Download Human Gene Therapy:

 Human Gene Therapy:
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The primary goal of human medical genetic research
is to develop treatments for each of the many different
genetic diseases
A few gene products, the characteristics of the
diseases caused by mutations in the genes that encode
these products and
the current treatments for these diseases are given in
Table 21.1 Glick 2nd Ed
With the discovery of molecular basis of DNA
transformation in bacteria, researchers have speculated
that
human genetic disease might be cured in an analogous
way by introduction of the normal gene into the
appropriate somatic cell type
During 1980s, the prospect of human somatic cell gene
therapy became more likely and less theoretical as:
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gene isolation techniques were developed
eukaryotic expression vectors were created and
transgenic experiments with mice became more
routine
By 1990s, after exhaustive reviews by various
regulatory panels, the first human gene therapy trial
was initiated with two young girls with SCID
Which is the consequence of the absence of the
enzyme adenosine deaminase (ADA)
In this protocol, a cloned complementary DNA
(cDNA) for ADA was introduced into lymphocytes
that had been removed from each patient
After culturing, each subject received transfusion
with her own genetically engineered lymphocytes,
which synthesized ADA, at regular intervals for 2
years
Four years after the onset of trial, integrated vector
and ADA gene expression was evident in both
patients and
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The SCID symptoms were alleviated
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However, it is not completely clear whether
replacement therapy with a protected form of the
enzyme ADA (polyethylene glycol-ADA) or
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gene therapy per se was responsible for this
improvement
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Regardless, the trial showed that this form of gene
therapy was safe and helpful
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Similar results were also observed with other SCID
patients who received both types of therapies
simultaneously
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Consequently, these initial trials have been expended
to include more patients
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In the USA, the development of therapeutic agent for
humans usually passes through the four clearly
defined levels of study and testing before it can be
sanctioned for use by FDA. These are:
 Pre-clinical trial:
 Phase 1:
that includes expensive in vitro
experiments and research on
laboratory animals
trials with small number (6-10)
of human subjects who often are
used to test the safety of
products
 Phase 2:
trials with an expanded number
of human subjects to examine
whether the product is helpful
 Phase 3:
that includes a large number of
human samples and fully
comprehensive analysis of the
safety and efficacy of the
product, implementing the
information gathered from the
previous trials
Table 21.2 Glick 2nd Ed
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Because gene therapy is so new and the number of
diseases that could be treated this way are so diverse
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A large number of different strategies are being
examined
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At present, all research on human gene therapy is
directed toward correcting genetic defects of somatic
cells
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For ethical, safety and technical reasons, human
germ line therapy is not being examined
experimentally at this time
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Which entails introducing DNA into cells that can
form part of a human germ line and be passed on to
successive generations
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In its broadest sense, human somatic cell gene
therapy can be described as the introduction of a
fully functional and expressible gene into a target cell
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With the results that a specific genetic disease will be
corrected permanently
However, this simple characterization masked some
critical biological considerations. For example:
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How will the cells that are to be targeted for
correction be accessed?
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How will the remedial gene be delivered?
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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 over-expression of the input gene cause
alternative physiological problems?
Will the cells with the input gene be maintained
indefinitely or
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Will repeated treatments be required?
