Therapy
of the Hereditary Disease
Medical Genetics
Operative Treatment
Operation correction
Tissue and organ transplant
Rules
Pharmacotherapy
Dietetic Treatment
Rules
Gene Therapy
Gene therapy is a technique for
correcting defective gene responsible
by transferring of a functional normal
copy of the gene into cells.
Gene Therapy
Gene correction,specific correction of
mutant gene without other changes in
target genome.
Gene augmentation,modifying content
or expression of defective cells by
introducing foreign normal gene
sequences.
Gene Therapy
Goal
Somatic gene therapy
Gene Therapy
Goal
Somatic gene therapy
Germ cell gene therapy
Gene Therapy
Key
Safety concerns
Effective expression
Understanding of the disease process
Structure/function of gene to be introduced
Efficient delivery of gene
Control of gene expression
Prevention/control of immune responses
Animal model and assessment of function
Clinical trial
Gene Therapy
1980,UCLA researcher (Dr,Martin
Cline) performed experiments without
institutional approval (done in Italy and
Israel).
Gene Therapy
Gene Delivery Method
In vivo
The genetic material is transferred directly
into the body of the patient
Gene Delivery Method
In vivo
More or less random process; small
ability to control; less manipulations
Only available option for tissues that
can not be grown in vitro; or if grown
cells can not be transferred back
Gene Delivery Method
Cells removed from body
Ex vivo
The genetic material is transferred into
the cells
Cells cultured in vitro
Cells returned to the body
Gene Delivery Method
Ex vivo
Controlled process; transfected cells
are selected and expanded; more
manipulations
Cells are usually autologous; they are
then returned back to the patient
Gene Delivery Method
Ex vivo
Physical Methods
Direct injections
Electroporation
Microparticle bombardment
(gene gun)
Chemical Methods
Coprecipitation with calcium
phosphate
Transfer efficiency,1% ~0.1%
Membrane Fusion
Artificial liposomes
Erythrocyte ghosts
Microcell
Spheroplast
Membrane Fusion
Advantages
Stable complex
Can carry large sized DNA
Can target to specific cells
Does not induce immunological reactions
Disadvantages
Low transfection efficiency
Transient expression
Inhibited by serum
Homologous Recombination
Xba I Bst I
neo
13.1 Kb
Xba I Xba IBst I
11.1 Kb
neo?
Xba I Bst I Xba I Bst I Xba I
16.5 Kb 7.7 Kb
Bst I
Virus Vectors
Virus mediated gene transfer
More efficient than other methods
Can infect every cell in a target
population
Advantages
High transfection efficiency,100%
Integration and persistent expression
Wide host cells
Disadvantages
Can only accomodate 7 kb maximum
Random insertion,influence host gene
Potential for carcinogenesis
Only infecting dividing cells
Retrovirus,RV
Moloney murine leukemia virus
(Mo-MLV)
5’ LTR LTR 3’
U3 R U5 U3 R U5
ψ
gag pol env
Retrovirus,RV
Moloney murine leukemia virus
(Mo-MLV)
5’ LTR LTR 3’
U3 R U5 U3 R U5
ψ
Exogenous gene
Retrovirus,RV
Retrovirus,RV
Packaging cell line
ψ+
RNA
ψ-Helper virus
Packaging cell RNA
Packaging
protein
病毒颗粒 重组病毒颗粒
Adenovirus,AV
Advantages
Can carry large sized DNA
No insertional mutagenesis
Infecting dividing and nondividing cells
Wide host cells
Disadvantages
Transient expression
Highly immunogenic
E1 E3
Adenovirus,AV
DNA
Cell 293
Packaging cell line
Adenovirus,AV
DNA E3 Cell 293
Packaging cell line
Adeno-Associated Virus,AAV
Advantages
Integration (chr19) and persistent expression
No insertional mutagenesis
Infecting dividing and nondividing cells
Safe
Disadvantages
Size limitation,4.9 kb
Immunogenic
Dependent on a helper virus for replication
Herpes Simplex Virus,HSV
Advantages
dsDNA viruses that infect a neurons
Wide host cells
Infecting dividing and nondividing cells
Disadvantages
Cell toxicity
Vaccinia Virus,VV
Advantages
Easy
Safe
Disadvantages
Highly immunogenic
Lentiviral
Advantages
Infecting dividing and nondividing cells
Persistent expression
Integration
Antisense Nucleic Acid
A nucleic acid molecular with a
nucleotide sequence complementary to a
specified mRNA.
Antisense RNA expression vector
Direct injection of antisense RNA/DNA
Antisense RNA delivered by liposome
Others
TdR d T M P d T D P dTTP1 TKH S V 1 TKH S Vc y t o s o l T K c y t o s o l d T M P - K
c y t o s o l d N D P - K
G C V G C V M P G C V D P G C V T P1 TKH S V 1 TKH S V
c y t o s o l d N D P - K
Suicide Gene
A gene that transforms a nontoxic
form of a drug (pro-drug) into a toxic
substance.
herpes simplex virus thymidine kinase gene
(HSV1TK)
Suicide Gene
By-stander effect
The toxic substance (transform from
pro-drug) diffuses out into wide
surrounding area and kills other
tumour cells.
