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Gene Therapy
张咸宁
[email protected]
Tel：13105819271; 88208367
Office: A705, Research Building
2012/09
Learning Objectives
1. Traditional managements
2. Gene therapy
Treatment of Genetic Disease by Metabolic Manipulation
Intervention
Substance or
Technique
Disease
Drug/diatary avoidance Antimalarial drugs
G6PD deficiency
Dietary restriction
Phe
Gal
Cholesterol
PKU
Galactosemia
Familial
hypercholesterolemia
Replacement of
deficient product
Thyroxine
Congenital
hypothyroidism
Protein drug therapy
Penicillamine
Wilson disease
Replacement of
deficient
enzyme/protein
Blood transfusion
SCID
Wilson disease: Cu toxicity, AR
Wilson SAK. Brain,
1912; 34:295-507
Wilson disease:Before/After therapy
Gene therapy
The medical procedure involves either
replacing, manipulating, or
supplementing nonfunctional genes
with healthy genes.
OR
“Everyone talks about the human genome,
but what can we do with it?”
Impact of the Genome Project on
Medicine
•Facilitate identification of genes associated
with complex disorders
ie. Cardiovascular disease, cancer
•provides more therapeutic targets-in turn
enhances our ability to treat cause of disease
instead of symptoms
•bioinformatics, array technology, proteomics
-enable a systems approach to biomedical
research
Monogenic Diseases Which May Be
Candidates For Gene Therapy
Sickle cell anemia/Thal
Bone Marrow
Congenital immune deficiencies Bone Marrow
Lysosomal storage and metabolic Bone Marrow
--------------------------------------------------------------Cystic fibrosis
Lung - airways
--------------------------------------------------------------Muscular dystrophy
Muscle
--------------------------------------------------------------Hemophilia A or B
Liver
Urea cycle defects
Liver
Familial hypercholesterolemia
Liver
Types Of Conditions That
May Be Treated By Gene Therapy
Monogenic Diseases (>1,000 known)
Cancer, Leukemia
Infectious (AIDS, Hep C)
Cardiovascular
Neurologic
Technical Requirements for
Successful Gene Therapy
1. Normal gene must be cloned.
2. Effective method for gene delivery to cells.
3. Inserted gene expressed at appropriate level.
4. Safe for patient and public.
5. More medically beneficial and/or cost-
effective than other treatments.
Gene Delivery Can Be:
I. Ex vivo – gene into isolated cells
II. In vivo – gene directly into patient
a) Systemic injection
+/- targeted localization
+/- targeted expression
b) Localized
1) Percutaneous
5) Bronchoscope
2) Vascular catheter
6) Endoscope
3) Stereotactic
7) Arthroscope
4) Sub-retinal
General considerations for the use of
somatic gene therapy (approved in 1988)
1. Compensate for a mutation resulting in the
loss of function
examples of monogenic disorders:
cystic fibrosis, hemophilia
General considerations for the use of gene
therapy
1. Compensate for a mutation resulting in the
loss of function
examples of monogenic disorders:
cystic fibrosis, hemophilia
stage of the research:
http://clinicaltrials.gov/ct2/results?term=gene+therapy+cystic+fibrosis
General considerations for the use of gene
therapy
1. Compensate for a mutation resulting in the
loss of function
2. Replace or inactivate a dominant mutant gene
General considerations for the use of gene
therapy
1. Compensate for a mutation resulting in the
loss of function
2. Replace or inactivate a dominant mutant gene
example: Huntington disease (expanded CAG repeat)
? Ribozymes or siRNA to degrade mRNA
General considerations for the use of gene
therapy
1. Compensate for a mutation resulting in the
loss of function
2. Replace or inactivate a dominant mutant gene
example: Huntington disease (expanded CAG repeat)
? Ribozymes or siRNA to degrade mRNA
state of research – no open studies for Huntington’s
General considerations for the use of gene
therapy
1. Compensate for a mutation resulting in the
loss of function
2. Replace or inactivate a dominant mutant gene
3. Pharmacologic gene therapy
example: cancer
General considerations for the use of gene
therapy
1. Compensate for a mutation resulting in the
loss of function
2. Replace or inactivate a dominant mutant gene
3. Pharmacologic gene therapy
example: cancer
state of research:
clinicaltrials.gov website currently lists 35624
gene therapy trials for cancer; 10649 are open to
enrollment
General considerations for the use of gene
therapy
1. Compensate for a mutation resulting in the
loss of function
2. Replace or inactivate a dominant mutant gene
3. Pharmacologic gene therapy
Yet, it is important to note that there is not yet a
single FDA-approved use of gene therapy!
