Exam IV: Lecture 15

Question 1

What are some examples of diseases caused by a defective gene? (5)

Answer 1

1. Cancer
2. DMD (Duchenne Muscular Dystrophy)
3. CF (cystic fibrosis)
4. ALS (amyotrophic lateral sclerosis)
5. SCID (severe combined immune deficiency)

Question 2

What gene is defected in SCID? What does that gene do? How does that contribute to the disease?

Answer 2

ADA
synthesis of nucleotides
Body can’t produce B and T cells

Question 3

What gene is defected in Hemophilia? What does that gene do? How does that contribute to the disease?

Answer 3

Factor IX, VII
Catalyze clotting
Can’t clot

Question 4

What gene is defected in Cystic Fibrosis? What does that gene do? How does that contribute to the disease?

Answer 4

Membrane Salt transporter

Build-up of salt in the cells > not fluid around the outside of the cells

Question 5

What gene is defected in DMD? What does that gene do? How does that contribute to the disease?

Answer 5

Dystrophin
Connects muscle cells togher
Muscle dysfunction

Question 6

What some examples of polygenic diseases? (3)

Answer 6

1. Cancer
2. Rheumatoid Arthritis
3. Neurological Diseases (Alzheimer’s, Parkinson’s)

Question 7

What are the 4 goals for building the ideal gene therapy vector?

Answer 7

1. It should be able to carry a full-length gene
2. It should be able to penetrate the plasma membrane
3. It should be able to transverse the cytoplasm and enter the nucleus
4. It should be able to express large amounts of the gene product when required

Question 8

If you are delivering genes with a viral vector, at what point to you want to block the normal viral life cycle?

Answer 8

You want to block replication

So, you have attachment > entry > delivery > transcriptions and translation of proteins. No replication and assembly.

Question 9

Why use retroviral vectors for gene therapy?

Answer 9

The genome of the “virus” goes into the nucleus and integrates into the genome and becomes a provirus

Question 10

How do you stop replication and assembly of retrovirus for gene therapy?

Answer 10

Remove the packaging signal so the viral proteins produced won’t be packaged and the virus particle won’t assemble

Question 11

If you remove the packaging signal, how do you make enough viral particles to treat a patient with since the virus can not replicate inside the body?

Answer 11

You create a packaging cell line/producer cell line

Question 12

Tell me about the packaging cell line

Answer 12

A cell line grown in the lab with the therapeutic gene and packaging signal > grow a bunch of viral particles with therapeutic gene inside > remove packaging signal (?) > treat patient

Question 13

What is Ex vivo gene therapy?

Answer 13

Cells are taken out of the patient (usually bone marrow stem cells) > transduced with virus > returned to patient via transfusion

Question 14

What are the advantages of ex vivo gene therapy? Disadvantages?

Answer 14

Advantages: efficeint
Disadvantages: only works in cells that can be grown outside of the body (limits the diseases that can be treated)

Question 15

What is In vivo gene therapy?

Answer 15

The virus is injected into the target tissue

Question 16

What are the advantages and disadvantages of in vivo gene therapy?

Answer 16

Advantage: less procedures for patient, less steps
Disadvantage: mechanims of viral entry and transport is still not clear, inefficient

Question 17

What are 2 advantages of retroviral vectors?

Answer 17

1. Provirus is inherited by daughter cells in a Mendelian fashion
2. Small virus (does not make too many proteins)

Question 18

What are 5 disadvantages of retroviral vectors?

Answer 18

1. Small size
2. Inserts randomly into genome
3. Can only be used in replicating cells, not in terminally differentiated cells
4. Recombination events between the two genomes can give rise to replication competent virus
5. Expression of the therapeutic gene is turned off after a few months (don’t know why)

Question 19

Who was the first successful gene therapy patient*? What did they have?

Answer 19

Ashanthi Desilva
SCID with ADA deficiency
Retroviral vector into bone marrow cells
*Also treated with soluble ADA

Question 20

What are 5 advantages to Adenoviral vectors?

