Nucleic Acid Drugs

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Nucleic Acid Drugs
Antisense oligonucleotides siRNAs
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Oligonucleotides

• Single-stranded segment of DNA or RNA
• Generally produced with a oligonucleotide
  synthesizing machine
• lt 100 bases in length
• Can be either sense or antisense
• Sense same sequence as mRNA
• Antisense complementary to the mRNA

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Antisense Oligonucleotides (ASO)

• Single-stranded oligonucleotide, most often DNA
• Antisense (complementary to mRNA, i.e., will bind
  to mRNA)
• Usually very short (12-30nt) length
• ASOs are specific to one unique mRNA sequence

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Antisense Oligonucleotides Hybridize to mRNA
mRNA
5 CUCAGCGUUACCAUCCUGCAA 3
3 GAGTCGCAATGGTAG 5
antisense oligo
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Antisense Oligonucleotide - Mechanism of Action
ASOs inhibit gene expression by blocking protein
synthesis
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Antisense oligonucleotides hybridize to mRNA
mRNA
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3
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oligonucleotide
X
Protein synthesis is blocked Two different
mechanisms of inhibition 1) destruction of mRNA
by ribonucleases 2) ribosome movement blocked
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How is protein synthesis inhibited by antisense
oligos? (1) Ribonucleases recognize and cleave
RNADNA duplex.
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ribonuclease
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What are ribonucleases?

• Endogenous enzymes that degrade RNA.
• RNase H - ribonuclease that recognizes and
  cleaves mRNA in an RNA/DNA duplex. Also cleaves
  RNA in an RNA/RNA duplex.
• RNase L - ribonuclease that recognizes and
  cleaves single-stranded RNA adjacent to hybrid
  RNA/RNA duplex.

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How is protein synthesis inhibited by antisense
oligos? (2)
An antisense oligo prevents ribosome from
proceeding along mRNA during translation.
mRNA
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Antisense oligo
Newly synthesized protein
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Advantages of Antisense Oligonucleotides over
Traditional Drugs

• Traditional drugs intervene after a disease
  causing protein is formed.
• Antisense therapeutics block mRNA translation and
  intervene before a disease causing protein is
  formed.
• Since antisense therapeutics target only one
  specific mRNA, they should be more effective with
  fewer side effects than current
  protein-inhibiting therapies.

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Antisense Oligonucleotide Design

• Important considerations
• Gene target
• Length of oligo
• Portion of mRNA to target

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Gene Target

• Target the mRNA from the disease causing gene.
• Cancer good target because there is often vast
  differences in gene expression between cancer and
  normal cell.

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Differences in Gene Expression Between Cancer and
Normal Cells

• Quantitatively, as shown for gene B, which is
  expressed at an abnormally high level, and gene
  A, which is not expressed at all.
• Qualitatively, as shown for gene C, which is
  mutated such that it produces an altered gene
  product.

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Choice of Target

• Design antisense to target the mRNA of the gene
  that is overexpressed in the cancer cell relative
  to normal cell. (Gene B in example)
• -gt downregulation of protein expression
• Target mRNA of mutant gene found only in cancer
  cells. (Gene C in example)

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Chromosomal Translocation

• Transposition of two segments of different
  chromosomes results from abnormal breakage and
  refusion of the segments. New sequence not found
  in normal cells.
• A mutation that often occurs in cancer.

translocation
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Antisense is unique to translocated gene.
Normal gene C
Gene C is altered due to translocation
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Optimal Length of Antisense Oligonucleotide
Complexity Total nucleotide bases Theoretical
oligo length to hybridize to one
unique sequence Human genome 3 x 109
bp of DNA 16mer Cellular RNA 1.5 x 107 nt
of RNA 12mer
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The length of the antisense oligonucleotide is
typically 16 nucleotides
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Targeting In theory Target AUG start
codon In practice Trial and error
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Limitations of Antisense Oligonucleotides

• Poor stability
• Poor cellular uptake
• Presence of Non-antisense effects

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Poor Stability of Antisense Oligonucleotides
Antisense contains phosphodiester bonds T1/2 1
hour in serum (T1/2 half life)
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Phosphodiester bonds are susceptible to
degradation
Cleaved by nucleases
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Oligonucleotide backbones are modified to
improve stability
O O P X O
X O-, normal phosphodiester bond found in RNA
and DNA X S-, phosphorothioate X CH3,
methylphosphonate
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Stability of RNA oligonucleotides in 95 serum at
37 C

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Normal RNA

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Modified RNA
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Currently all ASOs in clinical trials have a
chemically modified backbone

• Phosphorothioates - First generation
• Second generation modifications being developed

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Enhancing Cellular Uptake of ASOs

• Antisense are formulated with cationic
  surfactants to enhance cellular uptake.
• liposomes
• Microinjection

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Cationic Surfactants
Polar head ()
Hydrophobic Tail (Hydrocarbon chain)
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Liposomes
Polar head, neutralizes the negative charge from
the antisense.
Hydrocarbon tail, penetrates the cell membrane
(lipid bilayer).
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Nucleic acid
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ASO in Clinical Trials or Approved
Antisense Disease Target Gene
Target Fomivirsen(Vitravene) CMV retinitis CMV
G3139 Cancer Bcl-2 GEM92 HIV ISIS3521
(Affinitac) Cancer protein kinase
C GEM231 Cancer protein kinase
A Resten-NG restenosis c-myc EPI-2010 asthma
Alicaforsen psoriasis ICAM-1
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Vitravene (fomivirsen sodium injection)
Retina with fungus
Intra-occular injection
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Vitravene (fomivirsen sodium injection)

• Antisense to cytomegaloviral RNA
• Phosphorothioate antisense oligo
• IC50 in vitro to target is 0.06 µM
• comparison to IC50 of ganciclovir 2 µM
• First antisense oligo to gain FDA approval
• However, mechanism of action controversial

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G3139, Antisense Oligo in Clinical Phase III

• Antisense to Bcl-2 mRNA
• 18-mer phosphorothioate antisense oligo
  complementary to the first 6 codons of Bcl-2
  protein coding region.
• Clinical trials in both blood borne cancers and
  in melanoma.

