Simply, what is gene therapy?
Gene therapy is the delivery of nucleic acid polymers to a patient's cells as a drug to treat disease, usually to replace a mutated gene
What is the aim of gene therapy?
To replace, modify, or knock out defective, disease causing genes genes
What are the 3 routes by which gene therapy can be carried out?
1 Gene Addition: missing/mutant protein is supplied by expression of normal gene
2 Gene Correction/Alteration
3 Gene Knockdown
(last two are more challenging, use Zinc Finger Endonucleases - ZFN's)
What are the 4 barriers to successful gene therapy?
- Uptake of vector, transport and uncoating
- Vector genome persistence
- Transcriptional activity
- Immune response
What are the 2 types of cells that gene therapy can be applied to?
(again this is very broad)
- Somatic: for cells found in the body
- Germ-line: cells found in sperm and eggs (hereditary)
What are the 2 types of gene delivery approaches (vectors)?
Viral or Non-Viral
With gene therapy, what is the difference between insertion and transduction?
Insertion: integration of the DNA into the genome
Transduction: virus-mediated DNA transfer
How might gene therapy effects be short-lived?
Because it's hard to rapidly integrate therapeutic DNA into the genome, and the rapidly dividing nature of cells requires multiple rounds to make sure it's effective
What are the possible adverse responses to gene therapy?
Toxic, Immune and/or Inflammatory responses
May cause a new disease once inside
May induce a tumor if integrated in a tumor suppressor gene
May inactive an essential gene
Viral vector may infect surrounding healthy tissues
What is the difference between ex vivo and in vivo gene therapy?
Ex vivo: cells are removed and grown in tissue plate, then therapeutic gene is made customized for the person, then reinserted into the body
In vivo: new DNA is delivered directly to cells with a virus
What 4 types of viruses are used in gene therapy?
Herpes simplex viruses
What are the advantages of using a retrovirus for GT?
The coding region of the provirus is easily replaceable by the therapeutic gene
They infect cells at high efficiency, integrating a copy of their genome into the host cell
How might retroviruses be utilized for gene therapy?
What problems might occur?
Create double-stranded DNA copies from their RNA genome, using reverse transcriptase
Integrates into the human genome via integrase, which inserts the gene anywhere bc it has no specific site
-therefore this may disrupt the code of a gene, causing insertional mutagenesis-
Infectivity of retroviruses mostly limited to dividing cells
Only allows small length of code
What is the general map of the typical retrovirus genes?
(LTR = long terminal repeats)
What is the significance of the pol gene in retroviruses?
pol encodes for reverse transcriptase, RNase, and integrase
What is the significance of the env gene in the retrovirus?
Encodes for envelope glycoproteins, which mediate virus entry
What are lentiviral vectors?
Subgroup of retroviruses that infect both dividing and nondividing cells
So effective against neurons, muscle and liver cells, etc.
Potentially may lead to a cure for HIV
What are the advantages of using adenoviruses for GT?
-Can use very large inserts of DNA
-Can infect a broad range of mammal cells, both dividing and non-dividing
-High transduction efficiency
What are the disadvantages of using adenoviruses for GT?
-Transient expression! Viral DNA does not integrate
-So viral proteins can be expressed in host following vector administration
-Can be toxic with high dose
What are the advantages of using adeno-associated viruses (AAV) for GT?
-All viral genes are removed
-Does not stimulate immune response
-Enters both dividing and non-dividing cells
What are the disadvantages of using adeno-associated viruses (AAV) for GT?
-Small insertional size of DNA
-Status of genome not fully known
What are the advantages of using herpes simplex virus for GT?
-Allows large size of DNA
-Could provide long-term CNS gene expression
-Patient can take high dose
What are the disadvantages of using herpes simplex virus for GT?
Only infects cells of the nervous system
-Still under development
-Transient expression, currently
-Low transduction efficiency
What is the main barrier to using non-viral vectors in GT?
Currently, the nuclear membrane is difficult to bypass in order to alter DNA
So gene transfer is inefficient, and gene expression is transient
What is the strategy for knock-down of gene expression?