1
Antisense oligonucleotides
©Old ich Farsa 2012ř
2
Antisense RNA
•Expression vector that produces a
transcript, which is complementary to a
known transcript
•Expression vector is introduced into a host
cell by transfection and blocks translation of
target mRNA
3
Antisense RNA
4
Therapeutic Antisense RNA
•Insulin-like growth factor 1
• prevalent in malignant glioma (common brain
tumour) as well as prostate carcinomas
•Rat prostate carcinoma cells transfected
with antisense receptor cDNA
•Mouse injected with transfected cells
• small or no tumours compared to control rats
treated with non-transfected carcinoma cells
5
Why Antisense Oligonucleotides?
•Large antisense RNA is readily subjected
to degradation
• Human cells specifically target dsRNA for
turnover by nucleases
•DNA oligonucleotides more stable, more
readily delivered to target cells
•Directed to 5’ or 3’ ends of mRNAs, intronexon
boundaries, naturally ds regions
6
Antisense Oligonucleotides
7
Antisense Oligonucleotides - „mode of action“
8
Possibilities of RNA-triplex forming by action of antisense-oligos
9
Possibilities of RNA-triplex forming by action of antisense-oligos (continued)
10
Optimizing Antisense Oligos
• Natural oligos (A) susceptible
to cellular nucleases
• Alterations improve nuclease
resistance
• Replacement of free oxygen
with sulfur in phosphodiester
bond (B) particularly effective
• Replacement of deoxyribose
with morpholine ring together
with substitution of hydroxyl
group in phosphate moiety (D)
with dimethylamine led also to
active compounds
O
O
O
base
PO OH
O base
3'
5'
O
O
O
base
PO SH
O base
3'
5'
A B
O
O
NH
base
PO OH
O base
3'
5'
C D
O
base
O CH3
5'
E
N
N
NH2
O
CH2
F
(3')
(5')
O
base
NPN O
OCH3
CH3
O base
N
11
Phosphorothioate Antisense Oligos (B)
•Water-soluble in form of polysodium salts
•Complex with target mRNA activates
RNase H
•Some therapeutics in clinical trials
• eg. against cytomegalovirus infection of retinas
in patients with AIDS
•Decreased mRNA binding, increased
nonspecific protein binding wrt native oligos
12
Native oligos: Splice Site Correction
•β-thalassemia:
Mutations in
hemoglobin chains
lead to reduced
oxygen binding
(anemia)
•Binding of oligos
prevent inappropriate
splice activity
13
Oligos' Delivery
• Extremely hydrophilic
• Exactly tissue-targeted
delivery needed
• Liposome plus cell-specific
binding proteins could
allow targeted, efficient
delivery of appropriate
doses of therapeutic
antisense oligonucleotides
14
Compounds Used
fomivirsen
•21 deoxyribonukleotides, phosphorothioate
•docosasodium salt
•sequence 5'-G-C-G-T-T-T-G-C-T-C-T-T-C-T-T-C-T-T-G-C-G-3'
• cytomegalovirus replication inhibitor: complementary to RNA of IE2
(„immediate early region 2“) of HCMV, inhibits IE2 protein production
and thus virus replication
•treatment of CMV retinitis in patients with AIDS
•Vitravene™ intravitreal inj. (approved in USA cca 1998 - 2001)
15
Compounds in Clinical Trials
Phosphorothioate oligos
alicaforsen – therapeutic of ulcerative collitis, inh. of synt. ICAM (intercellular
adhesion molecule)
afovirsen – antivirotic & antineoplastic – inhibitor of human papillomavirus
replication
miravirsen, SPC-3649 – antivirotic – anti liver-specific miRNA - hepatitis C
aprinocarsen, Affinitak™ − antineoplastic – inhibitor of protein kinase Cα
- non
small cells lung & breast cancer
oblimersen, Genasense®
− antineoplastic - targeted to gene of Bcl2 anti-apoptic
protein – various tumours
trabedersen, AP-12009 – antineoplastic - inhibitor of overexpression of
Transforming Growth Factor β2
(TGF β2
) – brain cancers (gliome, astrocytome)
16
Compounds in Clinical Trials
