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Anti sense technology
Anti sense technology was first effectively used in plants to alter the
level of various degradative enzymes or plant pigments.
• Example -Tomato. Anti sense was used to block the one that is
involved in spoilage, thereby increasing the length of time a
tomato could be sold.
The technology was rapidly applied to mammalian cells in 1992
.
Making a Protein—Transcription
• First Step: Copying of genetic information from DNA to RNA
called Transcription
Why? DNA has the genetic code for the protein that needs to be made, but proteins are made by the ribosomes—ribosomes are outside the nucleus in the cytoplasm.
DNA is too large to leave the nucleus (double stranded), but RNA can leave the nucleus (single stranded).
• Part of DNA temporarily unzips and is used as a template to assemble complementary nucleotides into messenger RNA (mRNA). • mRNA then goes through the pores of the nucleus with the DNA code and attaches to the ribosome.
Making a Protein—Translation
• Second Step: Decoding of mRNA into a protein is called Translation.
• Transfer RNA (tRNA) carries amino acids from the cytoplasm to the ribosome.
These amino acids come from the food we eat. Proteins we eat are broken down into individual amino acids and then simply rearranged into new proteins according to the needs and directions of our DNA. •A series of three adjacent bases
in an mRNA molecule codes for a specific amino acid—called a codon.
•A triplet of nucleotides in tRNA
that is complementary to the codon in mRNA—called an anticodon.
•Each tRNA codes for a different
amino acid. Amino acid
Anticodon
• mRNA carrying the DNA instructions and tRNA carrying amino acids meet in the ribosomes.
• Amino acids are joined together to make a protein.
Polypeptide = Protein
BASIS FOR ANTISENSE TECHNOLOGY.
DNA carries all the instruction for building the protein.
DNA normally has two strand
•Sense strand
•Anti sense strand
The sense strand carries nucleic acid bases in an order that specifies which amino
acid should be assembled to produce the protein.
The complementary strand or “antisense” strand is used as a template for
assembling a complementary strand of messenger RNA (mRNA) in the process
called transcription.
The mRNA will have the same sequence as the sense strand of DNA but its
made up to ribose sugar instead of deoxy ribose sugar in the nucleoside
backbone.
The mRNA in further processed in the nucleus by capping and splicing into the
cytoplasm (spliced to remove non coding sequence and caping to protect from
cellular environment with 5’ cap and poly a tail.
In cytoplasm, the mRNA hooks up with ribosome’s where the protein
protection can start. Every three nucleotide in the mRNA molecule codes for
the specific amino acid and are appropriately called a codon.
The codon pair with anticodon of tRNA that has attached to an amino acid.
In this manner a polypeptide is formed.
How Antisense Works?
¾Traditional drug therapies -designing compounds that block or inhibit
disease-causing proteins,
¾Antisense therapies -preventing the production of disease-causing
proteins.
¾Antisense drugs are based on
¾Small DNA-like or RNA-like constructs that bind to the protein-coding
strand of genetic message (mRNA)
¾ Blocking the translation of the disease-causing protein.
¾ By binding to mRNA, antisense drugs prevent the genetic code from
being read by the ribosome, which is responsible for translating and
manufacturing proteins
¾ Additionally, the bound antisense/mRNA complex is enzymatically
degraded so the protein cannot be synthesized.
¾ Natural DNA will be digested by enzymes and cause immune response
¾ Synthetic DNA cannot be recognized by enzymes, so they are stable and
may not cause immune response
¾ So, synthetic DNA can selectively block gene expression
Modification in the base, sugar and phosphate moieties as well as in the
length of polymers - create molecules with enhanced or more selective
affinity for specific sites on the RNA,
to enhance nuclease stability,
to improve cellular uptake and distribution and optimize
distribution and clearance.
Design of Antisense drug
Satisfy many conditions
•It must be administered in a manner that the antisense drug delivers it to the
site of action in the body without the drug being degraded by the nucleases,
which are common in human cells.
•It must get into the cell of the target tissue and colocalize with its target
RNA at a sufficient concentration for a bimolecular reaction to occur.
•It must have structure that favours association with the target RNA and be
designed to bind to the RNA region that is vulnerable to binding.
Sense strand, non-coding strand
DNA
Antisense strand, coding strand,
template for mRNA synthesis
mRNA
Sense sequence
DNA drug
Antisense sequence
Why Can DNA Be Used as Drugs?