Minimal requirements that must be met:
• Identification of the affected gene
• A cDNA clone encoding the gene
Minimal requirements that must be met:
• Identification of the affected gene
• A cDNA clone encoding the gene
• A substantial disease burden and a favorable
risk-benefit ratio
• Sufficient knowledge of the molecular basis
of the disease to be confident that the gene
transfer will have the desired effect
Minimal requirements that must be met:
• Identification of the affected gene
• A cDNA clone encoding the gene
• A substantial disease burden and a favorable riskbenefit ratio
• Sufficient knowledge of the molecular basis of the
disease to be confident that the gene transfer will
have the desired effect
• Appropriate regulation of the gene expression: tissue
specific and levels
• Appropriate target cell with either a long half life or
high replicative potential
• Adequate data from tissue culture and animal studies
to support the use of the vector, regulatory sequences,
cDNA and target cell
Minimal requirements that must be met:
• Identification of the affected gene
• A cDNA clone encoding the gene
• A substantial disease burden and a favorable risk-benefit
ratio
• Sufficient knowledge of the molecular basis of the
disease to be confident that the gene transfer will have
the desired effect
• Appropriate regulation of the gene expression: tissue
specific and levels
• Appropriate target cell with either a long half life or
high replicative potential
• Adequate data from tissue culture and animal studies
to support the use of the vector, regulatory sequences,
cDNA and target cell
• Appropriate approvals from the institutional and
federal review bodies.
Gene therapy
• In most gene therapy studies, a "normal"
gene is inserted into the genome to replace
an "abnormal," disease-causing gene.
• A carrier molecule called a vector must be
used to deliver the therapeutic gene to the
patient's target cells. Currently, the most
common vector is a virus that has been
genetically altered to carry normal
human DNA.
Gene Transfer Methods
Non-viral: Expression plasmid or other nucleic acid
(mRNA, siRNA).
Challenge: Naked DNA or RNA does not enter cells.
a) Transfer into cells using physical methods such as
direct micro-injection or electroporation.
b) complex to carrier to allow cross of cell membrane
liposomes,
cationic lipids,
dextrans,
cyclohexidrins
(aka nanoparticles)
Gene Transfer Methods
Viral vectors = viruses that have been
adapted to serve as gene delivery vectors
include:
retrovirus
Lenti-virus
adenovirus
adeno-associated virus (AAV)
herpes virus
In Vivo Gene Transfer By AAV Vector
Characteristics of the Ideal Vector for Gene
Therapy
• Safe
• Sufficient capacity for size of therapeutic DNA
• Non-Immunogenic
• Allow re-administration
• Ease of manipulation
• Efficient introduction into target cells/tissues
• Efficient and appropriate regulation of
expression
•Level, tissue specificity, transient, stable?
Types of viral vectors
• Retrovirus
• Lenti-virus
• Adenovirus
• Adeno-Associated virus (AAV)
• Herpes virus
Types of viral vectors
stable/transient
infect non-dividing cells
• Retrovirus
stable
no
• Lenti-virus
stable
yes
• Adenovirus
transient
yes
• Adeno-Associated virus
• Herpes virus
?
transient
yes
yes
Which of the following gene-therapy
vectors preferentially infects nerve
cells?
A. Adeno-associated virus
B. Retrovirus
C. Herpes virus
D. Adenovirus
E. Liposome
Which of the following gene-therapy
vectors preferentially infects nerve
cells?
A. Adeno-associated virus
B. Retrovirus
√ C. Herpes virus
D. Adenovirus
E. Liposome
Which of the following vectors targets
both dividing and non-dividing cells?
A. Retrovirus
B. Adenovirus
C. Adeno-associated virus
D. Herpes virus
E. Liposome
Which of the following vectors targets
both dividing and non-dividing cells?
A. Retrovirus
√ B. Adenovirus
C. Adeno-associated virus
D. Herpes virus
E. Liposome
Choice of target cells is critical
Stem cells
Choice of target cells:
● Long life or substantial replicative potential
bone marrow
● Must express an additional proteins needed for
biological activity
● Some approaches employ neighboring cells
growth factors stimulating repair of nearby heart muscle
In vivo and ex vivo gene therapy
Two strategies for introducing foreign
genes into patients
In vivo gene therapy
Gene therapy vector +
therapeutic gene
Advantages: cells and organs not
available ex vivo (lining of the lung)
Disadvantages: virus could spread
to other cells/tissues
Less control over titer and
conditions of exposure
Two strategies for introducing foreign
genes into patients
Ex vivo gene therapy
Advantages:
More controlled infection
higher titer virus
Disadvantages:
technically difficult
Stem cells
Gene therapy vector +
Normal gene
Types of viral vectors
stable/transient
infect non-dividing cells
• Retrovirus
stable
no
• Lenti-virus
stable
yes
• Adenovirus
transient
yes
• Adeno-Associated virus
• Herpes virus
?
transient
yes
yes
Use of retroviral vectors to introduce
therapeutic genes into cells
Use of retroviral vectors to introduce
therapeutic genes into cells
Severe Combined Immunodeficiency Syndrome
(SCID)——adenine deaminase (ADA) deficiency
Severe Combined Immune Deficiency (SCID)
SCID is popularly known as “bubble baby disease” after
a boy with SCID was kept alive for more than a decade
in a germ-free room.
