This document summarizes a presentation on using aptamers to deliver siRNA for gene silencing. The presentation introduces siRNA and RNAi, and discusses the challenge of delivering siRNA to specific cells and tissues. It then describes a study that conjugated an anti-prostate specific membrane antigen (PSMA) aptamer to siRNA targeting GADPH or lamin A/C mRNA. This conjugate selectively delivered functional siRNA to PSMA-positive prostate cancer cells but not PSMA-negative cells. The aptamer-siRNA conjugate showed rapid internalization and gene silencing comparable to commercial reagents. This approach could provide a cell-specific siRNA delivery method without immunogenicity issues of other systems.

1. Paper presentation 20/1/14
Aptamer mediated siRNA delivery
Prepared by Toh
Saw Yi
Impact
factor: 8.278

2. Introduction
What are siRNA?
• Short interfering RNA
• A class of double-stranded RNA molecules
• ~20-25 base pairs in length
What is RNAi?
• RNA interference/ Post transcriptional gene silencing (PTGS)
• A biological process in which RNA molecules inhibit gene
expression, typically by causing the destruction of
specific mRNA molecules
• How does it work? *Video*

3. Why all the hype?
• siRNA has proven time and time again that it has
several advantages as a potential new bio-drug to
treat various diseases, including cancer and HIV
• However, the role of therapeutic siRNA in vivo
remains a big challenge for achieving the desired
RNAi effect.
• The key problem? Delivery to specific cells/ tissues

4. Hypothesis
• To determine whether anti-prostate specific
membrane antigen (PSMA) aptamer can
deliver functional siRNA to a cell.

5.  Aptamer synthesis
 Run-off transcription of a DNA template with T7 RNA
polymerase promoter.
 Template obtained from Lupold et al.
 Aptamer biotinylation
 siRNA synthesis
 2 types: targets mRNA of GADPH; mRNA of lamin A/C
 With addition of disulphide linkers and biotinylation
 Modification to sense strand doesn’t affect function of siRNA
 27mer siRNA
Methodology

6.  Aptamer and siRNA conjugation to streptavidin
 Ratio for aptamer:siRNA:streptavidin = 2:2:1
 Disulphide linker will be released upon entering reduced
environment of the cell
Methodology

7.  Transfection
 Androgen-sensitive human prostate adenocarcinoma (LNCAP)
cell line – PSMA positive
 And human prostate cancer (PC3) cell line – PSMA negative
 Cell confluence 70%
 22.5nM of conjugate (2x siRNA per conjugate)
 Duration: 3 days
 Real-time PCR
 Monitor gene expression – inhibition present/not?
Methodology

8. Results
• Significant inhibition only seen when both siRNA and
aptamer are present (in form of conjugate).
• Anti-PSMA aptamer targets PSMA cells specifically.
• siRNA that targets mRNA of GADPH only targeted
mRNA of GADPH. Vice versa for mRNA of lamin A/C.
• siRNA synthesized does not activate cellular immune
response (mRNA of interferon β-1a [INFB] is not
present).

9. Additional work done
• In order to compare how fast aptamers can reach the
target site and deploy the siRNA, they compare it with
commercial transfecting reagent, Oligofectamine
amalgams [Life Technologies].
Results:
• Rate of internalization is very high. Only required 30
mins for maximum inhibition.
• Comparable with commercial transfecting reagent.
• Oligofectamine is so wrapped up in ‘protective gear’ to
protect its cargo and itself against nucleases and other
dangers that aptamers can just ignore.

10. Significance of Aptamer-use in
Targeted siRNA Delivery
• Why is this advantageous over the other methods of
delivery?
 Lack of immunogenicity in vivo
 No additional components or preparations were necessary (to
assess the inhibition of gene expression)
 Relatively small in physical size
 Easy functional modifications (disulphide linkers)
 Rapid in vitro selection
 Rapid internalization by cells and are practically ‘naked’ to
serum nucleases in media
• Plus, it was successfully used as a method of delivery by A
LOT of researchers for a variety of targets

11. Disadvantage/s
• Quality control for the conjugation step is tedious –
requires simplification.
• Streptavidin was shown to elicit immunogenic
responses. – may limit its use as a part of the delivery
vehicle.
 Possible to conjugate aptamer and siRNA via other
linkers as well.

12. Others methods of delivery utilising
aptamers
• The cargoes are attached to aptamers through:
 Direct conjugation to the aptamer
 Assembly with functionalised groups appended to aptamer
and cargo
 Aptamer-based
origami

13. Conclusion
• Aptamers can replace current cell-specific transfection
reagent.
 Can be pre-appended onto streptavidin and, later, mixed with
any biotinylated siRNA to make a one-step, ready-to-use
transfection reagent.
 And it’s easier to be made as compared to the series of
complex packaging steps required to make current cell-specific
transfection reagents.
 More reproducible than when using lipid reagents for
transfection.
• The method described may pave way as a general
strategy for the delivery of specific siRNA to almost
any cell type.