Exam IV: Lecture 15 Flashcards
What are some examples of diseases caused by a defective gene? (5)
- Cancer
- DMD (Duchenne Muscular Dystrophy)
- CF (cystic fibrosis)
- ALS (amyotrophic lateral sclerosis)
- SCID (severe combined immune deficiency)
What gene is defected in SCID? What does that gene do? How does that contribute to the disease?
ADA
synthesis of nucleotides
Body can’t produce B and T cells
What gene is defected in Hemophilia? What does that gene do? How does that contribute to the disease?
Factor IX, VII
Catalyze clotting
Can’t clot
What gene is defected in Cystic Fibrosis? What does that gene do? How does that contribute to the disease?
Membrane Salt transporter
Build-up of salt in the cells > not fluid around the outside of the cells
What gene is defected in DMD? What does that gene do? How does that contribute to the disease?
Dystrophin
Connects muscle cells togher
Muscle dysfunction
What some examples of polygenic diseases? (3)
- Cancer
- Rheumatoid Arthritis
- Neurological Diseases (Alzheimer’s, Parkinson’s)
What are the 4 goals for building the ideal gene therapy vector?
- It should be able to carry a full-length gene
- It should be able to penetrate the plasma membrane
- It should be able to transverse the cytoplasm and enter the nucleus
- It should be able to express large amounts of the gene product when required
If you are delivering genes with a viral vector, at what point to you want to block the normal viral life cycle?
You want to block replication
So, you have attachment > entry > delivery > transcriptions and translation of proteins. No replication and assembly.
Why use retroviral vectors for gene therapy?
The genome of the “virus” goes into the nucleus and integrates into the genome and becomes a provirus
How do you stop replication and assembly of retrovirus for gene therapy?
Remove the packaging signal so the viral proteins produced won’t be packaged and the virus particle won’t assemble
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?
You create a packaging cell line/producer cell line
Tell me about the packaging cell line
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
What is Ex vivo gene therapy?
Cells are taken out of the patient (usually bone marrow stem cells) > transduced with virus > returned to patient via transfusion
What are the advantages of ex vivo gene therapy? Disadvantages?
Advantages: efficeint
Disadvantages: only works in cells that can be grown outside of the body (limits the diseases that can be treated)
What is In vivo gene therapy?
The virus is injected into the target tissue
What are the advantages and disadvantages of in vivo gene therapy?
Advantage: less procedures for patient, less steps
Disadvantage: mechanims of viral entry and transport is still not clear, inefficient
What are 2 advantages of retroviral vectors?
- Provirus is inherited by daughter cells in a Mendelian fashion
- Small virus (does not make too many proteins)
What are 5 disadvantages of retroviral vectors?
- Small size
- Inserts randomly into genome
- Can only be used in replicating cells, not in terminally differentiated cells
- Recombination events between the two genomes can give rise to replication competent virus
- Expression of the therapeutic gene is turned off after a few months (don’t know why)
Who was the first successful gene therapy patient*? What did they have?
Ashanthi Desilva
SCID with ADA deficiency
Retroviral vector into bone marrow cells
*Also treated with soluble ADA
What are 5 advantages to Adenoviral vectors?
- Can incorporate large pieces of DNA
- Does not cause serious disease in humans
- Broad host range
- Low cytotoxicity
- Easy to inactivate
How do you inactivate adenoviral vectors?
Inactivate E1 and E3
Insert therapeutic genes into E1 and E3 ORFs
What are 3 disadvantages of adenovirus vectors?
- Immunogenic (Capsid proteins are highly antigenic)
- Viruses containing E1 may transform cell (Recombination events between cellular E1 gene and viral genome > competent )
- Homologous recombination is not very efficient
How is the Adenovirus viral genome modified for gene therapy?
Homologous recombination
Talk to me about the delivery of therapeutic genes using adenoviral vectors
Attachment > entry > ciruclar DNA in nuclues (doesn’t integrate) > doesn’t get degraded because its circular > production of necessary protein
Who was Jesse Gelsinger? What did we learn from this patient?
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!
List adenovirus, Retrovirus, AAV, Lentivirus, and Herpesvirus in order of genome size
AAV: 4.5kb Lentivirus: 7.5kb Retrovirus: 8kb Herpesvirus: 30kb Adenovirus: 35kb
What are 2 advantages to AAV?
- Integrates specifically
2. Expresses for a long time
What is 1 advantage and 1 disadvantage of lentivirus vectors?
Advantage: can integrate into non-diving cells
Disadvantage: integrates randomly
What is 1 advantage and 1 disadvantage of herpesvirus vectors?
Advantage: expresses genes for long period of time in neurons
2. Highly cytotoxic in non-neuronal tissue
What promoter do Herpesvirus vectors target in neurons?
LAT promoter
What kind of changes would you make to HSV-1 to make it non-toxic?
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
What is an example of an oncolytic viruses that is being considered for gene therapy?
AAV
How can you use lipid capsids in gene therapy?
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
How can you use PEG in gene therapy?
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 ?
What are 3 “tricks of the trade” to help develop the therapeutic vector you need?
- Modify the surface glycoproteins to target the virus
- Use inducible promoters to turn on gene expression when needed
- Use tissue-specific promoters to express genes only in tissues where they are needed (DMD in muscle cells, MMTV LTR in mammary cells)
What are 5 general technical problems with gene therapy?
- Unregulated gene expression
- Immunogenicity (immune system kills off the cell harboing the gene therapy virus)
- Inability to achieve prolonged expression
- Virus toxicity (may still produce genes that kill the cell)
- Viral RNA can still trigger the antiviral response
What are 4 non-viral gene delivery methods?
- Cell-mediated gene transfer = modify the host cells then reinsert
- Liposome mediate gene transfer
- Direct DNA injection
- Gene guns with nanogold bullets that puncture the skin and cell membrane with the DNA/RNA bound to the gold