SCID is a fatal disease, with infants dying from
overwhelming infection due to the congenital absence
of a functioning immune system.
More than a dozen genes have been found to be
able to cause human SCID.
The first “SCID gene” to be identified in humans
is ADA, which makes an enzyme needed for
Immune cells to survive.
Somatic Therapy for SCID
Ex vivo
Severe Combined Immunodeficiency Disease (SCID) is due to a defective gene for Adenosine
Deaminase (ADA). A retrovirus, which is capable of transferring it's DNA into normal eukaryotic
cells (transfection), is engineered to contain the normal human ADA gene. Isolated T-cell stem
line cells from the patient are exposed to the retrovirus in cell culture, and take up the ADA gene.
Reimplantation of the transgenic cells into the patient's bone marrow establishes a line of cells with
functional ADA, which effecitvely treats SCID.
ADA deficiency (SCID): Ashanti de Silva，
1990
Father of GT: Anderson WF, 1990
Geneticist guilty of molestation, 2006
Clinical Trial of Stem Cell Gene Therapy
for Sickle Cell Disease
Bone Marrow Harvest
Add Normal
Hemoglobin Gene
Isolate Stem Cells
βAS3 Globin
ψ
SIN
LTR
β-Globin LCR
HS2
RRE cPPT
Myeloablate with
Transplant GeneBusulfan (16 mg/kg) Corrected Stem Cells
Freeze
Certify
HS3
HS4
WPRE SIN
LTR
Follow:
Safety
Efficacy
Gene Therapy Approaches To Cancer
a. Replace missing tumor suppressor genes.
b. Block over-active oncogenes (e.g. siRNA).
c. Insert “suicide genes” (e.g. HSV TK) into
tumors.
d. Insert genes to induce anti-tumor immune
responses (e.g. IL-2, GM-CSF, CD80).
e. Express genes which impede tumor neovasculature.
f. Add chemotherapy resistance genes to HSC
to allow chemotherapy dose intensification.
Suicide gene therapy for brain tumors in vivo
tk
• Inject HSV thymidine kinase (tk)
gene into tumor cells
gancyclovir
• Gancyclovir (nucleoside analog)
binds viral gene to block DNA
synthesis
• Bystander effect kills surrounding
tumor cells
• Takes advantage of the fact that
tumor cells are dividing
Cancer Vaccine Approach
Ex vivo gene therapy
Time of surgery
tumor cells
Irradiated tumor cells transduced with cytokine
gene
Gene therapy vector +
Cytokine (immune
modulator) gene
Other methods to introduce therapeutic DNA:
(approved in 1993)
• Naked DNA
• DNA packed in liposomes（脂质体）
• Protein-DNA conjugates (targeting to cell
surface receptor
++++ easy to prepare, inexpensive, avoids problems of
viral vectors, no size limitations
------- low efficiency in vivo, only transient expression
Risks of Gene Therapy
1. Adverse reaction to vector or gene
1999/9/17: reaction to an adenovirus caused death of
18-yo man, Jesse Gelsinger, Arizona, the first
victim of gene therapy. OTC (ornithine
transcarbamylase) important for metabolism of N
Injection of viral particles triggered massive
inflammatory response in an individual with mild
form of disease being treated with drugs and diet.
Subsequent FDA audit revealed protocol and IRB
violations.
Risks of Gene Therapy
2. Activation of harmful genes by viral
promoters/enhancers stably integrated
into the genome.
2002 retrovirus-induced leukemia
Children with otherwise fatal X-linked SCID injected with ex
vivo HSC modified by introduction of the g-c chain
cytokine receptor in 2000 (affects lymphocyte maturation)
Initial immune function was good
2/11 patients developed leukemia-like disorder at 2 years.
Clonal analysis shows insertion and activation of LMO2 gene.
FDA-cannot be used as first line therapy if BMT is an option
What factors have kept GT from becoming
an effective treatment for genetic disease?
•
•
•
•
Short-lived nature of gene therapy
Immune response
Problems with viral vectors
Multigene disorders
RNAi
Gene therapy using Autologous HSC
Made from Induced Pluripotent Stem Cells
Tissue Sample
(e.g. skin biopsy)
Autologous
Transplant
Patient
Differentiation to
Hematopoietic
Stem Cells (HSC)
Gene Addition or
Gene Correction
De-Differentiation
to Induced
Pluripotent Stem
cells (iPS)
Gene Therapy
Current
Future
Experimental
Proven
Limited Scope
Curative
High Tech
Off the Shelf
Gene Therapy Clinical Trials
Worldwide (updated list of all gene
therapy protocols)
www.wiley.com/legacy/wileychi/genmed/clinical/
Acknowledge（PPT特别鸣谢！）
• UCLA David Geffen School of Medicine
• www.medsch.ucla.edu/ANGEL/
• Prof. Kohn DB (Department of
Microbiology, Immunology and
Molecular Genetics (MIMG) ), Prof.
Gasson JC (UCLA Jonsson
Comprehensive Cancer Center ), et al.