Intercellular Adhesion Molecule (ICAM) Synthesis Inhibitors
Ulcerative Colitis = Inflammatory Bowel Disease (IBD) = chronic relapsing
inflammatory disease of the mucose layer of the intestine
•idiopathic = ethiology is unknown
•pathogenesis is believed to be multifactorial and to include genetic, environmental
and immunologic factors
•chronic inflammation is manifested namely due to dysregulation of the adaptive
immunity system, which leads to a change of tolerance to intestinal bacteria and to
an anomalous response to the normal luminal microflora ⇒ immunologic
imbalance ⇒ ↑ production of inflammatory cytokines and adhesion molecules
(ICAM, MadCAM), ↑ activation of polymorphonuclear monocytes (PMN); their
migration into the intestine and interaction with the epithelium influences epithlium
functions from the barrier one to electrolytes management
17
Mechanism of origin of IBD, biomolecules engage in it and therapeutic targets of selected biodrugs
ICAM-1 intercelullar adhesion molecule 1 EGF epidermal growth factor
MadCAM mucous address adhesion molecule PMN – polymorphonuclear monocyte
IFN interferon
IL interleukin
18
O
O
O
O
O
O
O
O
OH
O
O
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
O
NH2
O
O
N
N
H
N
N
O
O
NH2
N
N
H
O
O
O
O
CH3
P
O
S
-
N
N
N
N
NH2
O
P
O
S
-
P
O
S
P
O
S
-
P
O
S
-
P
O
S
-
P
O
S
-
N
N
O
NH2
O
O
N
N
O
NH2
O
O
P
O
S
-
N
N
H
N
N
O
O
NH2
P
O
S
-
P
O
S
-
P
O
S
-
N
N
O
NH2
O
O
N
N
N
N
NH2
O
P
O
S
-
N
N
H
O
O
O
O
CH3
P
O
S
-
P
O
S
-
P
O
S
-
P
O
S
-
P
O
S
-
N
N
N
N
NH2
O
N
N O
NH2
O
O
N
N
O
NH2
O
O
N
N
H
N
N
O
O
NH2
N
N
H
N
N
O
O
NH2
N
N O
NH2
O
O
N
N
H
N
N
O
O
NH2
N
N
H
O
O
O
O
CH3
N
N
N
N
NH2
O
N
N O
NH2
O
O
P
O
S
-
P
O
S
-
O
Alicaforsen
syn. ISIS 2302
•oligodeoxyribonucleotide modified by
thiophosphoric acid (phosphorothioate)
•20 bases
•binds (hybridizes) to the non-translated 3'region
of human ICAM-1 mRNA ⇒ activation
of nuclease RNasy-H ⇒ cleavage of the
heterodimer ⇒ attenuation of ICAM synthesis
•phase 3 clinical tests
19
Compounds in clinical trials: antiviral agents
afovirsen, syn. ISIS 8741 – free „acid“; ISIS 2105 – nonadecasodium salt
•Sequence: ttgcttccat cttcctcgtc
•Modification: residues of phosphoric acid substituted with those of thiophosphoric acid
•Usage: treatment of human papillomavirus infections (HPV; manifestations of the infection:
warts of female genitals – cervical cancer)
•mode of action: binding to viral mRNA
Effect of ISIS 2105 and ISIS 3925 (20
residue phosphorothioate oligonucleotide
designed to be complementary to
influenza A virus) to HPV DNA replication.
24 h after infection by electroporation,
cells of the line SCC-4 were treated with
increasing doses of either ISIS 2105 or
ISIS 3925. Cells were „harvested“ 24 h
after electroporation and their HPV DNA
content was determined. Data are plotted
in % of untreated control experiment;
mean of 3 assays.
20
Compounds in clinical trials: antiviral agents
miravirsen, SPC3649
•RNA, (P-thio)((2'-O,4'-C-methylene)m5C-dC-(2'-O,4'-C-methylene)A-dT-dT-(2'-O,4'-C-
methylene)G-(2'-O,4'-C-methylene)m5U-dC-dA-(2'-O,4'-C-methylene)m5C-dA-(2'-O,4'-Cmethylene)m5C-dT-(2'-O,4'-C-methylene)m5C-(2'-O,4'-C-methylene)m5C),
sodium salt
(1:14)
•16 bases, phosphorothioate groups, 8 methylene bridges between 2'-O and 4'-C atoms of
ribose
N
N
O
NH2
O O
O OH
N
N
O
NH2
O
O P
OH
S
OO
N
N
N
N
NH2
O
O P
OH
S
O
N
NH
O
O
CH3
O P
OH
S
O
O
N
NH
O
O
CH3
O
O O P
OH
S
O
NNH2
N
N
NH
O
O
OP
S
O
O
N
NH
O
O
CH3
O
OP
OH
S
O
N
OOP
OH
S
O
N
O
NH2
O
O
OP
OH
S N
N
N
N
NH2
N
N
N
N
NH2
O
O
O P
O
S
O
N
N
O
NH2
O
O P
OH
S
O
N
NH
O
O
CH3
O
O P
OH
S
O
N
N
O
NH2
O
O
O P
OH
S
O
O P
OH
SN
N
O
NH2
O
OHO
O
N
N
O
NH2
O
OP
