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Androgen receptor locked nucleic acid antisense oligos potently knocks down target gene
expression both in vitro and in vivo
Baisong Liao, Yixian Zhang, Jolanta Kosek, Wenzhe Liu, Stephen Castaneda, Lee M. Greenberger, Ivan D. Horak
Abstract #4631
Enzon Pharmaceuticals, Inc., Piscataway, NJ INTRODUCTION
In vitro results with LNCaP cells
In vitro results with 22Rv1 cells
In vivo results
Androgen-sensitive LNCaP cells were transfected with indicated LNA-ONs with 22Rv1 is a human prostate carcinoma epithelial cell line derived from a xenograft that Fig. 9. LNA-ONs inhibit mRNA of AR and PSA in 22Rv1 tumors.
Prostate cancer is the most common cancer and the second leading cause of
lipofectamine 2000 for 4 hrs. After transfection, cells were maintained in RPMI was serially propagated in mice after castration-induced regression and relapse of the cancer death in North American males. Growth of prostate cancer epithelial
parental, androgen-dependent CWR22 xenograft. Recently, a novel AR variant was medium containing 10% FBS. Messenger RNA of AR (Fig. 1), AR protein level cells is androgen-dependent during the initial stage, mediated by androgen
shown to promotes androgen-depletion resistant growth (2) .
(Fig. 1), cell proliferation (Fig. 2), PSA mRNA (Fig. 3) and secreted PSA (Fig. 4) receptor (AR), a major regulatory transcription factor for genes regulating
were determined between 24 hr to 48 hr after transfection.
Fig. 5. LNA-ONs inhibit mRNA of AR in 22Rv1 cells.
proliferation and survival of prostate cancer cells. Therefore, androgen
Fig. 1. LNA-ONs inhibit AR mRNA and protein levels in vitro.
deprivation is a mainstay therapy for prostate cancer. However, prostate
cancer that is resistant to existing therapies will emerge within 1-3 years.
Importantly, AR remains present and plays a key role in the progression of
cancer (1). Thus, mRNA antagonist of AR offer a promising therapeutic
(5 nM, 2d)
EZN4176-90 EZN4176MM-90
EZN4176-90 EZN4176MM-90
approach. To this end, locked nucleic acid (LNA) technology (third generation
antisense) was employed to design antisense oligonucleotides (ASONs)
against AR that has exceptional bio-stability and very high binding affinity for
Nude mice bearing 22Rv1 tumors were injected with 90 mg/kg of EZN-4176 and its complementary mRNA. In this study, we report the in vitro activities of two AR
mismatched control (q3d x 4, IV). Twenty four hours after the fourth dose and tumor LNA-ASONs, EZN-4176 and EZN-4187. Additionally, in vivo target and growth
samples were collected and processed for mRNA analysis by qRT-PCR. Data are inhibition is shown.
EZN3046-10 EZN4176-5 EZN4176-10 EZN4187-5 EZN4187-10 LNA oligo (nM)
EZN-4176 specifically down regulates mRNA of both AR (left) and
PSA (right) in 22Rv1 tumors
EZN-4176 and 4187 but not a scrambled oligonucleotide specifically
and potently knock down AR mRNA and protein
Cells were transfected with indicated LNA-ONs for 4 hr. After transfection, cells were Fig. 2. LNA-ONs inhibit the growth of LNCaP.
maintained in RPMI medium containing 10% FBS. Messenger RNA of AR was The target mRNA knockdown, growth inhibition, reduction of prostate specific
determined by qRT-PCR 24 hr after transfection antigen (PSA, an AR downstream target and biomarker of prostate cancer),
Fig. 10. LNA-ON inhibits the growth of tumors in CWR22 xenograft model
and apoptosis induction effects of EZN-4176 and EZN4187 were evaluated by
qRT-PCR, western blot analysis, ELISA, MTS assay, and caspase3/7 activity
Fig. 6. LNA-ONs inhibit the growth of 22Rv1 cells.
Casode x 150 m g/kg Q1dx21
assay, respectively, in two AR positive cancer cell lines (LNCaP and 22Rv1
EZN-4176 60 m g/kg q3dx10
prostate) after transfection. A scrambled LNA antisense oligo (EZN-3046) and
Inhibition of 22Rv1 growth
EZN-4176 3 m g/kg q3dx10
the AR negative cell line (15PC-3 prostate cancer) served as controls.
7 days post transfection
EZN-4176MM 60 m g/k g q3dx10
oligonucleotide on DHT-induced growth of LNCaP was evaluated in charcoal-
stripped serum (CSS) containing medium. In vivo, target knockdown efficacy
of EZN-4176 in 22Rv1 tumors was evaluated after intravenous administration.
LNA oligo (nM)
Tumor inhibition was examined with an androgen-dependent CWR22 xenograft
EZN-4176 and 4187 but not a scrambled oligonucleotide inhibit the
growth of LNCaP cells
No inhibitory effect found in an AR negative 15PC-3 cell line (data
not shown)
Fig. 3. LNA-ONs inhibit PSA mRNA and secreted PSA from LNCaP.
Nude mice bearing CWR22 tumors were injected with LNA-ONs (in saline) at indicated doses (q3d x 10, IV). Tumor volume was measured at indicated days and expressed as percent change (see graph). Data are means + SE (n = 7) Cells were transfected with indicated LNA-ONs. Cells growth was determined seven • EZN-4176 shows comparable efficacy to high-dose Casodex
EZN-4176MM is inactive in growth inhibition
EZN3046-10 EZN4176-5 EZN4176-10 EZN4187-5 EZN4187-10
LNA oligo (nM)
Fig. 7. LNA-ONs inhibit AR level in 22Rv1 cells .
5 nM 0.5 nM 5 nM 0.5 nM
EZN3046-10 EZN4176-5 EZN4176-10 EZN-4187-5 EZN4187-10
Selective and specific downmodulation of AR mRNA and protein in
LNA oligo (nM)
Cells were transfected with indicated LNA-ONs. Supernatant of the culture was β-tubulin
cells correlate with in growth inhibition
harvested and PSA determined with a ELISA kit (Abazyme) 24 hr after transfection.
Genes (i.e PSA)
Fig. 4. LNA-ONs inhibit DHT-induced growth of LNCaP cells.
Potent inhibition of AR and PSA mRNA in 22Rv1 xenograft model
DHT, 10 nM
Cells were transfected with indicated LNA-ONs. Cells were harvested 48 hrs after Oligos, μM
Significant tumor growth inhibition in CWR22 xenograft model
transfection and AR protein level detected by Western analysis.
Fig. 8. LNA-ONs inhibit PSA level in 22Rv1 cells .
AR oligos reduce secreted PSA levels in
In the absence of testosterone (T), or dihydrotestosterone (DHT), androgen
22RV1 culture (2d, 10 nM, ELISA)
receptor (AR) monomers are associated with heat shock protein complex
(HSP) and other proteins

