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Gene TherapyGene Therapy
Tel ： 13105819271; 88208367 Office: A705, Research Building
2012/09
Learning Objectives
1. Traditional managements
2. Gene therapy
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
Treatment of Genetic Disease by Metabolic Manipulation
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 MarrowCongenital immune deficiencies Bone MarrowLysosomal storage and metabolic Bone Marrow ---------------------------------------------------------------Cystic fibrosis Lung -
airways ---------------------------------------------------------------Muscular dystrophy Muscle ---------------------------------------------------------------Hemophilia A or B LiverUrea cycle defects LiverFamilial 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 gene3. 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 gene3. 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 gene3. 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 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
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 MethodsNon-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 vectorsstable/transient infect non-dividing cells
• Retrovirus stable no
• Lenti-virus stable yes
• Adenovirus transient yes
• Adeno-Associated virus ? yes
• Herpes virus transient 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 therapyGene 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 therapyStem cells
Gene therapy vector +
Normal gene
Advantages:More controlled infectionhigher titer virus
Disadvantages:technically difficult
Types of viral vectorsstable/transient infect non-dividing cells
• Retrovirus stable no
• Lenti-virus stable yes
• Adenovirus transient yes
• Adeno-Associated virus ? yes
• Herpes virus transient 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 forImmune cells to survive.
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.
Ex vivoSomatic Therapy for 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
β-Globin LCRβAS3 Globin
WPRERRE cPPT
ψ
SIN LTR
SIN LTR
HS2 HS3 HS4
Bone Marrow Harvest Isolate Stem CellsAdd Normal
Hemoglobin Gene
Myeloablate with Busulfan (16 mg/kg)
Transplant Gene-Corrected Stem Cells
FreezeCertify
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 neo-vasculature.
f. Add chemotherapy resistance genes to HSC to allow chemotherapy dose intensification.
Suicide gene therapy for brain tumors in vivo
• Inject HSV thymidine kinase (tk) gene into tumor cells
• 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
tk
gancyclovir
Cancer Vaccine Approach
Ex vivo gene therapy
tumor cells
Gene therapy vector +
Cytokine (immune modulator) gene
Irradiated tumor cells transduced with cytokine gene
Time of surgery
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
Patient
Tissue Sample (e.g. skin biopsy)
Gene Addition or Gene Correction
De-Differentiation to Induced Pluripotent Stem cells (iPS)
Differentiation to Hematopoietic Stem Cells (HSC)
Autologous Transplant
Gene therapy using Autologous HSC Made from Induced Pluripotent Stem Cells
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. KohnProf. Kohn DB (Department ofDepartment of Microbiology, Immunology and Microbiology, Immunology and Molecular Genetics (MIMG)Molecular Genetics (MIMG) ), Prof. Prof. Gasson JC ((UCLA Jonsson Comprehensive Cancer Center ), et al.), et al.
