GENE THERAPY

Adenovirus Vectors in Gene Therapy

o Adenoviruses
Gene Therapy - simple double-stranded DNA viruses
- terminal protein in each end of DNA
- genetic surgery
- 240 hexons proteins comprises the
- a technique that modifies a person’s genes to
capsid
cure or treat disease
- penton base fiber
- less permanent
- coxsackievirus adenovirus receptor
- cures the defect
(CAR)
- does not affect the germline (not inherited)

Entry to Target Cell

Genetic Engineering 1. Enters via CAR and Integrin
2. Goes into cytoplasm
- altering an organism permanently so that the
3. Inject its DNA, travels in the nuclear
changes will be stably inherited
pore, enters the nucleus
-permanent alteration of the germline
ADVANTAGES:
- relatively harmless
- non-oncogene
Principles of Gene Therapy - easy to culture
> To deal with a hereditary defect due to a - well-understood site cycle and
single gene – a recessive condition gene function
- complete DNA sequence is known
* a single good copy of the gene can cure the
defect o Cystic Fibrosis Therapy by Adenovirus
- Healthy CF gene successfully cloned
and inserted to crippled adenovirus
STEPS: - Introduced to patient via aerosol (nose
a) Identification and characterization of to lungs)
gene Successful only for 30 days
b) cloning - Cures only symptoms but does not
c) choice of vector correct the defect
d) method of delivery
e) Expression o Adeno-Associated Virus (AAV)
- AAV are just satellite virus
- found in cells infected with adenovirus
but they are completely harmless
Aggressive Gene Therapy
ADVANTAGE:
- provision of gene whose products may cure - does not stimulate inflammation
disease even they are not responsible for - not antigenic (stimulates immune
delivering disease. response)
- used in cancer treatment
 - infects wide range of animals with Retrovirus Gene Therapy (ver. 2.0)
appropriate helper virus
- can cater nondividing cells Ver 1.0 Ver 2.0
- unusually minute in size - classic SCID - variant form of SCID
- integrate its DNA into a single site - retrovirus as vector with defects on IL-7
- using of stem cell (both B & T cells
(umbilical cord) inactive)
Retrovirus Gene Therapy - rejuvenates with - retrovirus vector
o Retroviruses purified ADA + gene carries the gene for
therapy the missing subunit of
- infect many types of cells in mammals
IL-7 receptor after it
- need dividing cells for successful
has been cultured
infection
- rejuvenates with
- has nucleocapsid containing RNA
gene therapy only
genome

o Severe Combined Immunodeficiency

(SCID)
- defective T & B cells
- people with this disease live in a
special sterile bubble chambers
Nonviral Delivery in Gene Therapy
*safer but neglected due to inefficiency
Alternatives:

[Ada gene that encodes for the enzyme 1. Purified naked nucleic acid
adenosine deaminase needed for the incorporated in plasmids
production of purine bases and that 2. Particle bombardment
without prevents the development of 3. Receptor mediator uptake
lymphocytes] 4. Polymer complex DNA-binding to
positively charged polymer
5. Encapsulated cells in porous
Retrovirus Gene Therapy (ver. 1.0) polymeric coat and injected locally
6. Liposomes
Bacterium carrying
Genetically
plasmid with cloned
disabled retrovirus
normal human ADA gene

