Basic Science_20250704_095218_0000.pdf 00

PRESENTATION
By Isabel Mercado

BACKGROUND
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PROJECT GOALS
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Basic Science:
1.Genes contain the information necessary to produce proteins.
2.Protein production occurs in two phases called transcription and
translation.
3.In the transcription phase, the DNA strand is used as a template for
manufacturing an mRNA molecule.

4.mRNA is responsible for communicating the genetic message in the DNA
to the cell so that protein production can take place.
5.In the translation phase, the mRNA travels to the ribosome, and carries
out protein synthesis.

Overview
Currently, a total of ~4,000 genetic disorders are known.
The mutated genes produce proteins that cannot function properly,
leading to the occurrence of the diseases.
Examples: Sickle-cell anemia, Cystic fibrosis, Color blindness.
What are the ways?
1.Oligonucleotides
2.Ribozyme
3.RNAi
4.Nanotechnologies like LNA and S-ON

What is Antisense Technology?
➤In this technique Short segments of single stranded DNA ca oligo de
oxy nucleotides introduced.
➤ These oligonucleotides are complementary to the mRNA,
➤Antisense technology pre the synthesis of specific prote
➤Antisense technologies are a suite of techniques that, together form a
very powerful weapon for studying gene function and for discovering
more treatments of disease. specific

What are Antisense Oligonucleotides?
➤Oligonucleotides are unmodified or chemically modified single stranded
DNA molecules In general, they are relatively short (13-25 nucleotides)
➤The antisense effect of oligonucleotide sequence was first demonstrated in
1970s Zamecnik and Stephenson, in Rous sarcoma virus.
➤AS-ONs usually consist of 15-20 nucleotides, which are complementary to
their target mRNA.
➤When these AS-ON combi with target mRNA, a DNA/PNA hybrid form,
which degraded the enzyme RNase H.

RNase H
RNase H is a non-spe endonuclease, catalyzes cleavage of RNA via hydro
mechanism.
RNase H has ribonuclease activity cleaves the 3'-O-P bond of RNA in a
DNA/RNA duplex.
RNase H activity, leading to mRNA degradation,
induces translational arrest by steric hindrance of ribosomal activity,
interferes with mRNA maturation by inhibiting splicing or
destabilizes pre-mRNA in the nucleus, resulting in downregulation of
target protein expression

Blocking Translation
When mRNA forms a duplex with a complementary antisense RNA
sequence, translation is blocked.
This may occur because
1. The ribosome cannot gain access to the nucleotides in the mRNA
2. Duplex RNA is quickly degraded by ribonucleases in the cell.

Types Of AS-ON
➤First generation AS-ON
➤Second generation AS-ON
➤Third generation AS-ON

A successful AS-ON depends on the followin characteristics:
➤ Unique DNA sequence
➤ Efficient cellular uptake
➤ Minimal nonspecific binding
➤ Target specific hybridization
➤ Non-toxic antisense construct
➤ Nuclease resistant to protect AS-ON

First generation Antisense
oligonucleotides:
* First synthesized by Eckstein and colleagues
* Phosphoro-thioate-deoxy-nucleotides are the first generation
oligonucleotides and have a sulfur atom replacing the non-bridging oxygen
of the sugar phosphate backbone. It preserves the overall charge and can
also activate RNaseH for the degradation of mRNA.

Characterstics of First generation
Antisense oligonucleotides
* Better stability to nucleases but still degrades.
* Can activate RNase H.
* Are highly soluble and have excellent antisense activity.
* They were first used as Antisense oligonucleotides for the inhibition of HIV.
* Cannot cross the lipid bilayer because of their charge and polarity.
* Complement activation due to their polyanoinic nature.
* Once in the circulation they can be taken up by many cell types and not just
the cell targeted leading to potential side-effects.

* Observed side effects seen in clinical studies performed on
humans include
* Thrombocytopenia
* Fatigue
* Fever
* Rashes
* Leukopenia
* There is also a transient inhibition of the clotting times shown
by an increased activated partial thromboplastin time (aPTT).

Second generation Antisense
oligonucleotides
* Second generation Antisense oligonucleotides containing nucleotides with
alkyl modifications at the 2' position of the ribose.
* 2'-O-methyl and 2'-O-methoxy-ethyl RNA are the most important member of
this class.

Characterstics of second generation Antisense
oligonucleotides
* Mechanisms of action for the 2' modified oligonucleotides do not rely on
RNase H activation but on translation arrest by blocking 80S ribosome complex
formation as well as with splicing interference.
* They were developed to try and avoid the toxicity associated with the first
generation AS-ONs.
* Show high binding affinity to target mRNA.
* Best stability to nucleases.
4G+59%
* Less toxic than first generation AS-ON.
* Higher lipophilicity compared to first generation AS-Ons.

