gene therapy 2

Question 1

Can viruses replicate on their own?

Answer 1

NO

Question 2

Why are viruses the preferred method of gene transfer?

Answer 2

Viruses are highly efficient at introducing DNA into cells

Question 3

Viruses contain a N____ genome.

The genetic material is surrounded by a p____, known as a c_____

Answer 3

Viruses contain a Nucleic acid genome.

The genetic material is surrounded by a protein , known as a capsid

Question 4

The retrovirus genome is what?

Answer 4

ssRNA and 7-9 kb.

Question 5

Describe the retrovirus genome.

What are the parts?

Answer 5

LTR--long terminal repeat
Ψ-Packaging signal 
GAG- capsid protein
POL-reverse transcriptase
ENV-envelope protein

Question 6

Describe how the retrovirus enters a cell and is replicated in the body

Answer 6

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

Question 7

What are most retroviral vectors based on?

Answer 7

MoMLV- Moleney Murine Leukaemia Virus

Question 8

How do we package the viral vector?

Answer 8

Use a packaging cell line

Packaging cell lines contain an integrated retrovirus/provirus- with no Ψ

Question 9

When was the first trial using retroviral vectors in immune disorders?

Answer 9

1990- to treat ADA

Question 10

What is ADA deficiency?
Which gene is affected?

THIS CAUSES SCID

Answer 10

Inherited disorder
Defective adenosine deaminase gene
ADA encodes for a gene in the purine salvage pathway.
T-lymphocytes are severely affected by dATP

Question 11

How can retroviral vectors help ADA deficiency?

Answer 11

ADA+ retrovirus is added to T-lymphocytes. The ADA+ cells are transfused into the patient

Question 12

Name the two advantages of the retroviral vectors

Answer 12

High efficiency of gene transfer

High levels of expression

Question 13

Name the 3 disadvantages pf retroviral vectors

Answer 13

Max insert size 7.7-5kb
Only infects dividing cells
Toxic? Insertional mutagenesis

Question 14

What is insertional mutagenesis?

Answer 14

1/16 for SCID develop cancer as the treatment affects the proto oncogene

Question 15

Lentiviruses
What size is the RNA genome?
Which cells can it infect?

Answer 15

9Kb

Can infect both dividing and non dividing cells

Question 16

Name 2 ways lentiviruses are better than retroviruses

Answer 16

less prone to intergreation next to transcriptional start site
Remove strong promoters and enhancers in LTR

Question 17

What is the Adenovirus genome size ?

Answer 17

36kb of sd DNA

Question 18

Describe the life cycle of the adenoviruses

Answer 18

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

Question 19

Adenoviral vectors- first Gen
What is the level of transcription from viral genes
What is the cononing capacity
Why are E1 and E3 removed?

Answer 19

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

Question 20

Adenoviral vectors what is the difference between the 1st and second generation

Answer 20

2nd gen-all early genes were deleted as these induced host immune system
Decreased toxicity
Cloning capacity up to 35kn

Question 21

Name the advantages to Adenoviral vectors

Answer 21

Vectors don’t insert the genes into the host chromosome

Question 22

Name the disadvantages to the adenoviral vectors

Answer 22

Limited to only temporary protein expression
Doesn;t work well on dividing cells
Requires repeat treatments

Question 23

RNAi=

Answer 23

RNA interferance

Question 24

How can RNAi be used in gene slilencing?

Answer 24

Gain of function mutation

RNAi selectively inhibits the mutant gene from being expressed

Question 25

Describe the natural pathway gene silencing

Answer 25

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

Question 26

Describe direct siRNA therapy

Answer 26

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

Question 27

Describe short hairpin RNA-mediated siRNA therapy

Answer 27

shRNA = short hairpin RNA
Gene with inverted repeats
Transcribed in the nucleus
Formation of a hairpin = double stranded RNA
Dicer cleaves to create siRNA

Question 28

Name the 3 disadvantages of RNAi therapy

Answer 28

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