Transcript MOD ODN - Rutgers University

INTERPLAY OF POLYMER AND NUCLEIC ACID PROPERTIES ON EFFECTIVENESS OF
ANTISENSE OLIGONUCLEOTIDES
Mark
Sumati
2
Sundaram ,
of Biomedical Engineering, 2Department of Chemical & Biochemical Engineering, Rutgers University, Piscataway, NJ
METHODS
Antisense oligonucleotides present a potentially powerful means to inhibit the expression of a
specific gene that is central to a cellular phenotype, such as an oncogene in tumor growth.
The antisense mechanism involved comprises an mRNA-specific oligonucleotide complexed with
a polymeric carrier. Our research focuses on a synthetic cationic polymer, polyethylenimine
(PEI), as the delivery agent and the green fluorescent (GFP) gene as an easily quantified model
target. Fluorescence measurements indicate the level of downregulation of GFP, which in turn
is dependent on the mechanism of complex formation and dissociation that we have assessed
with biophysical measurements. Our experiments compare PEI of five molecular weights and
oligonucleotides with three different backbone structures. The results indicate that PEIoligonucleotide complexes with phosphodiester (PO) backbones dissociate much more rapidly
than those with phosphorothioate (PS) backbones. A third “end capped” ODN backbone
structure, where only the outer two nucleotides are connected by phosphorothioate linkages,
exhibits dissociation behavior similar to that of PO backbone oligonucleotides. Current work
focuses on evaluating the effectiveness of end-capped ODN using stably expressing GFP cells.
Vortex
Incubate for
~15 min
at
POLYMER
PEI – 5MW
MOD ODN
R.T.
Add OLIGREEN to
complex
5’…-A-G-G-U-C-A-C-U-U-U-G-C-A-A-C-G-…3’
• • • • • • • • • • • •
3’-C-A-G-T-G-A-A-A-C-G-T-T-5’
Normal Protein Production
target mRNA
antisense ODN
HO
SO
P O
O
O
O
Experiment 2 – COMPLEX DISSOCIATION:
Complexes are prepared at a single
concentration and oligreen is added.
Heparin in introduced last to mediate
complex dissociation. Oligreen binds to
released ODN.
BASE
PS ODN
60
25K
70K
40
600K
20
0
0.1
1.0
10.0
100.0
Results 2:
100
90
80
70
60
50
40
30
20
10
0
PEIPEI
MW
MW
10K
25K
70K
600K
• Molecular weight dependency is
insignificant in dissociation.
• Increasing heparin concentration
results in a more than
proportionate dissociation response
100 ug/ml
HEPARIN CONCENTRATION
PEI MW
No Protein
Production
CY-TAGGED ODN IN PEI/ODN COMPLEX V.
TIME
Antisense Oligonucleotide
Experiment 3 - ANTISENSE EXPERIMENT:
ODN is tagged with Cy5 dye (red) to
measure
cellular
uptake,
while
fluorescence level measures downregulation over time.
120
100
PEI MW
80
10K
25K
60
70K
40
600K
20
250
PEI MW
NORMALIZED
FLUORESCENCE
Protein
NORMALIZED FLUORESCENCE
FLUORESCENCE V. TIME FOR ALL PEI MW/ODN
200
10K
150
25K
100
70K
600K
50
0
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
TIM E (HR)
TIM E (HR)
O
PO ODN
O
P O
O
O
BASE
CHO blank
pd1EGFP blank
GFP down-regulation
ODN release
Results 3:
• Maximum down-regulation occurs 8 hours after introduction of AS ODN for all MW.
• Fluorescence levels return to normal by 30 hours.
• 10K MW PEI shows unexpected behavior at 4 hours in both plots.
O
O
10K
PEI POLYMER / ODN RATIO
SUGAR
BASE
SUGAR
1.2K
80
Results 1
• Complex formation increases
with polymer concentration.
• 1.2KDa MW PEI does not bind as
well to the ODN (high
fluorescence level)
DNA
mRNA
mRNA
DNA
PEI MW
PEI MW
100
25 ug/ml
Down-regulation duration depends on ODN backbone structure and its susceptibility to
BASE
nuclease degradation.
O
HO
5’…-A-G-G-U-C-A-C-U-U-U-G-C-A-A-C-G-…3’
PO ODN
• Rapidly degraded by cellular nucleases
• Transitory effects.
120
Antisense Inhibition
RATIONALE
O
Experiment 1 – COMPLEX FORMATION:
Complexes are prepared in vitro with
varied polymer concentrations and
allowed to form before a DNA binding
dye, oligreen (OG), is added to
interact with any unbound DNA. High
fluorescence levels indicate high levels
of unbound DNA.
PEI-MOD ODN COMPLEXATION
PEI-MOD ODN DISSOCIATION LEVEL @ T=2HR
HEPARIN MEDIATED COMPLEX DISSOCIATION - negatively charged heparin
binds competitively with the PEI carrier, displacing the ODN.
BACKGROUND
• Double stranded DNA separates and one
DNA strand is copied onto a single mRNA
“sense” strand during transcription. The
original template strand encodes the
antisense sequence.
• In antisense therapy a synthetic antisense
DNA strand enters the cell and binds to
target mRNA of complementary sequence,
preventing translation.
PEI-ODN complex solution
RESULTS

% FLUORESCENCE
ABSTRACT
What is antisense?
and Charles M.
1,2
Roth
% FLUORESCENCE
1Department
1
Hwang ,
5’…*A*G*G*U*C*A*C*U*U*U*G*C*A*A*C*G*…3’
PS ODN ( * bonds)
• Less susceptible to nuclease attack
• Longer lasting down-regulation, but displays a
• Non-specific effects due to its high charge
ratio. Binds to non-target molecules.
MOD ODN
By combining both backbone structures we
hope to extend the life of the AS ODN, while
decreasing non-specific effects. To the right
is a modified (MOD) hybrid ODN:
5’…*A*G-G-U-C-A-C-U-U-U-G-C-A-A-C*G*…3’
MOD ODN
MATERIALS
GFP F (FL1)
GFP F (FL1)
GFP F (FL1)
Cy5 F (FL4)
CONCLUSIONS
FUTURE DIRECTIONS
DNA backbone structure affects polymer-oligonucleotide complexation and dissociation. Comparing data from
each of the end-capped MOD sequence experiments to previous lab experiments on PO and PS sequences shows
that the hybrid backbone is influenced by both its PO and PS components.
Control experiments necessary to determine if non-specific effects accompany the
extended in-cell life of the MOD ODN. Experiments would compare the downregulation of scrambled control MOD ODN with that of anti-GFP MOD ODN sequences.
• Overlaying the MOD complex formation plot onto previous PO/PS complexation plots yields a greater match
with PO than PS plots. The PEI 1.2K MW does not complex significantly to either PO and MOD
oligonucleotides, while complexation occurs in the PS ODN.
• Experiments indicate that PEI-MOD complex dissociation following heparin addition is almost complete at
2hrs, compared to 15 minutes for PO and 8 hours for PS sequences.
SPECIFIC GOAL: evaluate effectiveness of end-capped MOD ODN sequence in antisense therapy.
• Anti-GFP MOD sequence causes maximum down-regulation at 8 hours.
ACKNOWLEDGEMENTS
We wish to thank Dr. Li Kim Lee and Sandra Viriyayuthakorn for useful images that
aided in highlighting key points.