Nucleic Acids as
Nucleic Acids as Therapeutic Agents
Many human disorders often results from the
overproduction of a normal protein.
Single stranded oligonucleotides could be used to
hybridize to genes (antigene) or mRNAs (antisense)
to reduce transcription or translation thereby
reducing protein production.
Synthetic RNA/DNA molecules called aptamers
bind to proteins and prevent them from functioning.
Ribozymes can be engineered to target mRNA.
RNAi may be used instead of antisense RNAs or
specific mRNAs by
Antisense RNA: This is the backwards
compliment of the normal RNA for a target
gene. It is designed to bind the target mRNA
and prevent translation.
Demonstration is done in maligant glioma and
prostate carcinoma cells which result from
over production of insulin-like growth factor 1
and insulin-like growth factor 1 receptor,
In both cases, cultured cells make large
tumors when injected into rats.
Cultured cells transfected with the proper
antisense gene in an expression vector do not
In both cases, the expression of the antisense
cDNA is controlled by a Zn sensitive promoter
from a metallothionein gene. Expression is
turned on by presence of ZnSO4.
Following transfection into tumor-causing cell,
when low level of ZnSO4 are added, the cell have
Antisense oligonucleotides: short sequences
designed to hybridize with mRNAs and prevent
Must hybridize to the target mRNA, be resistant to
degradation, and be delivered into cells easily.
Usually 15-24 nucleotides long.
Can be designed from nearly any portion of the
mRNA (5’ to 3’ ends of mRNAs, intron-exon
boundaries, and regions that form stem loops
have all been effective.)
Proteomic analysis of cellular proteins can be
used to determine if the protein production is
The oligodeoxynucleotides are susceptible to
degradation by intracellular nuclease.
Various modifications have been tried to make
the oligonucleotides more resistant to
degradation without affecting the ability of
antisense to hybridize to the target sequence.
Modification includes adding sulfur to the
phosphodiester backbone (phosphorthioate).
The RNA-DNA duplex activates the Rnase H
which cleaves hybrid molecules.
It is considered to be “first generation”
The second generation antisense therapeutic
agents typically contain alkyl modifications at the
2’ position of the ribose.
It is less toxic and more specific than
The third generation antisense oligonucleotides
contain a variety of modifications within the ribose
ring, and/or the phosphate backbone, as well as
being less toxic.
Some modifications are made to both enhance
stability and facilitate the binding to the target site.
Free antisense oligonucleotides are not easily
internalized by cells.
Delivery is often accomplished using liposomes
to facilitate cellular uptake.
Utility of antisense oligonucleotides has been
demonstrated in various cases.
Narrowing stenosis of coronary arteries:
alleviated by angioplasty but recurs quickly in
40% of patients because of a healing reaction.
When antisense oligonucletides that targeted
mRNA for proteins essential for cell cycle were
applied to rats, restenosis was reduced by 90%.
Smooth muscle cell proliferation implicated in
atherosclerosis, hypertension, diabetes mellitus,
and the failure of coronary bypassed grafted are
presumably controlled by similar antisense
Correction of a mutant splice site with an
antisense oligonucleotide can be used to treat
Ribozymes: naturally occurring catalytic RNA
molecules that are ~40-50 nucleotides in
length with separate catalytic and substrate
Comparing to protein therapeutics, it is less
likely to evoke an immune response.
The substrate-binding site combines by
The catalytic portion cleaves the target RNA at
a specific site, thereby protein production is
By altering the sequence, a ribozyme can be
engineered to cleave any mRNA sequence.
Representation of hammerhead (A) and hairpin
(B) ribozyme-mRNA substrate complexes.
Ribozymes may also be delivered directly by
injection or liposomes, but must be chemically
modified to avoid quick degradation.
Also being tested for utility in fighting viral
infections and prevent the accumulation of
Attempting to make synthetic DNA enzymes
(deoxyribozymes) because DNA is much more
Aptamers are nucleic acid sequences, RNA or
DNA, that bind tightly to proteins, amino acids,
drugs, or other molecules.
They are usually 15-40 nucleotides long, have
highly organized secondary and tertiary
structures, and bind with high affinity.
The advantages are their high specificity,
relative ease of production, low or no
immunogenicity, and long-term stability.
Aptamers are typically selected by a procedure
known as SELEX (systematic evolution of
ligands by exponential enrichment.)
Interfering RNAs (RNAi): Adding dsRNA
versions of a gene to a cell reduces the
expression of the native gene by gene silencing.
Occurs as a natural mechanism in many
organisms where it is thought to be involved in
protection from viruses and transposons.
Upon introduction od dsRNA into a cell, Dicer
complex binds to the RNA and cleaves it into an
siRNA containing ~21 bp.
The siRNA becomes part of RISC, directing the
cleavage of the complementary mRNA.
Antibody genes approach came from the
observation that rainbow trout could be
protected against hemorrhagic septicemia virus
by passive immunization.
Instead of administering a purified antibody, it is
possible to inject an animal with DNA encoding
The cloned gene-vector constructed was
injected into the circulatory system of rainbow
trout, survival increased when challenged.
The next step would be to create transgenic fish
with antibody genes that confer passive
immunity to various diseases.
The effectiveness of any therapeutic agent
depends upon the ability to deliver that agent to
tissues where it is required.
Systemic introduction often leads to the
accumulation in tissues where the agent is not
required and sometimes results in serious side
Viral vectors that deliver small nucleic acids to
specific cellular targets have been developed.
However, there are some safety concerns about
using viral vector.
There are several methods that have been
Local injection at the site of pathology
Packaging into cationic liposomes
Conjugated to another molecule
There are a number of concerns about human gene
How will the cell of target for correction be
How will the gene be delivered?
What proportion of the target cells must acquire
the input gene to counteract the disease?
Does transcription of the input gene need to be
precisely regulated to be effective?
Will it cause alternative physiological problems?
Will it be maintained or repeated treatments be
The viral vector are
because they do not
have a pol gene.
Despite the advantages of viral vectors, they are
often immunogenic, costly to maintain, and
difficult to produce on a large scale without
The least complicated non-viral gene delivery
system is the introduction of naked DNA.
To avoid degradation of introduced DNA, DNAmolecular conjugates ha.e been developed.
However, there are two major limitations.
The frequency of transfection is often too low to
The duration of gene expression is too brief.
With the cell receptor-binding sites arrayed on
the outside of the DNA-molecular conjugate, the
complexes bound specifically to the specified
A human artificial chromosome (HAC) as
The DNA carrying capacity would be very large.
This type of vector should have long-term
stability and sustained expression.
However, there are some issues must be
Will HACs be efficiently introduced into the nuclei
of target cells?
Will effective levels of therapeutic gene
expression be maintained for extended periods of
The efficacy of using
a particular gene
along with a specific
delivery system is
tested on a small
mice to ensure not
only the efficacy but
also no unexpected
A conjugate of cholesterol and an siRNA to
mediate the uptake of siRNA into cells.
Study are underway to improve the delivery of
siRNA to treat various diseases.
Use of a nonpathogenic strain of E. coli to deliver
siRNAs to certain tissues.
Negatively charged siRNA bind to positively
The complex greatly facilitates the delivery of
siRNAs to specific tissues.
A single-chain Fab fragment is fused to the
positively charged polypeptide protamine, which
binds to negatively charged siRNA.
The Fab fragment acts to deliver the siRNA to
Since aptamers also bind specifically to a target
protein, they can be used for targeting system.