Chapter 11:
1. Psoriasis is a disease of uncontrolled epidermal growth, and causes red and itchy patches on
the skin. In psoriasis, insulin-like growth factor I receptors are present in excess so the use
of an antisense oligonucleotide that could bind the insulin-like growth factor I mRNA and
decrease the amount of functional insulin-like growth factor I receptor protein that is
produced is a good therapeutic option.
2. A ribozyme is an RNA molecule that also has catalytic properties. These agents are
particularly useful because they are less likely to trigger an immune response than a
protein therapy. They have a binding site that complementarily binds the target mRNA and
a catalytic site that will cleave the mRNA rendering it non-functional. Ribozymes have
specific base sequences that they look for so one can design a ribozyme so that it will cut
only those mRNA’s that they want.
3. Interfering RNA’s are small pieces of RNA that are antisense to transcripts that the cell
wants to degrade. They are taken up by the RISC complex and are used as a template for
selecting mRNA’s for degradation. They are formed when the enzyme dicer cuts up a piece
of double stranded RNA. These can be used therapeutically because we can add our own
interfering RNA’s in order to attempt to degrade certain mRNA’s. When the piece becomes
incorporated into the RISC complex, the corresponding mRNA will be cleaved and the
target protein will be down regulated.
7. Antisense Oligonucleotides have the benefit of being very customizable. We can
synthesize almost any sequence that we want and since we design the sequence we can
choose to include sequences that will increase the therapeutic affect. However, they can be
degraded by nucleases so consideration must be taken to protect them and they have only
been shown to moderately decrease expression in most cases.
Ribozymes have the benefit of being customizable for a number of different targets as well
as having less of a chance of negative immune response after administration of the therapy.
Interfering RNA’s normally have a very dramatic affect on the gene expression but are less
specific than other methods because it is possible that the siRNA that you incorporate is
complementary to more than one mRNA transcript.
Aptamers have the benefit of being highly selective but can only be used when there are
surface receptor proteins that are specific to the target cell and that the siRNA can be
internalized into the cell to act.
For developing a new nucleic acid-based therapeutic agent, it is very important that they
are delivered to the particular tissue where it is required. Continuous introduction of such
agent may lead to accumulation of high levels in tissues where it might not be required and
may have serious side effects.
8. The key attributes of a therapeutic gene delivery system for humans would be
determining:
 If the gene will actually be incorporated into the target cells
 Will gene be degraded before it reaches nucleus?
 If it will be able to affect gene expression (target)
 If it will negatively affect the expression of other genes
 If it will act on other cells in ways that it should not
 How long therapeutic affects can be maintained
 Will repeated treatments be required?
Currently the two major limitations are the low frequency of transfection and the
brief duration of the therapy.
11. Age related macular degeneration can be limited by using an aptamer known as
pegaptanib. This targets Vascular Endothelial Growth Factor (VEGF), and binds with high
affinity to the protein which promotes the growth of new blood vessels by stimulating the
endothelial cells which form the blood vessel walls, and transport nutrients and oxygen to
the tissues.
Alternatively, it may be possible to treat age-related macular degeneration with RNAi by
silencing two mRNA transcripts that cause excessive blood vessel formation in the back of
the eye. The degeneration is normally caused by hemorrhaging that results from excessive
angiogenesis that ruptures vessels in the back of the eye. Adding siRNAs complementary to
the angiogenesis promoting mRNAs in the back of the eye restores the balance between
angiogenesis and degradation of blood vessels.
Chapter 12:
1. Some bacteria, for example in diphtheria, cause the disease by producing a toxin.
However, the vaccine against such disease, a toxoid, can be extracted from toxin with
reduced virulence. To prepare such a vaccine, the toxin is purified, and treated with
aluminum salts to produce a weakened form of the toxin called the toxoid. When the toxoid
is injected into the recipient, the toxoid mobilizes B cells which produce antibodies that can
adhere to, and inactivate the harmful toxin during a real attack.
2. There are a number of factors that limit the success of vaccines including:
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Not all infectious agents can be grown in culture. Those that can be grown
require animal cell culturing which is very costly. In addition, the yield is
often low.
Extreme safety precautions must be undertaken to protect those working to
develop the vaccine from the pathogen involved.
Killing/inactivation of pathogen may be incomplete.
Vaccines have a limited shelf-life and often require refrigeration to remain
potent, which may be a problem in undeveloped countries.
3. The major antigenic determinant capsid protein viral protein 2 can be used for cloning
for developing the vaccine against the bovine virus. Double stranded cDNA must be
synthesized from the RNA, the cDNA encoding the capsid protein must be identified and
cloned in an E. coli plasmid expression vector so as to produce a fusion protein with some
protein tag (to allow for purification of the fusion protein). The resulting plasmid construct
is used to transform E. coli and produce a stable fusion protein, which is isolated and used
as to inoculate animals against the bovine virus.
4. The idea with peptide vaccines is that short peptides may be able to mimic epitopes and
could therefore be used to induce production of antibodies. However, to be effective the
epitope must be a short stretch of contiguous amino acids, the peptide must be able to
assume the same exact conformation as the epitope and it will likely be necessary to use
multiple epitopes. In addition, these peptides must be bound to a larger molecule because
generally very small protein particles are degraded by the cell.
5. The vaccinia virus is well known as the virus that alleviated small pox. Vaccinia virus is
a sequenced virus having a double stranded genome with 187 kilobase pairs, encoding 200
different proteins. This virus replicates in the cytoplasm of infected cells. The vaccinia virus
DNA contains genes for DNA polymerase, RNA polymerase and different necessary
enzymes. It can be used for the production of unique live recombinant species, as insertion
of a foreign gene into the virus genome under the control of vaccinia virus promoter will be
expressed independently of host regulatory and enzymatic functions. This vector vaccine
delivers cloned genes that are expressed and encode antigens that elicit neutralizing
antibodies against pathogenic agents.
7. The advantages of live recombinant viral vaccine over killed virus or subunit vaccines are: 1.
the virus readily expresses antigen closely resembling a natural infection and 2. the virus can also
replicate within the host, increasing the quantity of antigen that activates the release of antibodies
from B cells, and stimulates the production of T cells.
15. A subunit vaccine against papilomavirus can be and was created by utilizing the virus-like
particles taken from major capsid proteins of major types of human papilomavirus. The gene for
the capsid protein can be cloned and expressed into yeast strain. After fermentation of the yeast
strains, the viral capsid proteins can be assembled into virus like particles which can purified and
combined to form the vaccine referred to as Gardasil.
16. To deal with tooth caries I would design a vaccine that contained sequences for a
alanine/proline rich peptide as well as the C-terminal domain of a glucosyltransferase
gene. These peptides are important for adhering to the tooth surface. I could also chose to
include CTLA4 and the Fc region of an immunoglobulin G molecule, which will amplify the
immune response by targeting the sequence to bind to immune cells (antigen presenting)
as well.