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19. Treatment of Genetic
Diseases
Somatic Cell Gene Therapy
a). Treatment strategies
i). Metabolic manipulation
ii). Manipulation of the protein
iii). Modification of the genome
b). Strategies for gene transfer
Three categories of somatic cell gene therapy:
1. Ex vivo – cells removed from body, incubated with
vector and gene-engineered cells returned to body.
2. In situ – vector is placed directly into the affected
tissues.
3. In vivo – vector injected directly into the blood stream.
Example of ex vivo somatic cell
gene therapy
• Usually done with blood cells because they
are easiest to remove and return.
• Sickle cell anemia
Examples of in situ somatic cell
gene therapy
• Infusion of adenoviral vectors into the
trachea and bronchi of cystic fibrosis
patients.
• Injection of a tumor mass with a vector
carrying the gene for a cytokine or toxin.
• Injection of a dystrophin gene directly into
the muscle of muscular dystrophy patients.
Example of in vivo somatic cell
gene therapy
• No clinical examples.
• In vivo injectable vectors must be
developed.
Barriers to successful gene therapy:
1. Vector development
2. Corrective gene construct
3. Proliferation and maintenance of target cells
4. Efficient transfection and transport of DNA to nucleus for
integration into genome
5. Expansion of engineered cells and implantation into patient
Types of vectors
• RNA viruses (Retroviruses)
1. Murine leukemia virus (MuLV)
2. Human immunodeficiency viruses (HIV)
3. Human T-cell lymphotropic viruses (HTLV)
• DNA viruses
1. Adenoviruses
2. Adeno-associated viruses (AAV)
3. Herpes simplex virus (HSV)
4. Pox viruses
5. Foamy viruses
• Non-viral vectors
1. Liposomes
2. Naked DNA
3. Liposome-polycation complexes
4. Peptide delivery systems
Advantages:
1. Randomly integrates into genome
2. Wide host range
3. Long term expression of transgene
Disadvantages:
1. Capacity to carry therapeutic genes is small
2. Infectivity limited to dividing cells
3. Inactivated by complement cascade
4. Safety
Adenovirus
Advantages:
1. Efficiency of transduction is
high
2. High level gene expression
3. Slightly increased capacity for
exogenous DNA
Disadvantages:
1. Expression may be transient
2. Cell-specific targeting difficult
to achieve
3. Virus uptake is ubiquitous
4. Safety
Other viral vectors
• Adeno-associated virus – infects wide range of
cells (both dividing and non-dividing), able to
integrate into host genome, not associated with
any human disease, high efficiency of
transduction.
• Herpes simplex virus, vaccinia virus, syndbis
virus, foamy viruses – not yet widely studied
• Onyx virus – limited replicating adenovirus that
replicates mainly in tumor cells.
Non-viral vectors
1. Liposome
2. Cationic polymers
3. Naked DNA
4. Peptide-mediated
gene delivery
May overcome limitations with viruses including small capacity for
therapeutic DNA, difficulty in cell-type targeting and safety
concerns.
Synthesis of a retroviral gene therapy vector
Selectable marker for
transduced cells
Site of insertion of therapeutic
gene
Percent effort
directed towards
different gene therapy
trials.
Examples of Gene Therapy Trials
• Adenosine deaminase gene transfer to treat Severe
Combined Immuno-Deficiency (SCID)
• CFTR gene transfer to treat Cystic Fibrosis (CF)
• Advanced Central Nervous System (CNS)
Malignancy
• Mesothelioma
• Ornithine Transcarbamylase Deficiency
• Hemophilia
• Sickle Cell Disease
Stem Cell Transplantation
Harvest
marrow
Donor
Infuse normal donor cells
Radiation/Chemotherapy
Patient
Stem Cell Gene Therapy
Make gene
Put into vehicle
for delivery into ce
Harvest
marrow
Introduce therapeutic gene
Reinfuse corrected cells
Radiation/Chemotherapy
The Molecular Basis of Sickle
Cell Anemia
a chains
z
a2
a1
Polymerization
Sickled red cell
Survives 15 - 25 days
a2 bs2
LCR
e
bs chains
g
g
bs
Preferential Survival of Normal
Red Blood Cells
in Sickle Cell Anemia
Normal
120 days
Sickle Cell
20 days
Gene Therapy for Sickle Cell
Anemia
a chains
z
a2
a1
No
polymerization
a2 bsg
LCR
e
g
bs chains
Non-sickled red cell
Survives 120 days
LCR
g chains
b g
g
bs
Mixed Chimerism following BMT
for b Thalassemia and Sickle Cell
Disease
• Occurs in a minority of patients (5 - 10%).
• A minority of donor-origin progenitors (10 20%) is sufficient to ameliorate disease.
Thus, it may be possible to achieve therapeutic
effects by gene transfer into only a fraction of
stem cells.
Preferential Survival of Normal
Red Blood Cells
TURNOVER
RATE
HIGH
LOW
20 / 120 = 1/6th
normal or
corrected
stem cells
=
50% corrected
mature red cells
Therapeutic effects from small
numbers of normal
stem and progenitor cells in the
marrow
BONE MARROW
BLOOD
N
120 days
SS
SS
SS
20 days
Approaches to Improving the
Efficiency of Gene Therapy Targeting
the Stem Cell
• Use selection to exponentially expand stem
cells carrying the therapeutic gene.
• Use repeated treatments to additively expand
stem cells carrying the therapeutic gene.
In Vivo Selection
Therapeutic gene
Selectable gene
Selectable gene =
MDR1 (taxol, navelbine, vinblastine)
DHFR (methotrexate)
Other (MGMT, aldehyde dehydrogenase,
cytidine deaminase)
In Vivo Selection of Genetically
Modified Bone Marrow
Drug Treatment
Gene Therapy for Sickle Cell
Disease
GCSF
Mobilize
Stem Cells
REPEAT
Introduce gene
Re-infuse
In vivo selection
One developing technology that may be utilized
for gene therapy is nuclear transfer (“cloning”).
What’s in a Name? – Nuclear Transplantation
vs. Therapeutic Cloning vs. Human
Reproductive Cloning.
Ethical Considerations
• Use of technology for non-disease conditions such
as functional enhancement or “cosmetic” purposes
– for example, treatment of baldness by gene
transfer into follicle cells , larger size from growth
hormone gene, increased muscle mass from
dystrophin gene.
• In utero somatic gene therapy – only serious
disease should be targeted and risk-benefit ratios
for mother and fetus should be favorable.
• Potential for real abuse exists by combining
human reproductive cloning and genetic
engineering.