Third generation Antisense
oligonucleonues
* Newest and most promising.
* Enhance binding affinity and biostability.
1.Peptide nucleic acids (PNAS)
2. Locked nucleic acid (LNA)
3. Tricyclo-DNA (tcDNA)
4.Cyclohexene nucleic acids (CeNA)

Peptide nucleic acids (PNA)
* In PNAs the deoxyribose phosphate
backbon replaced by polyamide
linkages.
* The property of high-affinity nucleic
acid binding can be explained by the lack
of electrostatic repulsion because of the
absence of negative charges on the PNA
oligomers.
* The antisense mechanism of PNAs
depends on steric hindrance

Locked nucleic acid (LNA)
* The ribose ring is connected by a methylene bridge
(orange) between the 2'-O and 4'-C atoms thus "locking"
the ribose ring in the ideal conformation for Watson-Crick
binding.
* Thus the Pairing with a complementary nucleotide
strand is more rapid and increases the stability of the
resulting duplex
.* LNA oligonucleotides exhibit unprecedented thermal
stability when hybridized to a complementary DNA or
RNA strand.
* LNA based hepatitis C drug called Miravirsen, targeting
miR-122, is in Phase II clinical testing as of late 2010.

Tricyclo-DNA (tcDNA)
* Chemically, tc-DNA deviates from natural DNA by three additional C-
atoms between C(5') and C(3

Cyclohexene nucleic acids (CeNA)
The replacement of the furanose moiety of DNA by a cyclohexene ring
gives Cyclohexene nucleic acids or CeNA.
CeNA is stable against degradation in serum and a CeNA/RNA hybrid is
able to activate RNase H, resulting in cleavage of the RNA strand.

What is Biodistribution?
Biodistribution = how a drug spreads and accumulates in different
tissues after administration.
For antisense oligonucleotides (ASOs), it’s crucial because their
effectiveness depends on reaching the right tissue (like liver, muscle,
brain).
Also helps predict and prevent off-target effects or toxicity.

Why Biodistribution is Critical in ASO
Therapy
Ensures the ASO reaches its target mRNA in the right tissue.
Influences dose required, therapeutic window, and side effects.
Determines whether drug is accumulating too much in non-target
organs like kidney/liver.
Helps in designing delivery systems (e.g., nanoparticles, ligands).

Factors That Influence Biodistribution of ASOs
1. Route of Administration
1.IV: Fast, systemic
2.SC: Slow, prolonged
3.Intrathecal: Direct CNS delivery
2. Molecular Properties
1.Size: ~15–25 nucleotides
2.Charge: Highly negative (limits passive diffusion)
3. Chemical Modifications
1.Phosphorothioate backbone → ↑ stability
2.2'-O-methyl or MOE → ↑ cellular uptake

4. Plasma Protein Binding
Alters tissue distribution and half-life
Enhancing Biodistribution – Smart Strategies
1. Chemical Modifications
Enhance nuclease resistance
Improve tissue uptake
2. Ligand Conjugation (e.g., GalNAc)
Targets liver-specific receptors
3. Nanoparticles & Carriers
Liposomes, dendrimers for tissue targeting
4. Direct Routes (e.g., Intrathecal for CNS)
Bypass barriers like BBB

TREATMENT FOR B-THALASSEMIA AND
CYSTIC FIBROSIS
1.Antisense oligonucleotides were first used to restore normal splicing of
the B-globin gene in ẞ-thalassemia patients.
2.In this context, the antisense sequences were targeted to block the use
of cryptic splice sites in patients.
3.Splicing of the cystic fibrosis transmembrane conductance regulator
(CFTR), a protein which is spliced incorrectly in some cystic fibrosis
patients, could likewise be re-established via AONs.
4. Both approaches do offer therapeutic potential by the redirection of
normal splicing in patients.

Delivery vectors
* Delivery vectors can take care of toxicity. The vector can also
protect the drug from degradation and also from rapid clearance
from the body.
The vector must:
* Be of small size to allow intercalation between tissues.
* To allow intracellular transport, they must be non-toxic and stable
in the blood stream
* They must retain the drug when in the circulati and
* Must release it at its target before elimination.

* These are quite challenging tasks but many ideas have been
developed such as liposomes, protein or peptide constructs and
polymers.
* Liposomes are small microscopic spheres of one or more
concentric, closed phospholipid bilayer.
* Polar drugs such as 1st and 2nd generation oligonucleotides
can be entrapped in the internal space.

CONCLUSION

Basic Science_20250704_095218_0000.pdf 00

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