Gene therapy Flashcards
1
Q
What is gene therapy
A
- Treatment of disease by genetic manipulation of the patient’s cells
- How?
- By insertion of a functional exogenous gene (transgene) into cells of a patient
- To correct an inborn error of metabolism
- Or to repair an acquired genetic abnormality
- Or to provide a new function to a cell
- By insertion of a functional exogenous gene (transgene) into cells of a patient
- It is a form of drug delivery in which altered cells produce the needed proteins continuously at the therapeutic level
2
Q
Examples of caused by single-gene defects
A
- Treatment options
- Administration of the deficient proteins to alleviate the symptoms (conventional therapy)
- Restore gene function that has been lost- gene therapy
3
Q
Some acquired disease amenable to gene therapy
A
4
Q
Approaches for gene therapy: gene replacement or addition
A
- Replacement
- Remove defective gene
- Incorporate functioning allele
- Requires recombination event (technically difficult)
- Addition
- A functional gene is integrated into genome
Or it exists independently
5
Q
Target of gene therapy: Germ cells and somatic cells
A
- Germ cells
- Modifies egg and sperm cells or a fertilised egg
- Inheritable change: the change is passed on to the next generation
- Somatic cells
- Only affected cells of body (Somatic): the changes is not passed on to the next generation
- May be In-Vivo or Ex-Vivo
6
Q
Germline therapy
A
7
Q
Somatic therapy
A
8
Q
Possible routes for administration of therapeutic genetic materials
A
- Dependent upon the type of delivery system and the cells targeted by the therapy
- Inhalation
- Oral administration
- Intramuscular injection
- Intravenous injection
- Ex-vivo administration
9
Q
How to transfer a functional gene into cells delivery vehicles
A
- Genes transfer involves the delivery of a functional gene to the target cells and sequences controlling it’s expression
- An ideal delivery vehicle
- Efficient delivery
- Relatively specific for target cells
- Limited immune response
- Alllow transgene eexpression
- Carry large pieces of DNA
10
Q
Delivery vehicles
A
- Direct injection of naked DNA: Used in germline therapy
- Viruses (infective but replication-defective) such as retrovirus and adenovirus
- Non-viral vehicles, such as liposomes
11
Q
Physical methods to gene delivery
A
-
Electroporation
- Uses short pulses of high voltage to carry DNA across the cell membrane
- This shock causes the temporary formation of pores in the cell membrane, allowing DNA molecules to pass through
-
Gene gun
- DNA is coated with gold particles and loaded into a device which generates a force to achieve penetration of DNA/gold into the cells
-
Sonoporation
- Ultrasonic frequencies to deliver DNA into cells. The process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to enter into the cells
-
Magnetofection
- DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer
12
Q
Viruses
A
- Very effective in transferring genetic materials into host cells and evading host-defence system, but it is destructive
- How to make them safe: By substituting therapeutic gene for genes involving in viral replication and virulence
13
Q
Retroviruses
A
- SingleStranded RNA viruses
- RNA converted to double-stranded DNA
- Reverse transcriptase
- Viral DNA incorporated into host cell chromosome
- Stable, permanent
- Infect only dividing cells
- Suitable for ex-vivo gene therapy
14
Q
Construction of retroviral vector for gene therapy
A
- The therapeutic DNA is first cloned into the retroviral vector
- A packaging cell line expressing retroviral genes necessary for replication and viral packaging then is transfected with the retroviral vector DNA
- The transfected DNA is transported to the nucleus of the transfected cell and the retroviral vector DNA integrates into the genome of the transfected cell
- The infective recombinant retroviral particles are released from transfected cells
- Purify and characterise the released recombinant retroviral particles to ensure they contain the therapeutic DNA and that they remain replication-deficient
- Once characterised, the therapeutic retroviral vector must be amplified to produce stocks of sufficient titre for therapeutic administration
15
Q
Retroviral-mediated gene delivery
A
- The retroviral vector encoding the therapeutic gene binds to specific cell receptors and enters the cell by endocytosis
- The viral vector then is released from the endosome and its RNA genome is converted to DNA before being transported into the nucleus of the cell
- Once inside the nucleus, retroviral DNA is integrated randomly into the genome of the infected cell
- The integrated retroviral and/or therapeutic DNA then is transcribed and translated into a functional protein using the transcriptional and translational machinery of the host cell
- As the recombinant retroviral vector is integrated into the genome of the infected cell the expression of the therapeutic DNA is stable and is passed onto resultant progeny
18
Q
Construction of adenoviral vector for gene delivey
A
- First, the therapeutic DNA is cloned into an adenoviral shuttle vector
- The adenoviral shuttle vector and replication-deficient adenoviral DNA are then co-transfected into a packaging cell line
- Once inside the cell, the process of homologous recombination allows DNA to be swapped between the adenoviral DNA and the shuttle vector, thus generating recombinant adenoviruses that encode the therapeutic gene
- Generated adenoviruses are translocated to the nucleus, where they are replicated and packaged and then released from the infected cells
- The recombinant adenoviruses are purified and characterised to ensure that they have remained replication-deficient and that they express the appropriate therapeutic DNA
- The characterised adenovirus is amplified to generate high-titre stocks required for in-vivo administration
19
Q
Adenoviral-mediated gene delivery
A
- The recombinant adenoviral vector enters the cell by receptor-mediated endocytosis
- In the endosome, the viral particle is released from this internal cellular compartment and the adenoviral capsid is translocated into the nucleus
- Once inside the nucleus, the therapeutic DNA is transcribed from the episomal recombinant adenoviral genome and if necessary, the therapeutic RNA in translated
- As the recombinant adenoviral vector is replication-deficient, the genome of the adenovirus is not replicated or packaged and therefore the infection is unable to spread to surrounding cells
23
Q
Liposome-mediated gene delivery
A
- Negatively charged DNA is first condended and then mixed the cationic lipid mixture
- The complexes of DNA and liposomes that form are then administered to the target tissue or cells, where the complexes are taken up by endocytosis
- Once inside the cell, the complexes are released from the endosomes and enter the target cell nucleus, where the therapeutic DNA is transcribed and translated to express the therapeutic gene product
27
Q
Cystic fibrosis- pathology
A
28
Q
Gene therapy for cystic fibrosis
A
- In-vivo gene therapy
- Insert transgene into liposome or lipoplexes or viral vector/vehicle systems
- Ex-vivo gene therapy
