to ~6 siRNA
molecules

11. 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

12. RESULT(northern blot
analysis)

13. 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

14. RESULT
 Absence of gene silencing
 untransfected gag p24(-) cells
 Irrelevant fas siRNA
 Unmodified F105 in place of F105-P
Loading
control

15. 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

16. 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)

17. HIV gag staining

18. ELISA for p24 Ag

19. 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

20. 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

21. Contd…
 F105-p-VEGF,MDM2&c-myc siRNA
 The greatest effect on gp160-B16 cells

24. 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

26. 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

27. RESULT

28. Single chain fragment variable
 2 variable domains-VH&VL
 High antigen specificity

29. 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

32. 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

34. 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