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Although somatic gene therapy is at an early stage of
development, some of these questions are beginning
to be answered for certain genetic disease
In addition, various strategies for implementing
somatic cell gene therapy are emerging and can be
grouped under two broad categories:
 Ex vivo gene therapy
 In vivo gene therapy
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In addition to gene therapy, other forms of nucleic
acid therapeutic agents that correct gene mutation in
vivo are being developed. Such as:
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Antisense oligonucleotides
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Genetically engineered RNA enzymes and
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Oliogonucleotides
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RNAi
 Ex-vivo Gene Therapy:
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Usually gene therapy involves the following procedure:
i. Collect cells from an affected individual
ii. Correct the genetic defect by genetic transfer into the
isolated cells
iii. Select and grow the genetically corrected cells
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iv. Either infuse or transplant them back into the patient
The use of a patient’s own cells (autologous cells)
ensures that no adverse immunological response occurs
after infusion or transplantation
Fig 21.1 Glick 2nd Ed
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It is important that the gene transfer protocol for ex vivo
gene therapy be efficient and
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That the remedial (therapeutic) gene be both stably
maintained and persistently expressed
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Currently, with human subjects, vectors derived from
mouse retrovirus are often used to meet these ends
However, because retroviruses can convert normal
cells into cancerous ones
It is essential that this possibility be at least
diminished and preferably completely abolished
In general, a wild-type retrovirus particle carries two
identical single-stranded RNA genomes that are each
organized into six regions
From 5’ end, these regions consist of a long terminal
repeat (5’-LTR) sequence
A non-coding sequence that is required for packing the
RNA strand inside a virus particles (encapsidation)
and is designated psi+ (ψ+, packing signal)
Three genes that encode for an internal capsid
structural proteins (gag), reverse transcriptase and
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and integrase function (pol) and an envelope protein
(env) and a 3’-LTR sequences
Fig 21.2 Glick 2nd Ed
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Briefly, the life cycle of a retrovirus includes the
following stages:
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Infection of target cell
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Production of DNA copy from RNA genome by
using the reverse transcriptase that is carried
within the virion
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Transport of viral DNA to the nucleus
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Integration of the viral DNA into the host
genome at one of a number of different
chromosomal sites
Transcription of mRNA from the viral DNA
that is driven by a strong promoter with in the
5’-LTR sequence
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Translation in the cytoplasm of the Gag, Pol and
Env proteins
Formation and packing of the viral capsid with two
RNA strands and reverse transcriptase molecules
Shedding of single-membrane-enveloped virions to
the exterior of the cell
Fig 6-23 Lodish 3rd Ed
After an entire sequence of retroviral DNA is cloned
into a plasmid, it can be constructed into vector
Fig 21.3 Glick 2nd Ed
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Although retroviral vector DNA can be used by itself
to transform cells, the efficiency of both delivery and
integration is very low
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Consequently, a methodology was devised to package
retroviral vector RNA into virus particles
The intact particles deliver the complete vector RNA
to a host cell at a high frequency
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Insert ~ 8kb
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Thereby virtually insuring that its DNA equivalent
will be integrated into the genome of a host cell
Fig 21.4 Glick 2nd Ed
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Retroviruses readily infect replicating cells, so
actively growing target cells are either treated with
purified packaged retroviral vector particles directly or
Co-cultivated with the packing cell line that is
producing the packaged retroviral vector particles and
Differentially selected to separate them from the
packing cells
In either case, the transduced target cells are tested to
ensure that
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The remedial gene product is synthesized
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Replication-competent retroviruses are not
being produced and
At one chromosome site within its
genome
At another chromosomal site
Both of these segments are
transcribed constitutively.
