Download PHRM 521: Molecular Biology and Biotechnology

Genomics, personalized medicine
and pharmacogenomics
 Genomics and personalized medicine are
changing the field of pharmacogenomics by
two ways:
 by optimizing drug therapies and
 by reducing adverse drug reactions.
In the near future, personalized medicine will
allow physicians to predict which diseases you
will develop, which therapeutics will work for
you, and which drug dosages are appropriate.
6.1.PHRM-521.
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Definition
 Today, the phrase personalized medicine is used to
describe the application of information from a patient’s
unique genetic profile in order to select effective
treatments that have minimal side-effects and to detect
disease susceptibility prior to development of the
disease.
 Pharmacogenomics is the study of how an individual’s
entire genetic makeup determines the body’s response
to drugs. The term pharmacogenomics is used
interchangeably with pharmacogenetics, which refers to
the study of how sequence variation within specific
candidate genes affects an individual’s drug responses.
 In pharmacogenomics, scientists take into account many
aspects of drug metabolism and how genetic traits affect
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these aspects.
Variations in patient response to drugs
 When a drug enters the body, it interacts with various
proteins including carriers, cell-surface receptors, transporters, and metabolizing enzymes. These proteins affect a
drug’s target site of action, absorption, pharmacological
response, breakdown, and excretion. Because there are so
many interactions that occur between a drug and proteins
within the patient, many genes and many different genetic
polymorphisms can affect a person’s response to a drug.
Figure: A general
summary of the
percentages of
patients for which
a particular class of
drugs is effective.
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Optimizing drug therapies
 On average, a drug will be effective in only about 50% of
patients who take it (see Fig. in previous slide), which means
that physicians often must switch their patients from one drug
to another until they find one that is effective.
 Not only does this waste time and resources, but also it may
be dangerous to the patient who is exposed to a variety of
different pharmaceuticals and who may not receive
appropriate treatment in time to combat a progressive illness.
 One of the most common current applications of personalized
pharmacogenomics is in the diagnosis and treatment of
cancers.
 Large-scale sequencing studies show that each tumor is
genetically unique, even though it may fall into a broad
category based on cytological analysis or knowledge of its
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tissue origin.
 One of the first success stories in personalized medicine was that
of the human epidermal growth factor receptor 2 (HER-2)
gene and the use of the drug Herceptin® in breast cancer.
 Because Herceptin will only act on breast cancer cells that have
amplified HER-2 genes, it is important to know the HER-2
phenotype of each cancer. In addition, Herceptin has potentially
serious side-effects. Hence, its use must be limited to those who
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could benefit from the treatment.
Determining the gene and protein status
of breast cancer cells
 A number of molecular assays have been developed to
determine the gene and protein status of breast cancer
cells. Two of the most commonly used tests are based
on immunohistochemistry (IHC) and fluorescence in situ
hybridization (FISH).
 In IHC assays, an antibody that binds to HER-2 protein
molecules is added to fixed tissue on a slide. The
antibody is bound to another molecule that reacts to
produce a visual stain.
 After washing and staining, the tissues are observed
under a microscope. The level of HER-2 staining is
assessed from “0” (fewer than 20,000 HER-2 molecules
per cell) to “+3” (approx. 2 million molecules per cell).6
Immunohistochemistry (IHC)
 IHC is a method to stain the tissue sections/cells and is perhaps
the most commonly applied immunostaining technique. While
the first cases of IHC staining used fluorescent dyes, other nonfluorescent methods using enzymes such as peroxidase and
alkaline phosphatase are now used.
 These enzymes are capable of
catalysing reactions that give a
coloured product that is easily
detectable by light microscopy.
Alternatively, radioactive
elements can be used as
labels, and the immunoreaction can be visualized by
autoradiography.
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Enzyme Linked ImmunoSorbent Assay (ELISA)
 ELISA is a diagnostic method for quantitatively or semiquantitatively determining protein concentrations from blood
plasma, serum or cell/tissue extracts in a multi-well plate
format (usually 96-wells per plate). Broadly, proteins in
solution are adsorbed to ELISA plates. Antibodies specific for
the protein of interest are used to probe the plate.
 Background is minimized
by optimizing blocking
and washing methods (as
for IHC), and specificity is
ensured via the presence
of positive and negative
controls. Detection
methods are usually
colorimetric or chemiluminescence based.
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 In FISH, DNA or RNA molecules with sequence complementarity to the HER-2 gene sequence are added to the fixed
tissue on the slide.
 These DNA or RNA probes are labeled with a fluorescent tag
molecule. After hybridizing the probes to the tissue and
washing off excess probe, the location and intensity of the
probe are determined by observing the tissue under a
fluorescence microscope.
 The number of HER-2 genes is assessed by comparing the
fluorescence signal of the HER-2 probe with a control signal
from another gene that is not amplified in the cells.
 Herceptin has had a major effect on the treatment of HER-2
positive breast cancers. When Herceptin is used in
combination with chemotherapy, there is a 25 to 50 percent
increase in survival, compared with the use of chemotherapy
alone. Herceptin is now one of the biggest selling biotechnology products in the world, generating more than $5 billion in
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annual sales.
