Download 13. Caner Gene Therapy

13.
การร ักษามะเร็งด้วยวิธย
ี น
ี บาบ ัด
(Cancer Gene Therapy)
ว ัตถุประสงค์


สามารถอธิบาย Methods of Gene Transfer ได้
สามารถอธิบายหล ักการพร้อมยกต ัวอย่างการร ักษามะเร็งโดยวิธ ี
ยีนบาบ ัด
้ หา
เนือ
13.1 Introduction
13.2 Types of gene therapy
13.3 General gene therapy strategies
13.4 Methods for Gene Transfer into animal cells
13.5 Cancer Gene Therapy
13.6 Problems and Ethics
13. Cancer Gene Therapy
13.1 Introduction
 Gene therapy is the insertion of genes into an individual’s cells and tissues to
treat a disease, and hereditary diseases in which a defective mutant allele is
replaced with a functional one.
 1990 - the first approved gene therapy, U.S.National Institutes of
Health
----> SCID (severe combined immunodeficiency)
- lacked a healthy immune system
- vulnerable to every passing germ or infection
 Basic process for gene therapy:
- A “corrected” gene is inserted into the genome to replace an “abnormal”
disease-causing gene.
- A carrier called a vector must be used to deliver the therapeutic gene to the
patient’s target cells.
13.2 Types of gene therapy
1. Somatic gene therapy (somatic cells - most cells of the body)
2. Germline gene therapy (sperm cells, ova, stem cell precursors
of sperm cells and ova)
- All gene therapy to date on humans has been directed at somatic
cells (whereas germline engineering in humans remains
controversial)
- For germline gene therapy, the introduced gene must be
incorporated into the chromosomes by genetic recombination.
- Somatic gene therapy
1. ex vivo - cells are modified outside the body and then transplanted
back in again
2. in vivo - genes are changed in cells still in the body
- Recombination-based approaches in vivo are especially uncommon
---> most DNA constructs recombination has a very low probability
13.3 General gene therapy strategies
1. Gene augmentation therapy
- for diseases caused by loss of function of a gene
- introducing extra copies of the normal gene
2. Targeted killing of specific cells
- popular in cancer gene therapies
- direct cell killing ---> the inserted gene produces a lethal toxin or
prodrug
- indirect cell killing ---> the inserted genes are immunostimulatory
genes to provoke or enhance an immune
response against the target cell
3. Targeted mutation correction
- the inherited mutation produces a dominant-negative effect
- gene targeting based on homologous recombination
- therapeutic ribozymes/therapeutic RNA editing
4. Targeted inhibition of gene expression
- disease cells display a novel gene product/ inappropriate
expression of a gene
- selectively inhibit the expression of a particular gene
=> blocking transcription--gene-specific oligonucleotide
=> blocking post-transcriptional processing--antisense RNA
=> blocking post-translational processing--antibodies
13.4 Methods for Gene Transfer into animal
cells (Transfection)
1) Non Viral Transfection System (physical transfection,
chemical transfection, bactofection)
2) Viral-based Transfection System (Transduction)
13.4.1 Non Viral Transfection System
1. Calcium phosphate precipitation (chemical transfection)
- Bacchetti S. and Graham L. (1977)
- mix DNA with calcium chloride in phosphate buffer
----> “DNA-CaPO4” complex
- DNA enters cell via “endocytosis”
2. DEAE-Dextran (chemical transfection)
- facilitate DNA binding to cell membrane
- DNA enters cell via “endocytosis”
- transient transfection
3. Cationic Liposome-mediated transfection (chemical transfection)
-Lipofectamine
-Fugene6
Calcium
Phosphate
DNA
Endocytosis
DEAEDextran
Endocytosis
DNA
Artificial
Liposomes
DNA
-Endocytosis
-Fusion
Liposome
4. Linear cationic polyethylenimine (chemical transfection)
-jetPEI
PEI = Polyethylenimine
Endocytosis
-เหมาะสาหร ับ non attached cells
Biomol company
5. Electroporation/electropermeabilization (Physical transfection)
้
-ทำให ้เกิดรู (pore) ทีเ่ ยือ
่ หุ ้มเซลล์โดยใชกระแสไฟฟ้
ำ
-pore เกิดได ้เมือ
่ แรงดันไฟฟ้ ำสูงกว่ำค่ำกำรต ้ำนทำนไฟฟ้ ำของเยือ
่ หุ ้มเซลล์
(dielectric strength)
-ใชไ้ ด้ผลดีก ับเซลล์หลายชนิด
6. Nucleofection (~ electroporation) (physical transfection)
- Nucleofector:-set optimum electrical parameter for specific cell type
- Nucleofector solution:-cell type specific
1.
3.
2.
4.