Because the ψ+ region is
absent and les than full size viral
RNA molecules are produced,
empty viral particles are formed
Genetically engineered
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The retroviral vector DNA has not been inserted into
site that either alters the growth properties of the cells
or interferes with normal culture functions
After these characterization, the transduced cells are
grown in culture, collected in large amount and
then introduced into the patient at various intervals
With hope that the cells will be maintained and
The disease that is being treated will be corrected
Individuals with genetic diseases that respond to bone
marrow transplantation are likely candidates for
ex vivo gene therapy
Fig 21.5 Glick 2nd Ed
 In-vivo Gene Therapy:
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In vivo gene therapy entails direct delivery of a
remedial gene into the cell of a particular tissue of
prospective patients
Fig 21.6 Glick 2nd Ed
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Retroviral vectors require that the target cells must be
dividing in order to be infected
However, in many of the tissues that might be treated
using gene therapy, the majority of cells are quiescent
Consequently, a number of strategies have been devised
which include using
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Viral and non-viral systems to deliver a
therapeutic gene to cells of the target tissue
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The variety of gene delivery systems for in vivo
therapy reflects both the diversity of potential target
tissues such as:
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skin, muscle, lung, brain, colon, spleen, liver
and blood cells and their location with in the
human body
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The attributes of an ideal in vivo gene delivery system
are high efficiency of uptake of the therapeutic gene
by:
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the target cells
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transportation of the therapeutic gene to the
nucleus of the target cell with a minimum of
intracellular degradation and
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sustained expression of the therapeutic gene at
a level that alleviates the condition
 Viral Gene Delivery Systems:
 Retrovirus Vector system;
 Adenovirus system;
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Infect a wide range of non-dividing human cells and
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have been used extensively used as live vaccines
against
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respiratory infections and
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gastroenteritis without side effects
These features make adenovirus a likely prospect for
delivering gene to target cells
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After infection of target cell with recombinant
adeno-virus, the DNA is passed into the cell nucleus,
where the therapeutic gene expressed
The recombinant DNA construct does not integrate
into chromosome and consequently
it does not persist for long periods
Therefore, adenovirus gene therapy requires periodic
administration with additional recombinant viruses
Fig 21.7 Glick 2nd Ed
 Adeno-associated Virus Vector System;
 It is small, non pathogenic, single-stranded human
DNA virus with size 4.7 kb
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That can integrate into the specific site on
chromosome 19
Productive infection by adeno-associated virus
depends on proteins from another virus (helper virus)
such as adenovirus
x from adenovirus
~ 7.5 kb insert
1
Target gene in place of E1
2
Co-
Recombination-overlapping
-It has been used for treating
cystic fibrosis but results
were not encouraging
- Very few patients were
transduce with CFTR and
multiple treatment with
recombinant adenovirusvirus
triggered severe
immunological responses in
patients because of low level
of expression of some of the
adenoviral genes that
destroyed the transduced
cells
Adenovirus DNA molecule lacks some
5’ region including E1 but share some
homology with 1
Packing cell line carries E1- essential
for replication
Viral production E1 and assembled
virus
99%
Transfer of non-viral DNA by a virus to a cell
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Hence the name adeno-associated virus
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After the adeno-associated virus enters the nucleus, the
polymerases of a host cell convert the adeno-associated
virus genome to double-stranded DNA that is then
transcribed
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Its absence of pathogenicity makes adeno-associated
virus a good candidate as a vector for the delivery of
therapeutic genes
Fig 21.8 Glick 2nd Ed
 Herpes Simplex Virus Vector System;
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Although it is possible to engineer retro-virus and
adenovirus vectors to infect specific cell types
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Some viruses naturally are directed to partial type of
cell
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Herpes simplex virus type I infects and persist within
non-dividing neuronal cells
Plasmid 1
Plasmid 2
~4.5 kb insert
Co-
Host cell line
-In absence of rep gene, the recombinant adenivirus-associated virus DNA is unlikely to
integrate into chromosome 19
-On the other hand, without any adenoassociated virus genes, the recombinant vector
will not evoke an immune response
- In phase I clinical trials with cyctic fibrosis
patients, no inflammatory response was
observed after adminstration of a CFTR-adenoassociated virus vector
- The vector persisted for but 70 days
- Further clinical trials will determine if
therapeutic levels of the CFTR gene product
are produced
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It is a common human pathogen that causes periodic