Genetically altered Applications of Gene Therapy

cells are
reimplanted, > cure diseases or improve body’s
Cloned ADA gene is
produce ADA ability to fight diseases
incorporate into > cancer
virus
T-cells disabled
> cystic fibrosis
Cells are grown in
ADA gene culture to ensure > Heart disease
isolated from ADA gene is active > Diabetes
SCID patient
Retrovirus infects > Hemophilia
T-cells, transfers > AIDS
ADA gene to cells
MOLECULAR BIOLOGY OF CANCER
o Cancer is genetic in origin
- we all have cancer gene. They are just
activated when triggered
Definition of Terms:
1. Somatic cell- body cells
o Genes that Affect Cancer
- comprises all parts of the body (except
germline
- always undergo mitosis
2. Germ line- egg/sperm/sex cells
3. Mutant- effect of mutation
4. Microtumor- onset development of
cancer and cannot be detected.
Localized
STAGES OF CANCER DEVELOPMENT
1. Control of normal cell division is lost
2. Abnormal (mutant) cell divides to form
microtumor
3. Many microtumors formed throughout
the body eventually leading to a tumor
o Types of Mutations that Generate
Can be
malignant
(cancerous)/be
nign (non-
cancerous)
Angiogenesis – formation of new blood vessel
Environmental Factors and Cancer
o Carcinogen
- agent that causes cancer
o Mutagen
- chemicals that causes mutations
(alters and damages the DNA)
Ex: radiation
Note: 80% of cancers are derived from
epithelial cells
NOT ALL CARCINOGENS ARE
MUTAGENS
2 types
- Oncogenes
- Tumor Suppressor Genes (anti-
oncogenes)
ONCOGENES
- mutated form of proto-oncogene
- cancer-causing genes and mutant
oncogenes
- mutations in oncogenes are dominant
*Proto-oncogenes – control cell division
- wild-type of oncogenes
-Detection by transformation – healthy
cells are checked upon insertion of
cancer cells
Oncogenes
- Mutations that generate oncogenes
are dominant from increased activity of
the cell.
Major Components (of cell division):
> Growth Factor
> Cell surface receptor
> Signal transduction proteins
> Transcription factors
*Cellular growth is signalled by a
growth factor that binds and activates
its cell surface receptor. The
 intracellular portion of the receptor
activates intracellular proteins that
move to the nucleus. Inside the nucleus,
the proteins activate a transcription
factor that turns on genes for growth.
o The Ras Oncogene – Hyperactive
Protein
- blocks action of all cyclins and freezes
the cell until damage is repaired
p16
- acts similarly with p21 but
only blocks cyclin E
- stops division at critical point
before S-phase

Ras Protein
Techniques in Genetic Analysis of Cancer:
- transmits signals concerning cell
- DNA sequence
division in human, flies, and even yeast.
- PCR
- result of a single alteration in the
- Microarrays
amino acid in the encoded protein
- Hyperactive Ras causes uncontrolled
cell division and beginning of possible
Formation of Tumor
cancer usually seen in lungs, colon,
General: (colon cancer)
pancreas and thyroid cancer.
1. Inactivation of APC anti-oncogene (both
copies)
o Tumor Suppressor Genes (Anti-
2. Activation of Ras oncogene
oncogene)
3. Inactivation of DCC anti-oncogene (both
- suppress division of cancer cells
copies)
4. Mutation of single copy of p53
Anti-oncogenes
- p16
Metastasis – spread of cancer to the other parts
- p21
of the body
- p53

Reason for Tumor to Metastasize

1. Loss of adhesion to neighboring cells
p53
and the home tumor
- found on the short arm of
2. Ability to penetrate the membrane
chromosome 17
surrounding other tissue
- has the protein T53
- acts as emergency blocking system for
Cancer-causing Viruses= HHV8, RSV, HPV
cell cycle
- shows effect in single mutation
despite the presence of second normal
Is Cancer Inherited?
gene copy
1. Possibility of inheriting one defective
copy of oncogene
p21
2. Mutations in certain special genes
- activated by normal p53 protein
affect rate at which further mutations
especially if cell’s DNA is damaged
occur during cell division
 3. Indirect effect of race or within - hydroxyl radicals
populations
Effects:
- damage protein
AGING & APOPTOSIS - damage lipids
- damage DNA
Terms:
o Age – amount of time during which
Reaction:
someone or something has lived or
- release of antioxidant enzymes
existed
Ex: superoxide dismutase and catalase
- accumulation of wear and tear
o Senescence – the condition of
deterioration with age
o Apoptosis – programmed cell death
Apoptosis and Necrosis

Apoptosis
Cellular Senescence
- programmed cell death
- a.k.a replicative senescence
- cells that undergo certain number of
Genes that initiates a controlled and deliberate
generations stopped from multiplying
death program: -eed-3, ced-4 and ced-9 and
despite addition of inducers
egl-1

3 Main Characteristics
> Senescent cells arrest in cell cycle in
Necrosis
G1, never enter S-phase
- happens to cells that are damaged by external
>Cells may become terminally
injuries, oxygen starvation or energy depletion
differentiated
> Senescent cells becomes resistant to
apoptosis
Advantage of Apoptosis over Necrosis
> proteins are recycled
> process can be stopped if necessary
Factors that Activate Senescence
> final result does not alter physiology of the
o Length of telomeres
entire organism
o DNA damage due to reactive oxygen
metabolites
o Presence of oncogenic mutation

Mitochondria and Aging

- Cellular senescence and aging can be
attributed to oxidative stress

Products of Oxidative Stress:

- superoxides ions
- peroxides