Answer 20

1. Can incorporate large pieces of DNA
2. Does not cause serious disease in humans
3. Broad host range
4. Low cytotoxicity
5. Easy to inactivate

Question 21

How do you inactivate adenoviral vectors?

Answer 21

Inactivate E1 and E3

Insert therapeutic genes into E1 and E3 ORFs

Question 22

What are 3 disadvantages of adenovirus vectors?

Answer 22

1. Immunogenic (Capsid proteins are highly antigenic)
2. Viruses containing E1 may transform cell (Recombination events between cellular E1 gene and viral genome > competent )
3. Homologous recombination is not very efficient

Question 23

How is the Adenovirus viral genome modified for gene therapy?

Answer 23

Homologous recombination

Question 24

Talk to me about the delivery of therapeutic genes using adenoviral vectors

Answer 24

Attachment > entry > ciruclar DNA in nuclues (doesn’t integrate) > doesn’t get degraded because its circular > production of necessary protein

Question 25

Who was Jesse Gelsinger? What did we learn from this patient?

Answer 25

Died in clinal trials of adenoviral vectors You need a high amount of viral vectors to actually cure the disease > developed a massive immune response > died We learned that AAV has low toxicity up to a point!

Question 26

List adenovirus, Retrovirus, AAV, Lentivirus, and Herpesvirus in order of genome size

Answer 26

``` AAV: 4.5kb Lentivirus: 7.5kb Retrovirus: 8kb Herpesvirus: 30kb Adenovirus: 35kb ```

Question 27

What are 2 advantages to AAV?

Answer 27

1. Integrates specifically | 2. Expresses for a long time

Question 28

What is 1 advantage and 1 disadvantage of lentivirus vectors?

Answer 28

Advantage: can integrate into non-diving cells Disadvantage: integrates randomly

Question 29

What is 1 advantage and 1 disadvantage of herpesvirus vectors?

Answer 29

Advantage: expresses genes for long period of time in neurons 2. Highly cytotoxic in non-neuronal tissue

Question 30

What promoter do Herpesvirus vectors target in neurons?

Answer 30

LAT promoter

Question 31

What kind of changes would you make to HSV-1 to make it non-toxic?

Answer 31

You could add the glycoprotien PS to the envelope of the virus > tells the immune system "I'm undergoing apoptosis" > immune system leaves it alone

Question 32

What is an example of an oncolytic viruses that is being considered for gene therapy?

Answer 32

AAV

Question 33

How can you use lipid capsids in gene therapy?

Answer 33

If a patient lacks a protein you can infect the persons with the protein in lipid capsids which are integrated into the cell membrane and so the protein is deposited into the cell

Question 34

How can you use PEG in gene therapy?

Answer 34

Polyethylene glycol (PEG)-mediated precipitation helps get protein into cell but can cause an immune response against protein which would cause the therapy to stop working ?

Question 35

What are 3 "tricks of the trade" to help develop the therapeutic vector you need?

Answer 35

1. Modify the surface glycoproteins to target the virus 2. Use inducible promoters to turn on gene expression when needed 3. Use tissue-specific promoters to express genes only in tissues where they are needed (DMD in muscle cells, MMTV LTR in mammary cells)

Question 36

What are 5 general technical problems with gene therapy?

Answer 36

1. Unregulated gene expression 2. Immunogenicity (immune system kills off the cell harboing the gene therapy virus) 3. Inability to achieve prolonged expression 4. Virus toxicity (may still produce genes that kill the cell) 5. Viral RNA can still trigger the antiviral response

Question 37

What are 4 non-viral gene delivery methods?

Answer 37

1. Cell-mediated gene transfer = modify the host cells then reinsert 2. Liposome mediate gene transfer 3. Direct DNA injection 4. Gene guns with nanogold bullets that puncture the skin and cell membrane with the DNA/RNA bound to the gold