EZN-4176MM LNCaP cells were plated in RPMI-1640 medium
Chen Y, et al. Targeting the androgen receptor pathway in prostate cancer,
containing 10% FBS. 24 h later, medium were replaced •Upon binding to T or DHT, AR becomes phosphorylated (P). Eventually, HSP
with 5% charcoal-stripped serum (CSS) and treated with indicated compounds without lipofection. mRNA Current Opinion in Pharmacology, 2008; 8:440-448
complex is dissociated and AR is able to enter the nucleus to regulated
assay was performed 3 d later while growth assay was specific target genes for growth and survival
2. Guo Z, et al. A novel AR splice variant is up-regulated during prostate cancer
EZN-4176MM = mismatched oligonuclotide
AR can also be activated in the absence of androgen through ligand-
progression and promotes androgen depletion-resistant growth, Cancer Res,
DHT induces robust growth of LNCaP cells
independent pathways (not depicted in the picture), which is an important
EZN-4176 but not a specifically designed mismatched oligonuclotide
mechanism leading to resistance to androgen ablation therapy
2009, 69:2305-2313
inhibits the growth of DHT-induced growth of LNCaP cells
Growth inhibition is associated with the down regulation AR mRNA
The design and discovery of EZN-4176 and EZN-4187 has been done in
Targeting AR represents an important therapeutic approach
MM Control is inactive in mRNA assay even at 10 μM (not shown)
Cells were transfected with indicated LNA-ONs (10 nM). Supernatant were harvested collaboration with Santaris Pharma A/S.
EZN-4176 and EZN-4187 are being
LNA-based technology see posters 4630, 4633, 4634
EZN-4176 is ineffective in multiple AR-negative cell lines tested
48 hrs after transfection and PSA determined by ELISA.
developed by Enzon under a license with Santaris Pharma A/S
AACR 100thAnnual Meeting, Denver, CO, 2009


Chapter 4 Conventional Medical Therapies “Today’s standard, AMA-approved medicine is rooted in treating symptoms, rather than causes. Its dependence on drugs and surgery is ruinously expensive to patients, insurance companies, “Why I Left Orthodox Medicine” Conventional medical treatments for FMS and CFS is a controversial topic. Consider the following statements

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