Antisense oligonucleotides

Nucleic Acid Chemistry

Third term 2022-2023

Randy Bryant

Department of Biochemistry and Molecular Biology

Johns Hopkins Bloomberg School of Public Health

 Antisense oligonucleotides (ASO)

Antisense oligonucleotides are designed to bind to

specific mRNA molecules

And inhibit the expression of targeted proteins that

are associated with specific diseases, thereby
resulting in a desired therapeutic effect

Two general types: Steric-blocking ASOs

RNase H-inducing ASOs
 Steric-blocking antisense oligonucleotides

Antisense oligonucleotides bind to a target mRNA to form

an ASO-mRNA hybrid duplex

The formation of the hybrid duplex may:

1. Prevent ribosomes from assembling around the

mRNA to initiate protein synthesis

2. Prevent the mRNA from being able to pass through

ribosomes to direct protein synthesis

Either mechanism may result in a reduced expression of

the targeted protein
Steric-blocking antisense oligonucleotide
 RNase H

Ribonuclease H (RNase H) is an enzyme found in all cells

Catalyzes the hydrolysis of the phosphodiester bonds of

the RNA strand in DNA-RNA duplex substrates

Produce 3´-hydroxyl and 5´-phosphate-terminated RNA

cleavage products

Normal cellular function is to remove RNA primers from

the lagging strand during DNA replication
 RNase H-inducing antisense oligonucleotides

Antisense oligonucleotide binds to a target mRNA to

form an ASO-mRNA hybrid duplex

RNase H degrades the mRNA strand of the ASO-mRNA

hybrid duplex

Results in a reduced expression of the targeted protein

RNase H-inducing antisense oligonucleotide

[Link]
 RNase H-inducing antisense oligonucleotides

Both the RNase H enzyme and the ASO remain intact

at the end of the hydrolysis reaction

A single ASO can therefore mediate the degradation

of multiple mRNA molecules

In contrast, a simple steric-blocking ASO can only block

one mRNA molecule
 Selectivity

There are 3 × 109 base pairs in the human genome

A DNA sequence of 17 nucleotides will be expected to

occur randomly at a frequency of 1/417 = 1/1.7 x 1010

So it may be feasible to target specific mRNAs using

ASOs that are 17 nucleotides long (or longer)
 Cellular nucleases

DNA oligonucleotides can be degraded by deoxyribonucleases

(DNases) that are present in cells

As a result, unmodified DNA oligonucleotides do not exhibit

a significant antisense effect in cells
 Deoxyribonucleases

Three general types: 5´-end

H2 O
5´-exonuclease

H2 O
endonuclease

H2 O
3´-exonuclease

3´-end
 Nuclease-resistant modifications

Antisense oligonucleotides can be modified chemically

to make them more resistant to nuclease degradation

Two general approaches:

Phosphodiester bond modifications

Deoxyribose sugar modifications

 Phosphodiester bond modifications

phosphorothioate methylphosphonate phosphoroamidate

[Link]
Phosphorothioate oligonucleotides

Most commonly used phosphodiester

modification in ASOs

Replacement of non-bridging oxygen

with sulfur makes the phosphorus
center less electrophilic

Increases resistance to hydrolysis by

cellular nucleases

[Link]
 Synthesis of phosphorothioate oligonucleotides

Phosphorothioate oligonucleotides can be synthesized

by the phosphoramidite method:

Replace the oxidation step (which converts the phosphite

linkage to a phosphate)

With a sulfurization step (to convert the phosphite linkage

to a phosphorothioate)
 Sulfurization reagent

tetraethythiuram disulfide
(TETD)

TETD can be used to produce phosphorothioate linkages

during phosphoramidite oligonucleotide synthesis
 Phosphoramidite sulfurization reaction

TETD

phosphite
(III)

phosphorothioate
(V)

[Link]
Stereochemistry of phosphorothioate linkages

RP SP

Phosphorothioate linkages are chiral centers

Synthetic phosphorothioate oligonucleotides consist of
a mixture of stereoisomers
[Link]
Phosphorothioate antisense oligonucleotides

Are are easy to prepare using automated DNA

oligonucleotide synthesizers

Have good resistance to degradation by cellular

nucleases

Do not interfere with the degradation of the mRNA

strand in ASO-mRNA duplexes by RNase H
 Fomivirsen

Is a first-generation phosphorothioate antisense drug

Was the first antisense drug approved by the FDA

Registered in 1998 as a treatment for cytomegalovirus

(CMV)-induced retinitis in AIDS patients

Kole et. al. Nature Reviews Drug Discovery 11, 125 (2012)
 Fomivirsen

Is a 21-mer oligonucleotide with phosphorothioate linkages:

GsCsGsTsTsTsGsCsTsCsTsTsCsTsTsCsTsTsGsCsG

black = deoxyribose
blue = phosphorothioate

Is complementary to the mRNA of the major immediate-early

region proteins of human cytomegalovirus
Inhibits expression of the CMV protein IE2 (a transcriptional
activator of viral gene expression)
Fomivirsen

Brand name: Vitravene

Produced by Isis Pharmaceuticals

Used to treat cytomegalovirus retinitis

Administered by intraocular injection

 Deoxyribose sugar modifications

2´ 2´

2´-O-methyl-ribose 2´-methoxyethyl-ribose
(2´-OMe) (2´-MOE)

[Link]
2´-O-methyl-ribose

Most commonly used deoxyribose

modification in ASOs

The 2´-O-methyl sugars adopt the

C3´-endo conformation

Increases the stability of ASO-mRNA

duplexes

Also increases resistance to hydrolysis

by cellular nucleases
 2´-O-methyl phosphoramidites

DMT-2′-O-methyl-rA(bz) DMT-2′-O-methyl-rG(ib)