Suicide Gene
By-stander effect
Suicide Gene
By-stander effect
Suicide Gene
By-stander effect
Suicide Gene
By-stander effect
MDR Gene
Multidrug-Resistance Gene
Tumor Suppressor Gene
Anti-oncogene
Gene replacement therapy
A method for replacing a mutated or
missing gene (usually a tumor suppressor
gene) that serves to keep cell growth and
division under control with a normal copy of
that gene.
Gene Replacement Therapy
Gene Replacement Therapy
Apoptosis
Target Cell
Target Cell
Gene Therapy Trials
ADA gene therapy (MIM 102700)
Severe combined immunodeficiency
(SCID) caused by mutation in
adenosine deaminase gene in T cells,
Gene Therapy Trials
ADA gene therapy
French Anderson (NIH)
LTR ADA SV40 neo LTR
September 14,1990
Retrovirus (Mo-MLV)
Gene Therapy Trials
Ashanti de Silva
Ashanti was the first
patient to be treated with
gene therapy.
Injections repeated 7 times in first 10.5 months
ADA,1% 25%
Gene Therapy Trials
Hemophilia B
Xue Jinglun (Fudan university)
1991,Retrovirus
Skin fibroblast
Factor IX,5%
2003,rAAV2-hF.IX
Gene Therapy Trials
Solid tumors
Cytokine
1984-1987,
lymphokine activated killer cell (LAK)
1986,
tumor infiltrate lymphocytes (TIL)/IL-2
1991,tumor necrosis factor (TNF)
Gene Therapy Trials
Solid tumors
Suicide gene
1992,HSV-TK/GCV (brain tumor)
Gene Therapy Trials
Solid tumors
Tumor suppressor gene
pRB,p53,p16,p21,p27
Gene Therapy Trials
Solid tumors
Nanoparticle,NP
Vascular endothelial cell growth factor
(VEGF)
Gene Therapy on Trial
Persistent expression
High-efficiency expression
Safe
Gelsinger Case
1999,Death of Jesse Gelsinger in Penn
OTCD trial
OTCD,orthinithin transcarbamylase (a
urea cycle enzyme) deficiency.
OTCD,X-linked,1/40 000 birth
Jesse Gelsinger,18 yr-old from Arizona,
died on 9/17/1999,4 day after gene
transfer.
Gene Therapy on Trial
Persistent expression
High-efficiency expression
Safe
Tissue-specific promotor
,Ideal” vector
of the Hereditary Disease
Medical Genetics
Operative Treatment
Operation correction
Tissue and organ transplant
Rules
Pharmacotherapy
Dietetic Treatment
Rules
Gene Therapy
Gene therapy is a technique for
correcting defective gene responsible
by transferring of a functional normal
copy of the gene into cells.
Gene Therapy
Gene correction,specific correction of
mutant gene without other changes in
target genome.
Gene augmentation,modifying content
or expression of defective cells by
introducing foreign normal gene
sequences.
Gene Therapy
Goal
Somatic gene therapy
Gene Therapy
Goal
Somatic gene therapy
Germ cell gene therapy
Gene Therapy
Key
Safety concerns
Effective expression
Understanding of the disease process
Structure/function of gene to be introduced
Efficient delivery of gene
Control of gene expression
Prevention/control of immune responses
Animal model and assessment of function
Clinical trial
Gene Therapy
1980,UCLA researcher (Dr,Martin
Cline) performed experiments without
institutional approval (done in Italy and
Israel).
Gene Therapy
Gene Delivery Method
In vivo
The genetic material is transferred directly
into the body of the patient
Gene Delivery Method
In vivo
More or less random process; small
ability to control; less manipulations
Only available option for tissues that
can not be grown in vitro; or if grown
cells can not be transferred back
Gene Delivery Method
Cells removed from body
Ex vivo
The genetic material is transferred into
the cells
Cells cultured in vitro
Cells returned to the body
Gene Delivery Method
Ex vivo
Controlled process; transfected cells
are selected and expanded; more
manipulations
Cells are usually autologous; they are
then returned back to the patient
Gene Delivery Method
Ex vivo
Physical Methods
Direct injections
Electroporation
Microparticle bombardment
(gene gun)
Chemical Methods
Coprecipitation with calcium
phosphate
Transfer efficiency,1% ~0.1%
Membrane Fusion
Artificial liposomes
Erythrocyte ghosts
Microcell
Spheroplast
Membrane Fusion
Advantages
Stable complex
Can carry large sized DNA
Can target to specific cells
Does not induce immunological reactions
Disadvantages
Low transfection efficiency
Transient expression
Inhibited by serum
Homologous Recombination
Xba I Bst I
neo
13.1 Kb
Xba I Xba IBst I
11.1 Kb
neo?
