Gene Therapy Vectors Flashcards
What are the rules for gene therapy?
Gene expression should be permanent
There should be no immune response to the ‘foreign’ gene product.
Gene should not be passed to subsequent generations.
Why do we need gene therapy vectors?
The delivery of a DNA sequence to treat or correct a genetic disease.
Gene delivery requires a ‘vehicle’ to carry the therapeutic gene to the cell of the host.
What are the characteristics of an ideal vector for gene therapy?
Administered through a non-invasive route
Only target desired cells within the target tissue
Express therapeutic amount of protein
Allow regulated expression
Express for a defined length of time
What are the in vivo barriers to gene delivery?
Cell membrane Dilution Passage through vascular bed Cell surface Lysosomal degradation after endocytosis Intracellular trafficking Nuclear entry and expression
What are the advantages of non-viral vectors (lipoplexes, oligo/polyplexes)?
Unlimited DNA-packaging capacity Low toxicity Low immunogenicity Not infectious Easy (chemical) production Targetable
What are the disadvantages of non-viral vectors?
Low efficiency
Only transient persistence
What are the characteristics of adenoviruses which is good for gene therapy?
Ubiquitous occurs in many animal species
Broad host range so good for infecting several animal models
Strong gene expression
Transient expression
Low pathogenicity in humans causing only mild symptoms of the common cold
Can infect non-proliferating cells
East to manipulate
Not associated with malignancy
Describe the adenovirus structure.
The capsid contains 3 types of proteins:
Fibre is involved in binding to the CAR-receptor
Penton base contains an RGD-sequence for stronger cell binding to integrins and cell internalisation
Hexon makes up the structure of the virus capsid.
What is the genome structure of adenoviruses?
Double stranded DNA genome
Genes are arranged as early (E1-E4) or late (L1-L5) regions on the genome and based on their expression before or after virus DNA synthesis.
E1-E4 are transcription regulators for virus expression leading genome replication
After this L1-L5 drives virus transcription
Regions of the genome are described as cis and trans elements
Cis-sequences for replication origin or virus packaging (psi) and carried on the backbone
Trans-can be replaced and complimented on separate genome (E1).
Describe the first generation vectors.
Removal of the E1 and E3 to prevents virus replication -causes immune responses which leads to destruction of cells expressing the virus.
Describe the second generation vectors.
Deletions of E1, part of E3 and part of E4 and have lower toxicity and longer gene expression.
Describe the third generation vectors.
These vectors need ‘helper virus’ because they are ‘gutted’. Human embryonic kidney cells are used. They are transfected with the E1a and E1b genes. Structural proteins are provided in trans to allow virus replication.
What are the uses of adenoviruses?
Vector for gene transfer
Cancer gene therapy