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Bcl-2, A Target for ASO

• Inhibitor of apoptosis (programmed cell death)
• Discovered from a chromosomal translocation in
  B-cell non-Hodgkins lymphomas
• Gene is overexpressed in many blood borne cancers
  leading to pathogenesis of the disease (i.e.,
  cancer) and resistance to anticancer drugs

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Bcl-2 Prevents Caspase-9 Activation
p53
Bax
Apaf-1 Cytochrome C Caspase-9
Bcl-2
p53
Bid
Caspase-3
Caspase-8
apoptosis
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BCL-2 is Down-regulated
Fig. 1. BCL2 down-regulation after 5 days of
G3139 treatment in melanoma biopsy samples from
patient 12. BCL2 protein is 70 lower on day 5
by scanning densitometry, normalized against
changes in the actin band.
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Results of Clinical Trial with G3139

• Melanoma patients treated with G3139 and
  dacarbazine (alkylating agent)
• Found to induce apoptosis in patients tissue
• First antisense trial in which downregulation of
  target protein (Bcl-2) in target tissue was shown
  
• No dose-limiting toxicity
• 6 of 14 patients showed antitumor responses
• (1 complete, 2 partial and 3 minor)

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Response of G3139 in Melanoma Patient 12
A skin metastases B computed tomography of
pelvis.
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Non-antisense Action of Oligonucleotides

• Immune stimulation is an undesirable side-effect
  of certain antisense oligos.
• Immunostimulation strongly dependent upon
• 1) Presence of unmethylated CpG dinucleotides
  (e.g., GTCGTT) within the antisense oligo.
• 2) Phosphorothioate backbone, regardless of
  the sequence.

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DNA Methylation
Methylated cytosine
Unmethylated cytosine
CH3
A C G T A T C G T G T T A T G T
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T G C A T A G C A C A A T A C A
CH3
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DNA Methylation

• Formation of 5-methylcytosine by methylation of a
  cytosine base in DNA.

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What is the role of DNA methylation in vertebrate
cells?

• Methylation of CpG islands frequently occur in
  the promoters of many genes.
• The DNA of inactive genes is more heavily
  methylated than the DNA of active genes.
• High methylation low transcription

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What does DNA methylation have to do with
antisense oligos?

• Presence of unmethylated CpG dinucleotides within
  the antisense oligo causes immunostimulation.
• Evolutionary conserved immune defense mechanism
  to destroy bacterial DNA that will not undergo
  methylation.
• Mechanism is exploited to design antisense oligos
  containing CpG dinucleotides that will trigger
  immune system to destroy cancer cells.
• G3139 is an example of an antisense oligo that
  contains CpG dinucleotide.

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RNA Interference (RNAi) Aka Gene Silencing
Long double-stranded RNAs (gt200 nt) can be used
to silence the expression of target genes. Upon
introduction, the long dsRNAs enter the RNA
interference (RNAi) pathway.
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RNAi - Mechanism of Action Long dsRNA fragments
are cut into smaller pieces gt siRNAs
RISC RNA-induced silencing complexes
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The Mechanism of RNA Interference (RNAi)
First, the dsRNAs get processed into 20-25
nucleotide (nt) small interfering RNAs (siRNAs)
by an RNase III-like enzyme called Dicer. Then,
the siRNAs assemble into endoribonuclease-containi
ng complexes known as RNA-induced silencing
complexes (RISCs). The siRNA strands are then
unwound to form activated RISCs. The siRNA
strands subsequently guide the RISCs to
complementary RNA molecules, where they cleave
and destroy the cognate RNA.
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RNAi Continued
Problem In mammalian cells, introduction of long
dsRNA (gt30 nt) initiates a potent antiviral
response (activates the interferon system) ---gt
nonspecific inhibition of protein synthesis and
RNA degradation. Solution The mammalian
antiviral response can be bypassed by the
introduction or expression of siRNAs.
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Outline of siRNA Mechanism
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First siRNA Clinical Trial
In November 2004, Sirna Therapeutics - worlds
first clinical study of a chemically optimized
siRNA, Sirna-027. Sirnas Phase I trial is
testing Sirna-027 in the treatment of patients
with the "wet" form of age-related macular
degeneration (AMD).
Sirna-027 is a chemically modified short
interfering RNA (siRNA) targeting Vascular
Endothelial Growth Factor Receptor-1
(VEGFR-1). Sirna's siRNA treatment inhibits
destructive blood vessel growth inside the
corneas of mice. By targeting VEGFR-1,
Sirna-027 is designed to shut down activation of
pathologic angiogenesis initiated by VEGF.
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ASO vs. siRNA
ASO siRNA ssDNA dsRNA 16mer 21-23bp Sim
ple MOA Complex MOA - oligo binds to mRNA -
siRNA complex binds - no protein synthesis or -
RISC forms mRNA degraded - siRNA binds to
RNA target - mRNA degraded