gene therapy 2 Flashcards
Can viruses replicate on their own?
NO
Why are viruses the preferred method of gene transfer?
Viruses are highly efficient at introducing DNA into cells
Viruses contain a N____ genome.
The genetic material is surrounded by a p____, known as a c_____
Viruses contain a Nucleic acid genome.
The genetic material is surrounded by a protein , known as a capsid
The retrovirus genome is what?
ssRNA and 7-9 kb.
Describe the retrovirus genome.
What are the parts?
LTR--long terminal repeat Ψ-Packaging signal GAG- capsid protein POL-reverse transcriptase ENV-envelope protein
Describe how the retrovirus enters a cell and is replicated in the body
Enters the cell and looses the lipid envelope, the RNA is converted into DNA by reverse transcriptase and integrated into host DNA
The DNA is transcribed and translated and the virus is assembled
What are most retroviral vectors based on?
MoMLV- Moleney Murine Leukaemia Virus
How do we package the viral vector?
Use a packaging cell line
Packaging cell lines contain an integrated retrovirus/provirus- with no Ψ
When was the first trial using retroviral vectors in immune disorders?
1990- to treat ADA
What is ADA deficiency?
Which gene is affected?
THIS CAUSES SCID
Inherited disorder
Defective adenosine deaminase gene
ADA encodes for a gene in the purine salvage pathway.
T-lymphocytes are severely affected by dATP
How can retroviral vectors help ADA deficiency?
ADA+ retrovirus is added to T-lymphocytes. The ADA+ cells are transfused into the patient
Name the two advantages of the retroviral vectors
High efficiency of gene transfer
High levels of expression
Name the 3 disadvantages pf retroviral vectors
Max insert size 7.7-5kb
Only infects dividing cells
Toxic? Insertional mutagenesis
What is insertional mutagenesis?
1/16 for SCID develop cancer as the treatment affects the proto oncogene
Lentiviruses
What size is the RNA genome?
Which cells can it infect?
9Kb
Can infect both dividing and non dividing cells
Name 2 ways lentiviruses are better than retroviruses
less prone to intergreation next to transcriptional start site
Remove strong promoters and enhancers in LTR
What is the Adenovirus genome size ?
36kb of sd DNA
Describe the life cycle of the adenoviruses
Early genes expressed E1a and E1b
Expression of E1-E4 followed by viral DNA replication
Expression of late genes
Packaging of new virus particles and cell lysis
Adenoviral vectors- first Gen
What is the level of transcription from viral genes
What is the cononing capacity
Why are E1 and E3 removed?
E1 and E3 removed to allow insertion of transgene and to stop viral replication
Low level of transcription from viral genes
Led to innate host response
Cloning capacity 7.5Kb
Adenoviral vectors what is the difference between the 1st and second generation
2nd gen-all early genes were deleted as these induced host immune system
Decreased toxicity
Cloning capacity up to 35kn
Name the advantages to Adenoviral vectors
Vectors don’t insert the genes into the host chromosome
Name the disadvantages to the adenoviral vectors
Limited to only temporary protein expression
Doesn;t work well on dividing cells
Requires repeat treatments
RNAi=
RNA interferance
How can RNAi be used in gene slilencing?
Gain of function mutation
RNAi selectively inhibits the mutant gene from being expressed
Describe the natural pathway gene silencing
Triggered by double stranded RNA
Key components= Dicer ribonuclease converted dsRNA to siRNA then sense strand degrades off
Complex of the anti-sense strand with RISC binds to mRNA to degreade mRNA
Describe direct siRNA therapy
Synthesis of RNA oligonucleotides with sequence complementary for the target transcript
Formation of a double stranded siRNA duplex
Packaging into liposomes for delivery across the lipid bilayer and into the cell
Describe short hairpin RNA-mediated siRNA therapy
shRNA = short hairpin RNA Gene with inverted repeats Transcribed in the nucleus Formation of a hairpin = double stranded RNA Dicer cleaves to create siRNA
Name the 3 disadvantages of RNAi therapy
Often target gene is ‘knocked down’ instead of ‘knocked out’ as complete gene silencing is difficult to achieve with this approach
Risk of off-target effects
Delivering sufficient RNA to target cells can be difficult