OH
OH
OH
S
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•microRNAs are short (21-23 nucleotide) non-coding regulatory RNAs that influence gene
expression at a post-transcriptional level
• microRNA-122 (miR-122) is a major regulatory RNA in liver that fine-tunes the expression of
over 100 cellular genes and enhances hepatitis C virus (HCV) replication through interaction
with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′
UTR) of the viral RNA
•„locked nucleic acid“ SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a
and 1b infection in vivo
•phase 1 – 2 clinical tests against HCV infection have been finished
22
Compounds in clinical trials: antitumour agents
Proteinkinase Cα
inhibitors
•proteikinases C: serine-threonine kinases taking part in cell signál transduction
(phosphorylation of signal molecules); their altered expression is linked with cancerogenesis
aprinocarsen, syn. ISIS 3521, LY900003, CGP 64128A
Affinitak™
•deoxyribonucleotide, 20 bases, phosforothioate
•A-C-T-T-T-G-A-G-T-G-G-T-C-G-C-T-C-T-T-G
•phase 2 clinical tests against non-small cells lung cancer (inoperable or metastasizing)
alone or combined with carboplatin, gemcitabin or paclitaxel; also against breast cancer
treated previously by an other drug have been finished
•phase 3 against lung cancer: addition of aprinocarsen to gemcitabin or carboplatin has
neither prolonged patients survival nor increased treatment efectiveness evaluated by other
measures
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Compounds in Clinical Trials
Morpholine oligos
eteplirsen, AVI-4658
•treatment of Duchenne muscular dystrophy (DMD)
•DMD: a fatal muscle degenerative disorder, caused by misreading and bad
translation of DMD (dystrophin) gene to dystrophin peptide due to mutations of this
gene
•1 per 3500 newborn boys is affected
•sequence CTCCAACATCAAGGAAGATGGCATTTCTAG; 30 bases; Mr
=
10369.83
•targeted to 51. exone in dystrophine mRNA
•enables partial restoration of reading and translation of DMD gene by „patching“ of
its bad splicing ⇒ production of truncated, but functional chain of dystrophine
•applied locally by i.m. injection
•2 phase 2 clinical studies proceed
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N
N
N
NH
O
NH2
O NH
O
N
N
N
N
NH2
O N
P
N O
CH3CH3
O
N
NH
O
O
CH3
N
P
N O
CH3CH3
O
O
N
N
O
NH2
N
P
N O
CH3CH3
O
O
N
NH
O
O
CH3
N
P
N O
CH3CH3
O
O
O O
N
NH
O
O
CH3
N
P
NO
CH3 CH3
O
O
N
NH
O
O
CH3
N
P
NO
CH3 CH3
O
N
N
N
N
NH2
ON
P
NO
CH3 CH3
O
N
N
O
NH2
N
P
NO
CH3 CH3
O
O
N
N
N
NH
O
NH2
ON
P
NO
CH3 CH3
O
N
N
N
NH
O
NH2
ON
P
NO
CH3 CH3
O
O
N
N
N
N
NH2
O N
P
N O
CH3CH3
O
N
N
N
NH
O
NH2
O N
P
N O
CH3CH3
O
N
N
N
N
NH2
O N
P
N O
CH3CH3
O
N
N
N
N
NH2
O N
P
N O
CH3CH3
O
N
N
N
NH
O
NH2
O N
P
N O
CH3CH3
O
N
N
N
NH
O
NH2
O N
P
N O
CH3CH3
O
N
N
N
N
NH2
O N
P
N O
CH3CH3
O
O
N
N
N
N
NH2
ON
P
NO
CH3 CH3
O
N
N
O
NH2
N
P
NO
CH3 CH3
O
O
N
NH
O
O
CH3
N
P
NO
CH3 CH3
O
O
N
N
N
N
NH2
ON
P
NO
CH3 CH3
O
N
N
O
NH2
N
P
NO
CH3 CH3
O
O
N
N
N
N
NH2
ON
P
NO
CH3 CH3
O
O
N
N
O
NH2
N
P
N O
CH3CH3
O
O
N
N
O
NH2
N
P
N O
CH3CH3
O
O
N
NH
O
O
CH3
N
P
N O
CH3CH3
O
O
N
N
O
NH2
N
P
N O
CH3CH3
ON
N
O
O
OH
O
O
P
N
O
O
CH3CH3
ON
P
NO
CH3 CH3
O
O
N
NH
O
O
CH3
N
P
NO
CH3 CH3
O
N
N
N
N
NH2
25
26
Compounds in Clinical Trials
Morpholine oligos
radavirsen, AVI-7100
•phosphorodiamidate morpholino antisense oligomer with positive charges on
selected subunits; 20 nucleotide bases
27
•intended for influenza A
•specifically targets viral messenger RNA sequences
•phase 1 clinical tests to characterize the safety, tolerability and pharmacokinetics of
escalating single-administration doses of AVI-7100 in healthy human subjects.