Amaxa, a Lonza company
7. Magnetofection (physical transfection)
-DNA + magnetic particles
-----> apply magnetic force
-Poly MAG, Combi MAG
8. Microinjection (physical transfection)
-inject DNA directly into nucleus of cells using
glass micropipettes
-Cells are grown on glass slides
-500-1000 cells/hr
-success 50-100%
9. Protoplast fusion (Bactofection)
www.research.uci.edu/tmf/images/pronuc1800.jpg
Bacterial cells containing plasmids of interest
Treat with lysozyme-remove cell wall
(generate protoplast)
Fuse bacterial protoplast to mammalian cells
by using polyethylene glycol
- contaminated bacteria are killed by antibiotic in the medium
- Not widely used
13.4.2 Viral-based Transfection System
- Viruses: intracellular parasites (cell type specific)
: transfect their own DNA/RNA into the host
cell ---> produce new viral particles
- Increased transfection efficiency
- Potentially infectious particles
1) Retrovirus Vector
- Virus = ssRNA genome
- Moloney murine leukemia virus
(Mo-MLV)
- ~10 kb
- At least 3 genes
gag -coding for core proteins
pol -coding for Reverse transcriptase
env -coding for viral envelope protein
- LTR = long terminal repeat
(promoter/enhancer/integration)
- infect only dividing cells
Retroviral based Transfection
LTR
Reporter gene
LTR
http://biology.kenyon.edu/slonc/gene-web/Lentiviral/HIVvector.jpg
2) Lentivirus Vector
- Virus = a subclass of retrovirus
- Able to infect both proliferating & non-proliferating cells
- More complicated than simple retrovirus (..contains additional
6 genes; tat, rev, vpr,vpu,nef,vif)
3) Adenovirus Vector
-Adenoviruses
: non-enveloped viruses
: linear dsDNA genome
: >40 serotype strains
: cause benign respiratory
tract infection in human
: does not integrate into host
genome
: ~35 kb (~upto 30 kb can be
replaced with foreign DNA)
: very efficient viral vectors
: ITR=Inverted Terminal
Repeat
4) Adeno-associated virus (AAV) Vector
-AAV
: non-pathogenic human parvoviruses
: linear ssDNA genome ~5kb
(insert – not larger than 4.7 kb)
: dependent on helper virus for
replication (..require Adenoviruses
or Herpes viruses)
: integrate into the host genome
: 145 bp Terminal Repeat (TR)
: very efficient viral vectors
: ITR=Inverted Terminal Repeat
-AAV vector
: cumbersome method, low yield,
contamination
5) Herpes Simplex virus (HSV) Vector
-HSV-1
: Human Neurotropic virus
: Vector for gene transfer to the
nervous system
: linear dsDNA , 152 kb (40-50 kb
foreign DNA can be inserted)
6) Other Viral Vectors
- Vaccinia and poxviruses: can accommodate large foreign therapeutic genes
- Reoviruses: have oncolytic capabilities --- dsRNA preferentially infect and
kill cells with an activated RAS signaling pathway
- Alphaviruses: ssRNA--- can infect many cell types, high level of replication
and gene expression
13.5 Cancer Gene Therapy
13.5.1 General Approaches
1) Artificial killing of cancer cells
: Insert a gene encoding a toxin (e.g. diphtheria A chain) or a gene conferring
sensitivity to a drug (e.g. herpes simplex thymidine kinase) into tumor cells
gcv = analogue of 2'-deoxy-guanosine
2) Stimulate natural killing of cancer cells
: Enhance the immunogenicity of the tumor by, for example,
inserting gene encoding foreign antigens or cytokines
(IL-2 and interferon-alfa 2b are two cytokines approved by the
FDA for treatment of cancer. )
: Increase anti-tumor activity of immune system cells by, for
example, inserting genes that encode cytokines
: Induce normal tissues to produce anti-tumor substances (e.g.
interleukin-2, interferon)
: Production of recombinant vaccines for the prevention and
treatment of malignancy (e.g. BCG-expressing tumor antigens)
BCG = bacillus Calmette-Guérin
3) Protect surrounding normal tissues from effects of
chemotherapy/radiotherapy
: Protect tissues from the systemic toxicities of chemotherapy (e.g.
multiple drug resistance type 1 gene)
13.5.2 Treatment of tumors resulting from oncogene
activation
1) Selectively inhibit the expression of the oncogene
2) Deliver gene-specific antisense oligonucleotide or ribozyme to
bind/cleave oncogene mRNA
3) Inhibit transcription by triple helix formation following delivery
of a gene-specific oligonucleotide
4) Use of intracellular antibodies or oligonucleotide aptamers to
specifically bind to and inactivate the oncoprotein
13.5.3 Treatment of tumors arising from inactivation of
tumor suppressor
1) Gene augmentation therapy
2) Insert wild-type tumor suppressor gene
Targets for Antisense Therapy
13.6 Problems and Ethics
 Short-lived nature of gene therapy
 Immune response
 Problems with viral vectors
---> toxicity, immune and inflammatory responses,
gene control and targeting issues
 Multigene disorders
 Chance of inducing a tumor (insertional mutagenesis)
 Religious concerns