Gene Therapy Guest Lecture

Question 1

What is Gene Therapy?

Answer 1

Used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized, to improve a genetic disorder
The way you deliver a normal gene into the patient’s cell is by using a vector
The most common vectors used in gene therapy are viral vectors

Question 2

Why are viral vectors used?

Answer 2

Viruses are very efficient at introducing their own genetic material into the host cell
Viruses have evolved to infect cells with great specificity
Virus is engineered to disable its ability to cause disease
Viral methods have proven to be the most efficient to date

Question 3

What needs to be considered for human gene therapy?

Answer 3

1) The nature of the disease: monogenetic, cancer, infectious?
2) How to access the target cells?
3) How to deliver the remedial gene? Which vector to use?
4) What proportion of target cells must acquire the gene?
5) Does the transcription of the input gene need to be regulated?
6) Will over expression cause additional physiological problems?
7) Will cells with the input gene be maintained indefinitely or will repeated treatments be necessary

Question 4

How can transgenes be delivered?

Answer 4

1) Ex vivo: Cells removed, transgene delivered to cultured cells and cells returned to the body
2) In vivo: Transgene delivered directly in to host

Question 5

What are the types of viral vectors?

Answer 5

1) Adenoviruses
2) Adeno-Associated Virus
3) Retro/Lenti viruses

Question 6

What is the biology of the Adenovirus?

Answer 6

Linear, dsDNA, episomal
Causes respiratory infections and conjunctivitis
Early and late gene expression
Infects non-dividing cells
Entry through the CAR receptor and integrin co-receptor

Question 7

How can the adenovirus be used to deliver a transgene?

Answer 7

Gutless Adenovirus genome and the transgene are co-transfected

Question 8

What are the advantages of the adenovirus as a viral vector?

Answer 8

Used extensively for live vaccines 
High transduction efficiency 
Genome is easily manipulated 
Cloning capacity is high 
High viral titer
Infects both dividing and non-dividing cells 
No insertional mutagenesis 
Efficiency and specificity of a gene delivery can be enhanced by reengineering

Question 9

What are the disadvantages of the adenovirus viral vector?

Answer 9

Immunogenic
Many people have pre-existing immunity
Transient expression, must be re-administer

Question 10

What is the biology of the retrovirus?

Answer 10

Diploid RNA, enveloped
Reverse transcribed into dsRNA and inserted into the host chromosomal DNA
Potential for oncogenesis
Simple retroviruses can only infect dividing cells
Lentiviruses can infect non-dividing cells

Question 11

What does the lentivirus comprise of?

Answer 11

Packaging signal, Gag, Pol, Env

Question 12

How can the lentivirus be used to deliver a transgene?

Answer 12

You deliver the transgene with the packaging signal
Deliver the Gag and Pol genes in trans
Deliver the envelope

Question 13

What are the advantages of the lentivirus vectors?

Answer 13

1) Integrate into host gene in stable fashion
2) High efficiency infection of dividing and non-dividing cells
3) Long term stable expression of transgene
4) Low immunogenicity
5) Insert up to 8 kb
6) Target specific cells
7) Modified to abolish activation of oncogenes
8) Well studied

Question 14

What are the disadvantages of the lentivirus vector?

Answer 14

1) Site of integration is unpredictable
2) Low titers
3) In vivo delivery is poor

Question 15

What is the Adeno-Associated Virus Biology?

Answer 15

4.5 kb, ssDNA genome
Smallest human virus: Rep and Cap
Requires “helper” adenovirus
Remains episomal: vector integrates at low frequency
Specific integration at the Ch19 in wild type

Question 16

How is the transgene delivered using AAV Vectors?

Answer 16

1) Helper genes
2) AAV Vector: Rep + Cap
3) Transgene

Question 17

How can AAV vectors be generated to express a protein of interest?

Answer 17

1) Transgene, helper plasmid, and the Rep/Cap protein

The three are co-transfected into a cell

Question 18

What are the advantages of the AAV Vector?

Answer 18

1) Non pathogenic in humans
2) Recombinant AAV can be easily purified
3) Transduce both dividing and non-dividing cells
4) Transduce multiple tissues
5) Mediates long term gene expression
6) Episomal form: don’t integrate
7) Random integrations unlikely
8) Minimal toxicity
9) Low immunogenicity

Question 19

What are the disadvantages of the AAV Vector?

Answer 19

1) Limited cloning capacity

2) Humoural immunity