DMT-2′-O-methyl-rC(ac) DMT-2′-O-methyl-rU
 2´-OMe and 2´-MOE modifications

The 2´-OMe and 2´-MOE modifications do increase the

resistance of ASOs to degradation by cellular nucleases

However, mRNAs that are bound to 2´-OMe or 2´-MOE-

modified ASOs are not degraded efficiently by RNase H

Therefore, these modifications are generally used only

at specific locations in ASOs
 Antisense gapmer oligonucleotides

Typically about 20-nucleotides long with phosphorothioate linkages

Five nucleotides at each flank are modified with 2´-OMe or 2´-MOE

groups to increase resistance to exonucleases

Leaves a central 10-nucleotide gap with unmodified 2´-deoxy nucleotides

that allows the targeted mRNA to be cleaved by RNase H
Antisense gapmer oligonucleotides

Kole et. al. Nature Reviews Drug Discovery 11, 125 (2012)
 Mipomersen

Is a second-generation gapmer antisense drug

Was the second antisense drug approved by the FDA

Registered in 2013 for the treatment of homozygous

familial hypercholesterolemia

Kole et. al. Nature Reviews Drug Discovery 11, 125 (2012)
 Mipomersen

Is a 20-mer gapmer antisense oligonucleotide:

GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC

green = 2´-OMe-ribose
black = deoxyribose
blue = phosphorothioate

Is complementary to a sequence in the mRNA for apolipoprotein

B-100 (APOB-100)
Inhibits expression of APOB-100 and reduces the formation of
low-density lipoprotein cholesterol in the liver
Mipomersen

Brand name: Kynamro

Developed by Isis Pharmaceuticals

(licensed to Genzyme Corporation)

Used to treat homozygous familial

hypercholesterolemia

Administered by weekly injection

 Nusinersen

Is a splicing modulation antisense drug

Was approved by the FDA in 2016

Is licensed for the treatment of spinal muscular atrophy

associated with a loss-of-function mutation in the
SMN1 (survival of motor neuron 1) gene

Wurster and Ludolph, Ther Adv Neurol Disord 11, 1 (2018)

 Nusinersen

Is an 18-mer antisense oligonucleotide:

UsCsAsCsUsUsUsCsAsUsAsAsUsGsCsUsGsG

green = 2´-MOE-ribose
blue = phosphorothioate

Is complementary to a sequence in the mRNA for the SMN2

(survival of motor neuron 2) gene
Does not induce RNase H degradation of the SMN2 mRNA
Instead, blocks an alternative splicing of SMN2 mRNA and
increases levels of functional SMN protein
SMN2 splicing
Nusinersen

Brand name: Spinraza

Developed by Ionis Pharmaceuticals

(licensed to Biogen)

Used to treat spinal muscular

atrophy associated with mutations
in SMN1 gene

Administered by injection into

cerebrospinal fluid
 Locked nucleic acids (LNA)

Base
Have a methylene bridge between
4´ the 2´-oxygen and the C4´-carbon
2´
of a ribose group

Base
3´ The methylene bridge locks the ribose
in the C3´-endo sugar conformation
2´
 Sugar pucker: DNA and LNA

DNA LNA
C2´-endo C3´-endo
 LNA oligonucleotides

The methylene bridge locks the ribose in a C3´-endo sugar

conformation

As a result, LNA oligonucleotides adopt the A-form helix

(which has a high duplex stability)

Entropic constraints in LNAs also result in significantly

stronger binding to complementary RNAs

The incorporation of LNAs into ASOs increases the affinity

for the RNA target and resistance to cellular nucleases
Locked nucleic acid phosphoramidites

DMT-LA(bz) DMT-LG(df)

DMT-LmC(bz) DMT-LT

[Link]
 Miravirsen

Is a third generation LNA-containing antisense drug

Is being developed by Santaris Pharma as a treatment

for hepatitis C

Targets a specific liver miRNA that is required for

hepatitis C virus (HCV) replication

First antisense drug to target a miRNA

 Miravirsen

Is a 15-mer LNA antisense oligonucleotide:

mC C A T T G T C A mC A mC T mC mC
s s s s s s s s s s s s s s

red = LNA (mC = 5-methylcytosine)

black = deoxyribose
blue = phosphorothioate

Is complementary to the liver-specific miRNA: miR-122

Prevents miR-122 from functioning in the replication of HCV

Peptide nucleic acids (PNA)

Have a backbone consisting of repeating

N-(2-aminoethyl)-glycine units linked by
peptide bonds

Bases are connected to the backbone

by methylene carbonyl linkers

[Link]
Peptide nucleic acid structure

A G C

methylene carbonyl
base linker

peptide bond

N-(2-aminoethyl)-glycine
unit

[Link]
 DNA and PNA oligonucleotides

DNA PNA

Inter-base distances in PNA are similar to those in DNA

 PNA-DNA duplex formation

PNA DNA

Base pairing occurs readily between PNA and DNA oligonucleotides

 PNA oligonucleotides

The uncharged backbone reduces the electrostatic repulsion

between oligonucleotides in PNA-DNA and PNA-RNA duplexes

As a result, PNA oligonucleotides bind to complementary

DNA and RNA oligonucleotides with higher affinity than do
normal DNA and RNA oligonucleotides.

Are not degraded by cellular nucleases or proteases

But also do not induce RNase H activity – so are only used

as steric blocking ASOs
 Limitations of PNA oligonucleotides

PNA oligonucleotides have low solubility in aqueous solution

(because they have an uncharged backbone)

PNA oligonucleotides also tend to form aggregates in solution

due to hydrophobic interactions between the bases

To overcome these limitations, PNA oligonucleotides have

been conjugated to hydrophilic molecules (such as charged
amino acids, DNAs, and polyethylene glycol polymers)