1. การรักษามะเร็งด้วยวิธียีนบำบัด
13.
การรักษามะเร็งด้วยวิธียีนบำบัด
(Cancer Gene Therapy)
วัตถุประสงค์
สามารถอธิบาย Methods of Gene Transfer ได้
สามารถอธิบายหลักการพร้อมยกตัวอย่างการรักษามะเร็งโดยวิธียีนบำบัด
เนื้อหา
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

2. 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.
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.

3. 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

4. 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

5. 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

6. 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)
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

7. Endocytosis Fusion Endocytosis Endocytosis Calcium DEAE- Phosphate
DNA
DEAE-
Dextran
Artificial
Liposomes
Endocytosis
Endocytosis
Endocytosis
Fusion

8. Liposome

9. 4. Linear cationic polyethylenimine (chemical transfection)
-jetPEI
PEI = Polyethylenimine
Endocytosis
-เหมาะสำหรับ non attached cells
Biomol company

10. 5. Electroporation/electropermeabilization (Physical transfection)
-ทำให้เกิดรู (pore) ที่เยื่อหุ้มเซลล์โดยใช้กระแสไฟฟ้า
-pore เกิดได้เมื่อแรงดันไฟฟ้าสูงกว่าค่าการต้านทานไฟฟ้าของเยื่อหุ้มเซลล์ (dielectric strength)
-ใช้ได้ผลดีกับเซลล์หลายชนิด

11. 6. Nucleofection (~ electroporation) (physical transfection)
- Nucleofector:-set optimum electrical parameter for specific cell type
- Nucleofector solution:-cell type specific 3. 1. 2. 4.
Amaxa, a Lonza company

12. 7. Magnetofection (physical transfection)
-DNA + magnetic particles
-----> apply magnetic force
-Poly MAG, Combi MAG

13. -inject DNA directly into nucleus of cells using glass micropipettes
8. Microinjection (physical transfection)
-inject DNA directly into nucleus of cells using
glass micropipettes
-Cells are grown on glass slides
cells/hr
-success %
9. Protoplast fusion (Bactofection)
Bacterial cells containing plasmids of interest
Fuse bacterial protoplast to mammalian cells
by using polyethylene glycol
- contaminated bacteria are killed by antibiotic in the medium
- Not widely used
Treat with lysozyme-remove cell wall
(generate protoplast)

14. 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

15. Retroviral based Transfection
LTR
Reporter gene

16. 2) Lentivirus Vector 3) Adenovirus 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

17. 4) Adeno-associated virus (AAV) Vector
: 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)

18. 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

19. 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

20. 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)

22. 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
Treatment of tumors arising from inactivation of
tumor suppressor
1) Gene augmentation therapy
2) Insert wild-type tumor suppressor gene

23. Targets for Antisense Therapy

24. 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