cold sores and rarely fatal in encephalitis
The virus remains latent in neurons
Reactivation of the lytic cell initiated by stress and
hormonal changes
There are many different disorders that affect the CNS
and PNS including tumors, metabolic and
immunological
defects
and
neurodegenerative
syndromes such as Alzheimer and Parkinson diseases
Neurological disorders are responsible for more
hospitalizations and chronic care than all other diseased
combined
Because of its preference for nerve cells, HSV is a
suitable candidate as a gene therapy vector for treating
these kinds of diseases
Fig 21.9 Glick 2nd Ed
Fig 21.10 Glick 2nd Ed
(E. Coli plasmid)
To supply viral proteins for
replication and assembly of viral
component
~ 30 kb insert
Modified
Alternative procedure
 Non-Viral Gene Delivery Systems;
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A virus-mediated delivery system utilizes cell receptor
sites to enter a target cell
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This avoids lysosomal degradation of vector and leads
to the deposition of vector DNA into the nucleus of the
cell
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However, besides these important advantages, viral
vectors are costly to maintain and
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in many instances, have limited cloning capacity
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In addition, viral proteins may induce an inflammatory
response that nullifies repeated administration of
vector
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As an alternative to viral vectors for gene therapy, a
number of non-viral gene delivery systems have been
devised
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Least complicated non-viral gene delivery system is the
introduction of pure DNA constructs directly into the
cells of a target tissue
When plasmid DNA injected into mouse skeletal
muscle, some of the cells took up the DNA and
Reporter gene was expressed for more than 50 days
Although appealing in its simplicity, this approach is
limited to accessible tissue and requires large amounts
of DNA
Pure DNA constructs that cover the surface of gold
particles (1-3 µm diameter) have been propelled into
skin cells and after incision, into subcutaneous tumor
cells with gene gun
Therapeutic genes delivered in this way are expressed
in the targeted tissues and gene products are released
into the circulatory system
Theoretically, secretion of a therapeutic protein into the
circulatory system should facilitate its conveyance to a
target tissue that is difficult to access directly
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However, most proteins that are not normally part of
circulatory system are inactivated or degraded by cells
or proteins in the blood
Special strategies need to be developed to over come
this problem
Liposome Delivery Method – Surrounding a DNA
construct with artificial lipid layers to form lipid sphere
(liposome) with an aqueous core facilitates the passage
of the DNA through the cell membrane
Various liposome formulation have been devised. For
example:
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Cationic liposomes have a positive charge on
the outside and bind to negatively charged DNA
molecules to form a lipid-DNA complex
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These are simple to form, relatively non-toxic
and non-immunogenic
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Unfortunately, the efficiency of gene transfer
is not high
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Why?
Because after cellular internalization a large
amount of the DNA is taken up by lysosomes
and degraded
DNA Molecular Conjugate Methods – DNA
molecular conjugates such as poly L-lysine have been
developed to deliver large DNA (>10 kb) constructs by
the endosome cellular transport pathway
Thereby avoiding lysosomal degradation of the DNA
Fig 21.11 Glick 2nd Ed
 Prodrug Activation Therapy;
 Despite intensive treatment with surgery, radiation
therapy and chemotherapy, cancer of all types is still
leading cause of human deaths
 The combination of the herpes simplex virus thymidine
kinase genes (HSVtk) and ganciclovir [GCV;9-(1,3
dihydroxy-2-propoxymethyl)guanine] has been used to
eradicate proliferating tumor cells
Fig 21.12 Glick 2nd Ed
 Nucleic Acid Therapeutic Agents;
 For the most part, protocols for ex-vivo and in-vivo gene
therapy use a cloned DNA construct to supply a
functional form of a protein which is either lacking or
defective
 However, a number of human disorders such as cancer,
inflammatory conditions and both viral and parasitic
infections result from the over-production of a normal
protein
 Therapeutic systems using nucleotide sequences are
being devised to treat these types of conditions
 In Vivo Antisense RNA Production
Fig 21.13a Glick 2nd Ed
 Antisense Oligonucleotides
• Sequence-specific effectiveness of chemically
synthesized antisense oligodeoxynucleotides relies:
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on hybridization to an accessible site on the
target mRNA
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resistance to degradation by cellular nucleases
and
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ready delivery into the cells
Fig 21.13b Glick 2nd Ed
Fig 21.14 Glick 2nd Ed
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Free antisense oligonucleotides are not readily
internalized by cells
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However, cellular uptake occurs when an
antisense oligonucleotide is encapsulated within
a liposome
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With an effective delivery system, low, non
toxic concentrations of the antisense molecule
can be used to inhibit translation of the target
mRNA
-Most extensively used antisense oligonucleotide contains sulfur
group in place of the free oxygen of phosphodiester bond. This
modification is called phoporothionate linkage.
