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Gene therapy and viral vectors
Lecture 5
Classification of Genetic Disorders
Level 1: Single Gene Disorders
 Disorders which result when a mutation causes
the protein product of a single gene to be altered,
differentiated, or missing.
Level 2: Chromosome Abnormalities
 Entire or whole chromosomes, or large segments
of the chromosomes, are missing, duplicated, or
altered.
Level 3: Multifactorial Disorders
 Multifactorial disorders are those which result
from mutations in multiple genes. They’re
complex, often coupled with environmental
causes.
Cancer
A term for diseases in which abnormal cells divide without
control and can invade nearby tissues.
There are several main types of cancer.
 Carcinoma is a cancer that begins in the skin or in tissues
that line or cover internal organs.
 Sarcoma is a cancer that begins in bone, cartilage, fat,
muscle, blood vessels, or other connective or supportive
tissue.
 Leukemia is a cancer that starts in blood-forming tissue,
such as the bone marrow, and causes large numbers of
abnormal blood cells to be produced and enter the blood.
 Lymphoma and multiple myeloma are cancers that
begin in the cells of the immune system.
 Central nervous system cancers are cancers that begin
in the tissues of the brain and spinal cord. Also called
malignancy.
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"Drivers" of Cancer
The genetic changes that contribute to cancer tend to
affect three main types of genes:
 Proto-oncogenes are involved in normal cell
growth and division. However, when these genes are
altered in certain ways or are more active than
normal, they may become cancer-causing genes (or
oncogenes), allowing cells to grow and survive when
they should not.e.g. HER2, Ras, Myc
 Tumor suppressor genes are also involved in
controlling cell growth and division. Cells with certain
alterations in tumor suppressor genes may divide in
an uncontrolled manner.e.g. p53, p10
 DNA repair genes are involved in fixing damaged
DNA. Cells with mutations in these genes tend to
develop additional mutations in other genes. Together,
these mutations may cause the cells to become
cancerous.e.g. BRCA1, BRCA2
Stages of Cancer
Staging helps describe where a cancer is located, if or where it
has spread, and whether it is affecting the other parts of the
body.
 Plan treatment, including the type of surgery and
whether chemotherapy or radiation therapy are needed
 Predict the chance that the cancer will come back after the
original treatment
 Predict the chance of recovery
 Talk about the diagnosis in a clear, common language with the
entire health care team
 Determine treatment effectiveness, and
 Compare larger populations with the same diagnosis to
research new, more effective cancer treatments.
TNM
How large is the primary tumor? Where
is it located? (Tumor, T)
 Has the tumor spread to the lymph
nodes? If so, where and how many?
(Node, N)
 Has the cancer spread to other parts of
the body? If so, where and how much?
(Metastasis, M)
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Tumor (T)
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The letter "T" plus a number (0 to 4)
describes the size and location of the tumor,
including how much the tumor has grown
into nearby tissues.
A larger tumor or one that has grown more
deeply into the surrounding tissue receives a
higher number.
For some types of cancer, lowercase letters,
such as “a,” “b,” or "m" (for multiple), are
added to the “T” stage category to provide
more detail.
Node (N)
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The letter "N" plus a number (0 to 3) describes whether
cancer has been found in the lymph nodes.
It may also describe how many of the lymph nodes contain
cancer.
Lymph nodes are tiny, bean-shaped organs that help fight
infection.
Regional lymph nodes are located closest to where the
cancer began.
Distant lymph nodes are located in other parts of the body.
Most often, the more lymph nodes with cancer, the larger the
number assigned.
However, for some tumors, the location of the lymph nodes
with cancer may determine the “N” stage category.
Metastasis (M)
The letter "M" indicates whether the
cancer has metastasized, or spread, to
other parts of the body.
 If the cancer has not spread, it is labeled
M0.
 If the cancer has spread, it is considered
M1.
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Cancer Stage Grouping
Stage 0. This stage describes cancer in situ, which means “in
place.” Stage 0 cancers are still located in the place they
started and have not spread to nearby tissues. This stage of
cancer is often highly curable, usually by removing the entire
tumor with surgery.
 Stage I. This stage is usually a small cancer or tumor that
has not grown deeply into nearby tissues. It also has not
spread to the lymph nodes or other parts of the body. It is
often called early-stage cancer.
 Stage II and III. These stages indicate larger cancers or
tumors that have grown more deeply into nearby tissue. They
may have also spread to lymph nodes but not to other parts
of the body.
 Stage IV. This stage means that the cancer has spread to
other organs or parts of the body. It may also be called
advanced or metastatic cancer.
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Prognostic factors
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Grade. The grade describes how much cancer cells look like
healthy cells under a microscope. It also helps predict how quickly
the cancer will spread. A tumor with cells that look more like
healthy cells is called well-differentiated or low-grade. A tumor with
cells that look less like healthy cells is described as poorly
differentiated, undifferentiated, or high-grade. Different types of
cancer have different methods to assign a cancer grade.
Tumor markers. Tumor markers are substances found at higher
than normal levels in the blood, urine, or body tissues of some
people with cancer. Doctors and researchers have been discovering
tumor markers for many types of cancer that can help determine
the best treatment. For some cancers, certain tumor markers may
be more helpful than stage in predicting how well a specific
treatment will work or the chance that the cancer will spread.
Tumor genetics. Many genes in cancer cells may help predict if
the cancer will spread or what treatment(s) will work. Recent
research studies have found ways to determine the genes involved
in many types of cancer. In the future, this information may also
help doctors target treatment to each person’s cancer. Learn more
about personalized medicine.
Treatment of cancer
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Surgery
Chemotherapy
Radiation Therapy
Targeted Therapy
Immunotherapy
Stem Cell Transplant
Hyperthermia
Photodynamic Therapy
http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/treatment-typeslanding
Herpes Simplex Virus
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Herpes Simplex Virus (HSV) is a
double stranded DNA virus that
belongs to Herpesviridae family.