Mono-genetic disorder
¾ Currently, a total of ~4,000 genetic disorders are known
¾ Some are single genetic disorder
¾ Changes (mutations) of the sequence of one gene (DNA)
9 Point mutation
9 Deletion
9 And more
¾ The mutated genes produce proteins that cannot function properly,
diseases occur
¾ Examples: Sickle-cell anemia, Cystic fibrosis (1/3900, most common, difficult
breathing, die in 20s-30s, no cure), Color blindness
¾ Mutations occur in many genes
¾ Do not have a clear cut of inheritance
¾ But do “run in families”
¾ The mutated genes produce proteins that cannot function properly,
diseases occur
¾ Difficult (not impossible) to study and treat because direct cause is
unknown
¾ Examples: Heart disease, hypertension, diabetes, obesity, cancers,
low IQ
¾Antisense drugs are based on small DNA like or RNA like constructs
that bind to the protein coding strand of genetic message (mRNA)
blocking the translation of the disease causing protein.
¾The DNA drug binds to the mRNA (A-U, G-C)by Watson crick base
paring in doing so, they induce a nuclease (RNase H) that cleaves the
mRNA at the site of binding or can physically block translation or other
steps in the mRNA processing and transport.
¾Because no disease-causing protein, disease is cured
Technical advantages
¾Mature technology (20 years in development)
¾Drug discovery and research is faster andmore predictable
¾Compounds are potentially more selective,effective and less toxic
¾Broad disease application
¾Dosing advantages (route and frequency)
“0”Generation
“1st”Generation
“2nd”Generation
Phosphodiester(P=O)
Phosphorothioate(P=S)
2’MOE (modifiedP=S)
Characteristics:
(1) Highly unstable to nucleases
(2) Good affinity to target
mRNA
Characteristics:
(1) Better stability to nucleases
but still degrades
(2) Decreased affinity to target
mRNA
Characteristics:
(1) Best stability to nucleases
(2) Increased affinity to target
mRNA
Result:
(1) Rapidly degrades
(2) Never advances to human
trials
Result:
(1) Unknown tissue
concentration
(2) Unknown target regulation
(3) Daily dosing
Result:
(1) Confirmed tissue
concentration
(2) Confirmed target regulation
(3) Once a week dosing
The first successful backbone chemistry modified the phosphodiester
structure in the DNA backbone to a phosphorothioate structure.
O
O
O
Base
O
O
P
O
O
O
O
O
O
Base
O
O
O
Phosphodiester
O
O
Base
O CH2CH2 OCH3
P
P
O
S
S
Base
O
O
O
Base
O
Phosphorothioate
O
2'MOE
Base
O CH2CH2 OCH3
PHOSPHOROTHIOATE CLASS
Phosphorothioate class of compounds are
¾ Negatively charged
¾Chiral at each phosphorothionate and
¾Much more resistant to nuclease digestion than the parent phosphodiester
compound.
¾Compound length has been determined empirically.
¾Molecules must have at least 7 to 8 complementary bases and the
compounds with 18 or 20 bases generally considered to be optimum.
Disadvantages
¾
Plasma half lives only one hour,
¾
Low affinity for their target mRNA and
¾
Toxic side effects, especially the immune stimulation and clotting
abnormalities.
SECOND GENERATION DRUGS
Second generation drugs are composed of both RNA like and DNA like
nucleotides, while first generation drugs are entirely DNA like. With
increased potency, second generation drugs are more active at lower doses
Antisense drugs are being researched to treat
cancers (including lung cancer, colorectal
carcinoma, pancreatic carcinoma, malignant
glioma and malignant melanoma), diabetes,
Amyotrophic lateral sclerosis (ALS), Duchenne
muscular dystrophy and diseases such as asthma
and arthritis with an inflammatory component
Current Research
Current Status of Antisense Drugs
2007
¾Fomivirsen-First antisense drug marketed
-21-mer phosphorothioate oligodeoxy nucleotide.
Use: Cytomegalovirus (CMV) retinitis in patients with AIDS.
The agents that are in clinical trials
¾ALICAFORSEN (ISIS 2302)
¾APRINOCARSEN (AFFINITAK)
Aprinocarsen is a phosphorothionate antisense agent showed activity in
phase I and II clinical trials against cell lung cancer when used in
combination with Cisplatin.
¾OBLIMERSEN SODIUM (GENASENSE)
It is investigated for use in the treatment of myeloma.
¾MIPOMERSEN
Mipomersen is in phase III clinical trial for some type of high cholesterol.