HER-2 gene and protein assays. (a) Normal and breast cancer
cells within a biopsy sample, stained by HER-2 immunohistochemistry. Cell nuclei are stained blue. Cancer cells that overexpress
HER-2 protein stain brown. (b) Cancer cells from the same tumor
assayed for HER-2 gene copy number by FISH. Cancer cell nuclei
appear green under the fluorescence microscope and the HER-2
gene DNA appears bright yellow. Large clumps of yellow stain
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indicate HER-2 gene amplification (>20 copies per nucleus).
Reducing adverse drug reactions
 Every year, about 2 million people in the United States suffer
serious side-effects from pharmaceutical drugs, and approximately 100,000 people die from that adverse side-effects.
 The costs associated with these adverse drug reactions
(ADRs) are estimated to be $136 billion annually.
 Although some ADRs result from drug misuse, others result
from a patient’s inherent physiological reactions to a drug.
 Sequence variations in a large number of genes can affect
drug responsiveness (see Table in the next slide).
 Of particular significance are the genes that encode the
cytochrome P450 families of enzymes. These family
members are encoded by 57 different genes.
 The products of the CYP2A6, CYP2B6, CYP2C9, CYP2C19,
CYP2D6, CYP2E1, and CYP3A4 genes are responsible for
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metabolizing most clinically important pharmaceutical drugs.
Examples of variant gene products that
affect drug responses
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Personalized medicine and disease diagnosis
 Growth of gene tests and testing laboratories from 1993 to
2009. (from the GeneTests Web site at www.ncbi.nlm.nih.gov/).
 As of 2009, there were genetic tests for approximately 2000
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different diseases.
Table: Some single-gene defects for which genetic
tests are available.
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 Although these genetic tests are extremely useful for
detecting some future diseases and guiding treatment,
it is clear that most disorders are multifactorial and
complex.
 It is likely that diseases such as diabetes, Alzheimer’s,
and heart disease are caused by interactions between
many genes, as well as by factors contributed by
epigenetic effects, lifestyle, and environment.
 These diseases tend to be chronic and have a
significant burden on health-care systems.
 Genome sequencing, SNP identification, and genomewide association studies (GWAS) are beginning to
reveal some of the DNA variants that may contribute to
the risk of developing multifactorial diseases such as
cancer, heart disease, and diabetes.
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The Pharmacogenomics Knowledge Base (PharmGKB):
Genes, Drugs, and Diseases on the Web
 PharmGKB is a publicly
available Internet database and
information source developed
by Stanford University. It is
funded by the National
Institutes of Health (NIH)
 On the PharmGKB Web site
(http://www.pharmgkb.org),
you may search for genes and
more than 650 variants that
affect drug reactions,
information on a large number
of drugs, diseases and their
genetic links, pharmacogenomic pathways, gene tests,
and relevant publications.
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Pharmacogenomics and Rational Drug Design
Fig. Different individuals with the same disease, in this case childhood leukemia, often respond
differently to a drug treatment because of subtle differences in gene expression. The dose of
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anticancer drug (6-MP) that works for one person may be toxic for another person.
 Several methods are being developed for expanding the uses of
pharmacogenomics. One promising method involves the
detection of SNPs.
 Perhaps researchers will be able to identify a shared SNP
sequence in the DNA of people who also share a heritable
reaction to a drug.
 If the SNP segregates with a part of the genome containing the
gene responsible for the drug reaction, it may be possible to
devise gene tests based on the SNP, without even knowing the
identity of the gene responsible for the drug reaction.
 In the future, DNA microarrays may be used to screen a patient’s
genome for multiple drug reactions.
 Knowledge from genetics and molecular biology is also
contributing to the development of new drugs targeted at specific
disease-associated molecules.
 Most drug development is currently based on trial-and-error
testing of chemicals in lab animals, in the hope of finding a
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chemical that has a useful effect.
Fig. Microarray
analysis for analyzing
gene-expression
patterns in a tissue.
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Rational Drug Design (RDD)
 RDD involves the synthesis of specific chemical substances
that affect specific gene products.
 An example of a rational drug design product is the new drug
imatinib, trade name Gleevec, used to treat chronic
myelogenous leukemia (CML).
 Geneticists had discovered that CML cells contain the
Philadelphia chromosome, which results from a reciprocal
translocation between chromosomes 9 and 22.
 Gene cloning revealed that the t(9;22) translocation creates a
fusion of the C-ABL proto-oncogene with the BCR gene. This
BCR-ABL fusion gene encodes a powerful fusion protein that
causes cells to escape cell-cycle control.
 The fusion protein, which acts as a tyrosine kinase, is not
present in non-cancer cells from CML patients.
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 To develop Gleevec, chemists used high-throughput
screens of chemical libraries to find a molecule that
bound to the BCR-ABL enzyme.
 After chemical modifications to make the inhibitory
molecule bind more tightly, tests showed that it
specifically inhibited BCR-ABL activity.
