Nucleic Acids as
Therapeutic Agents
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
ribozymes.
 Inhibition of
translation of
specific mRNAs by
antisense nucleic
acid molecules.
 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,
respectively.
 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
develop tumors.
 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
decreased tumorigenicity.
 Antisense oligonucleotides: short sequences
designed to hybridize with mRNAs and prevent
translation.
 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
reduced.
 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”
therapeutic agents.
 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
phosphorothioate-modified molecules.
 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
therapeutics.
 Correction of a mutant splice site with an
antisense oligonucleotide can be used to treat
β-thalassemia.
 Ribozymes: naturally occurring catalytic RNA
molecules that are ~40-50 nucleotides in
length with separate catalytic and substrate
binding domains.
 Comparing to protein therapeutics, it is less
likely to evoke an immune response.
 The substrate-binding site combines by
complementarity.
 The catalytic portion cleaves the target RNA at
a specific site, thereby protein production is
reduced.
 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
chemical/antibiotic resistance.
 Attempting to make synthetic DNA enzymes
(deoxyribozymes) because DNA is much more
stable.
 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.
RNAi
RNAi
 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 antibody.
 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
effects.
 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
developed.
 Intravenous injection
 Local injection at the site of pathology
 Packaging into cationic liposomes
 Physical methods
 Conjugated to another molecule
 There are a number of concerns about human gene
therapy.
 How will the cell of target for correction be
accessed?
 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
required.
 The viral vector are
replication defective
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
high-level expertise.
 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
be effective.
 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
cells .
 A human artificial chromosome (HAC) as
therapeutic vector.
 Pros
 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
addressed.
 Will HACs be efficiently introduced into the nuclei
of target cells?
 Will effective levels of therapeutic gene
expression be maintained for extended periods of
time?
 The efficacy of using
a particular gene
along with a specific
delivery system is
tested on a small
animals, typically
mice to ensure not
only the efficacy but
also no unexpected
side effects.
 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
charged atelocollagen.
 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
specific cells.
 Since aptamers also bind specifically to a target
protein, they can be used for targeting system.