Xba I Bst I Xba I Bst I Xba I
16.5 Kb 7.7 Kb
Bst I
Virus Vectors
Virus mediated gene transfer
More efficient than other methods
Can infect every cell in a target
population
Advantages
High transfection efficiency,100%
Integration and persistent expression
Wide host cells
Disadvantages
Can only accomodate 7 kb maximum
Random insertion,influence host gene
Potential for carcinogenesis
Only infecting dividing cells
Retrovirus,RV
Moloney murine leukemia virus
(Mo-MLV)
5’ LTR LTR 3’
U3 R U5 U3 R U5
ψ
gag pol env
Retrovirus,RV
Moloney murine leukemia virus
(Mo-MLV)
5’ LTR LTR 3’
U3 R U5 U3 R U5
ψ
Exogenous gene
Retrovirus,RV
Retrovirus,RV
Packaging cell line
ψ+
RNA
ψ-Helper virus
Packaging cell RNA
Packaging
protein
病毒颗粒 重组病毒颗粒
Adenovirus,AV
Advantages
Can carry large sized DNA
No insertional mutagenesis
Infecting dividing and nondividing cells
Wide host cells
Disadvantages
Transient expression
Highly immunogenic
E1 E3
Adenovirus,AV
DNA
Cell 293
Packaging cell line
Adenovirus,AV
DNA E3 Cell 293
Packaging cell line
Adeno-Associated Virus,AAV
Advantages
Integration (chr19) and persistent expression
No insertional mutagenesis
Infecting dividing and nondividing cells
Safe
Disadvantages
Size limitation,4.9 kb
Immunogenic
Dependent on a helper virus for replication
Herpes Simplex Virus,HSV
Advantages
dsDNA viruses that infect a neurons
Wide host cells
Infecting dividing and nondividing cells
Disadvantages
Cell toxicity
Vaccinia Virus,VV
Advantages
Easy
Safe
Disadvantages
Highly immunogenic
Lentiviral
Advantages
Infecting dividing and nondividing cells
Persistent expression
Integration
Antisense Nucleic Acid
A nucleic acid molecular with a
nucleotide sequence complementary to a
specified mRNA.
Antisense RNA expression vector
Direct injection of antisense RNA/DNA
Antisense RNA delivered by liposome
Others
TdR d T M P d T D P dTTP1 TKH S V 1 TKH S Vc y t o s o l T K c y t o s o l d T M P - K
c y t o s o l d N D P - K
G C V G C V M P G C V D P G C V T P1 TKH S V 1 TKH S V
c y t o s o l d N D P - K
Suicide Gene
A gene that transforms a nontoxic
form of a drug (pro-drug) into a toxic
substance.
herpes simplex virus thymidine kinase gene
(HSV1TK)
Suicide Gene
By-stander effect
The toxic substance (transform from
pro-drug) diffuses out into wide
surrounding area and kills other
tumour cells.
Suicide Gene
By-stander effect
Suicide Gene
By-stander effect
Suicide Gene
By-stander effect
Suicide Gene
By-stander effect
MDR Gene
Multidrug-Resistance Gene
Tumor Suppressor Gene
Anti-oncogene
Gene replacement therapy
A method for replacing a mutated or
missing gene (usually a tumor suppressor
gene) that serves to keep cell growth and
division under control with a normal copy of
that gene.
Gene Replacement Therapy
Gene Replacement Therapy
Apoptosis
Target Cell
Target Cell
Gene Therapy Trials
ADA gene therapy (MIM 102700)
Severe combined immunodeficiency
(SCID) caused by mutation in
adenosine deaminase gene in T cells,
Gene Therapy Trials
ADA gene therapy
French Anderson (NIH)
LTR ADA SV40 neo LTR
September 14,1990
Retrovirus (Mo-MLV)
Gene Therapy Trials
Ashanti de Silva
Ashanti was the first
patient to be treated with
gene therapy.
Injections repeated 7 times in first 10.5 months
ADA,1% 25%
Gene Therapy Trials
Hemophilia B
Xue Jinglun (Fudan university)
1991,Retrovirus
Skin fibroblast
Factor IX,5%
2003,rAAV2-hF.IX
Gene Therapy Trials
Solid tumors
Cytokine
1984-1987,
lymphokine activated killer cell (LAK)
1986,
tumor infiltrate lymphocytes (TIL)/IL-2
1991,tumor necrosis factor (TNF)
Gene Therapy Trials
Solid tumors
Suicide gene
1992,HSV-TK/GCV (brain tumor)
Gene Therapy Trials
Solid tumors
Tumor suppressor gene
pRB,p53,p16,p21,p27
Gene Therapy Trials
Solid tumors
Nanoparticle,NP
Vascular endothelial cell growth factor
(VEGF)
Gene Therapy on Trial
Persistent expression
High-efficiency expression
Safe
Gelsinger Case
1999,Death of Jesse Gelsinger in Penn
OTCD trial
OTCD,orthinithin transcarbamylase (a
urea cycle enzyme) deficiency.
OTCD,X-linked,1/40 000 birth
Jesse Gelsinger,18 yr-old from Arizona,
died on 9/17/1999,4 day after gene
transfer.
Gene Therapy on Trial
Persistent expression
High-efficiency expression
Safe
Tissue-specific promotor
,Ideal” vector