 Clinical trials revealed that Gleevec was effective
against CML, with minimal side effects and a higher
remission rate than that seen with conventional
therapies.
 Gleevec is now used to treat CML and several other
cancers.
 With scientists discovering more genes and gene
products associated with diseases, rational drug design
promises to become a powerful technology within the
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next decade.
Gene Therapy
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Although drug treatments are often effective in controlling symptoms of
genetic disorders, the ideal outcome of medical treatment is to cure
these diseases.
In an effort to cure genetic diseases, scientists are actively investigating
gene therapy - a therapeutic technique that aims to transfer normal
genes into a patient’s cells.
In theory, the normal genes will be transcribed and translated into
functional gene products, which, in turn, will bring about a normal
phenotype.
Human gene therapy began in 1990 with the treatment of a young girl
named Ashanti DeSilva who has a heritable disorder called severe
combined immunodeficiency (SCID). Individuals with SCID have no
functional immune system and usually die from what would normally be
minor infections.
Ashanti has an autosomal form of SCID caused by a mutation in the
gene encoding the enzyme adenosine deaminase (ADA). Her gene
therapy began when clinicians isolated some of her white blood cells,
called T cells. These cells, which are key components of the immune
system, were mixed with a retroviral vector carrying an inserted copy of
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the normal ADA gene.
(a) Ashanti DeSilva, the first person to be treated by gene therapy. (b) To treat SCID
using gene therapy, a cloned human ADA gene is transferred into a viral vector, which
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is then used to infect white blood cells removed from the patient.
 To date, gene therapy has successfully restored the health of
about 20 children affected by SCID. Although gene therapy
was originally developed as a treatment for single-gene
(monogenic) inherited diseases, the technique was quickly
adapted for the treatment of acquired diseases such as
cancer, neurodegenerative diseases, cardiovascular disease,
and infectious diseases, such as HIV.
 In the case of HIV, scientists are exploring ways to deliver
immune system-stimulating genes that could make
individuals resistant to HIV infection or cripple the virus in
HIV positive persons.
 There are nearly 1000 gene therapy trials actively underway
in the United States alone.
 Over a 10-year period, from 1990 to 1999, more than 4000
people underwent gene therapy for a variety of genetic
disorders. These trials often failed and thus led to a loss of
confidence in gene therapy.
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Gene replacement approaches
 Scientists are also working on gene replacement approaches
that involve removing a defective gene from the genome.
 Recent work with enzymes called zinc-finger nucleases
have shown promise in animal models and cultured cells.
 These enzymes can create site-specific cleavage in the
genome and when coupled with certain integrases may lead
to gene editing by cutting out defective sequences and
introducing normal homologous sequences into the genome.
 Encouraging breakthroughs have taken place in this area
using model organisms such as mice; however, this
technology has not advanced sufficiently for use in humans.
 Attempts have been made to use antisense oligonucleotides in order to inhibit translation of mRNAs from defective
genes, but this approach to gene therapy has generally not
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yet proven to be reliable.
Gene silencing approaches
 The recent emergence of RNA interference as a powerful genesilencing tool has reinvigorated gene therapy approaches by gene
silencing. RNA interference (RNAi) is a form of gene-expression
regulation.
 In animals, short and double stranded RNA molecules are
delivered into cells where the enzyme Dicer chops them into 21-nt
long pieces called small interfering RNAs (siRNAs).
 siRNAs then join with an enyzme complex called the RNA inducing
silencing complex (RISC), which shuttles the siRNAs to their
target mRNA, where they bind by complementary base pairing.
 The RISC complex can block siRNA-bound mRNAs from being
translated into protein or can lead to degradation of siRNA-bound
mRNAs so they cannot be translated into protein.
 A main challenge to RNAi-based therapeutics so far has been in
vivo delivery of double-stranded RNA or siRNA. However, several
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RNAi clinical trials to treat blindness are underway in USA.
Fig. Mechanisms
of gene regulation
by RNA-induced
gene silencing.
 RNA-induced
silencing
complex (RISC).
 RNA-induced
initiation of
transcription
silencing
complex (RITS).
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What is Bioinformatics?
 In today’s world, computers are as likely to be used by
biologists as by any other professionals - bankers or flight
controllers, for example.
 Many of the tasks performed by such professionals are
common to most of us: we all tend to write lots of memos and
send lots of e-mails; many of us use spreadsheets, and we all
store immense amounts of never-to-be-seen-again data in
complicated file systems.
 However, besides these general tasks, biologists also use
computers to address problems that are very specific to
biologists, which are of no interest to bankers or flight
controllers.
 These specialized tasks, taken together, make up the field of
bioinformatics. More specifically, we can define bioinformatics
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as the computational branch of molecular biology.
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What bioinformatics can do for us?
 Analyzing DNAs
 Analyzing RNAs
 Analyzing proteins
 Analyzing others such as complex pathways,
in silico simulation, bioimaging, etc.
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How most people use bioinformatics?
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