It contains three main structural
components.
A central core holds the viral
DNA, an inner core is
surrounded by an envelope that
is made of viral glycoproteins
and a capsid.
The tegument is located between
the capsid.
the envelope and various envelop
proteins.
The herpes family of viruses consists of three
families
of
viruses;
Alphaherpesviridae,
Betaherpesviridae, and Gammaherpesviridae.
 Among these families are many commonly known
viruses that are causative agents of many diseases,
such as HSV-1 and HSV-2 for cold sores and
genital warts, varicella zoster for chickenpox.
 Herpes virsues also tend to have latent, recurring
infections in the infected organisms, where the
virus remains in some part of the infected
organism.
 Herpes family viruses are incredibly common,
affecting upto 80% of population around the
world.
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Life Cycle of Herpes Virus
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http://www.sumanasinc.com/webcontent/animations/content/herpessimplex.
html
Herpes Simplex Viral Vector
Some of the important drawbacks
 Immunogenecity of the particle
 Packaging constraints
 Long term maintenance of the genetic
material
 Random integration
Cancer cell therapy with HSV
HSV has many traits that make it desirable as a treatment vector;
 HSV can infect a wide variety of cell types.
 The normal replication cycle of HSV causes cells to lyse (killing cancerous
cells).
 The HSV genome has many genes that are non-essential to replication that
can be replaced with therapeutic genes, there are already many
pharmaceutical options that can be used to control against unwanted
replication of the virus, and the viral genome remains as an intact plasmid
within the cell nucleus which protects against unwanted insertion of viral
DNA into the host genome.
 HSV viruses were re-engineered to express cytotoxic genes or genes to
stimulate immune response.
 The effectiveness of HSV as a treatment vector increased when used in
conjunction with other cancer therapies such as radiation treatment and
chemotherapy.
 Three strains of re-engineered HSV, designated G207, 1716, and NV1020,
were put through Phase I clinical trials in 2002. There were no negative
effects attributed to the virus. These strains were effective in the treatment
of a variety of tumors in mice, including breast, prostate, colon, and
pancreatic cancer (Varghese, 2002).
Oncolytic virus HF10 project
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HF10 is an attenuated, replication-competent mutant strain of Herpes
Simplex Virus type 1 (HSV1).
HF10 a spontaneously occurring mutant of HSV1 newly established by
Professor Yukihiro Nishiyama, Nagoya University, School of Medicine, and it
does not contain any foreign DNA.
Live viruses such as HSV infect human cells, replicate, and destroy the
infected cells.
Using this property of viruses, the new approach to cancer therapy termed
"oncolytic virotherapy" has been developed and investigated.
HF10 is one of the promising new strains for oncolytic virotherapy.
HF10 shows a strong killing effect against tumor cells because of its high
replication competence in these cells.
Moreover, administration of HF10 also induces a strong immune response
which is expected to further enhance the anti-tumor activity.
The virulence of HF10 is significantly reduced compared to wild-type
HSV1 because it carries mutations in genes controlling virulence.
Oncolytic Virus
An oncolytic virus is a virus that
preferentially infects and kills cancer cells.
As the infected cancer cells are destroyed
by oncolysis, they release new
infectious virus particles or virions to
help destroy the remaining tumour.
 Oncolytic virotherapy is an emerging
treatment modality which uses replication
competent viruses to destroy cancers.
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TCR gene therapy project
TCR gene therapy project
T-cell receptor (TCR) gene therapy, is one of the new
adoptive immunotherapies, which get attention as
"tailor-made therapy" against cancers. In this gene
therapy, the T-cell receptor genes which recognize
cancer antigens are transduced into lymphocytes of a
cancer patient for treatment. The gene-modified
lymphocytes are cultured in a large scale, and infused
back into the patient. The lymphocytes express the Tcell receptors on the surface, and these receptors
recognize the peptides derived from the cancer
antigens. So the gene-modified lymphocytes can
specifically attack tumor cells expressing the cancer
antigens and kill them finally.
 https://clinicaltrials.gov/ct2/show/NCT02272855?term
=HF10&rank=2
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MazF gene therapy project
MazF gene therapy project
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MazF gene therapy uses MazF, an endoribonuclease derived
from Escherichia coli, as a retroviral technology for HIV gene
therapy. In HIV disease, HIV infects a type of immune cell
called helper T-cells or macrophages and subsequently
replicates, causing deficiencies in the helper T-cells and the
entire immune system. MazF gene therapy uses expression
vector to transduce autologous helper T-cells ex vivo with
genes that express MazF conditionally upon HIV infection.
The MazF-modified T-cells that are infused back into the
patients are expected to block the replication of HIV when it
infects the transduced T-cells, thereby maintaining sufficient
immune functions. Thus, autologous transplantation of MazFmodified T-cells is an attractive strategy for HIV gene therapy.
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https://clinicaltrials.gov/ct2/show/NCT01787994
Population size in clinical trials
Lecture prepared from
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http://www.sciencedirect.com/science/arti
cle/pii/S0168170209002421
Assignment
Group 1: AIDS/HIV
 Group2: Arthritis
 Group 3: Hemophilia
 Group 4: Hypertension
 Group 5: Diabetes
 Group 6: Hepatitis
 Group 7: Cancer (Any type)
 Group 8: Cancer (Any type)
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Topics to be covered in the
assignment
Introduction of the disease or disorder
Type(s) of mutations involved
Conventional treatment
Type of vector
The procedure of gene therapy
History of clinical trials
At which phase of clinical trial the treatment
is undergoing?
 Is it commercially available?
 Deadline : 30 Oct
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