These
phosphorothioate antisense oligonucleotides are water soluble
poly anionic and resistant to nuclease degradation
- In addition, when these oliginucleotide hybridizes to its target
site , the DNA-RNA duplex activates the endogenous enzyme
ribonucleae H, which cleaves the m RNA component of the
hybrid molecule.
- C and D are very resistance to nuclease degradation as they
have phosphoramitide linkage and polyamide (peptiode)
linkage
- E and F enhance the stability and facilitate the binding of
antisense oligonucleotides to its target site because of
addition of chemical groups like 2’ carbon of sugar moiety
and the 5 carbon of pyrimidines
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Moreover, liposomes that are conjugated with
cell-specific binding sites direct an antisense
oligonucleotide to a particular tissue
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Several preclinical trials have shown the
usefulness of antisense oligonucleotides as
therapeutic agents. For example:
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The narrowing (stenosis) of coronary and
carotid arteries that leads to heart attacks and
strokes respectively
Ribozymes
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Naturally occurring catalytic RNA molecules
(RNA enzymes)
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That have separate catalytic and substrate
binding domains
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The substrate binding sequence combines by
nucleotide complementarily and possibly nonhydrogen bond interactions with its target
sequence
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The catalytic portion cleaves the target RNA at
a specific site
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The substrate domain of a ribozyme can be
engineered to direct it to a specific mRNA
sequences
Fig 21.15 Glick 2nd Ed
 Oligonucleotide Correction of Genetic Conditions:
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The ability to convert a mutant base pair of a
gene to the wild-type (normal, correct) version
would reverse the consequences of many
different genetic conditions
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A strategy using a modified RNA-DNA
oligonucleotide with 68 nucleotides (chimeric
oligonucleotide) has been devised for this
purpose
Fig 21.16 Glick 2nd Ed
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The rationale for this particular arrangement is
based on various experimental observations:
i.
Combined RNA-DNA molecules
participate more readily than duplex
DNA in homologous nucleic acid
pairing reactions
ii.
Hairpin caps, which do not interfere
with the pairing of homologous nucleic
molecules, protect the molecule from
exonucleases
iii.
The 2’-O methylation of the ribose units
shield the molecule from degradation by
RNase
iv.
In addition, the organization of the
nucleotides of a chimeric
oligonucleotide is important i.e.
v.
Ten nucleotide flank a central core of
five deoxyribonucleotides and
vi.
Except for the correct base pair, this
segment of the chimeric oligonucleotide
has the same sequence as the target
sequence
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The feasibility of base pair correction with a
chimeric oligonucleotide was examined with
both a mutated cDNA sequence carried by a
plasmid and
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a mutant site within a chromosomal sequence
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In both instances, with high frequencies, the
mutant sites were replaced by the correct base
pair
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Aberrant splicing of a mRNA occurs when a
mutation in an intron is recognized by the
RNA processing system an authentic splice
site and
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Consequently, a portion of the intron is
included as part of the processed mRNA
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The presence of part of an intron disrupts the
reading frame and
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A truncated protein is produced
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A disease condition may result from a
diminished level of normal protein
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This mutation in fact is responsible for one
form of thalassemia, which is an inherited
blood disorder that leads to loss of red blood
cells (anemia)
Fig 21.17 Glick 2nd Ed
 Interfering RNAs
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The addition of double-stranded RNA
to animal cells reduces the expression
of the gene from which double –
stranded sequence is derived
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This gene silencing, which specifically
reduces the concentration of a target
mRNA by up to 90% , is reversible
since there is no change in the target
cell’s DNA
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This phenomenon has been termed
RNA interference or RNAi and occur
naturally in flies, trypanosomes,
earthworms, hydras, zebra fish and
mice
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Moreover, RNAi in animals appears to
be related to gene silencing or
co-suppression in plants
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Although its biological role remains
to be established, RNAi may protect
both animals and plants from viruses or
from the accumulation of transposons
Fig 10.34 Glick 3rd Ed