Viral Vectors (Gene Therapy) - Dr. Whitt

Question 1

There are 5 types of vectors. Name them and discuss the advantages and disadvantages of each.

Answer 1

Adenovirus: - Episomal - High transduction efficiency - Infects replicating & non-replicating cells - Insert capacity: 8 - 36kb - Elicits immune response

Adeno-associated virus (AAV): - Integrates genome into specific region on human chromosome 19 - Low immunogenicity - No associated disease - Infects replicating and non-replicating cells - LIMITED insert capacity: ~5kb

Herpesvirus - Large insert capacity - Broad host range - Infects replicating and non-replicating cells

Liposomes (Naked DNA) - No limit to size of gene to be delivered - Low immunogenicity - Poor levels of gene transfer - Requires high dose level

Retrovirus - Inserts genome into host cell’s DNA - non-pathogenic to humans - Long term expression - Insert capacity of 8kb - Stably transduces replicating cells but not non-replicating - Inactivated by human complement

Question 2

discuss the advantages and disadvantages of Adeno-associated virus (AAV) vectors

Answer 2

• Integrates genome into specific region on human chromosome 19
• Low immunogenicity
• No associated disease
• Infects replicating and non-replicating cells - LIMITED insert capacity: ~5kb

Question 2

Explain the mechanism of retrovirus vectors for gene therapy. What is a disadvantage of this vector?

Answer 2

Binds to target cell, endocytosed, uncoats, reverse transcriptase makes dsDNA out of RNA, integrates into host genome

• Allows for long-term expression and ability of therapeutic gene to be maintained during replication
• Need to delete structural proteins: receptor binding proteins and capsid proteins
• Replaced with therapeutic gene of interest
• Disadvantage: (Murine) - need replicating cells for integration

Question 2

What is Glybera?

Answer 2

• Gene therapy AAV1 carrying lipoprotein lipase gene (approved in Europe)
• Targets skeletal muscle muscle cells - To treat familial LPL deficiency
• Disease is noted in childhood with: abd pain, acute & recurrent pancreatitis, skin lesions, liver and spleen involvement - hepatosplenomegaly

Question 3

What are two types of intervention strategies for gene therapy?

Answer 3

1) Therapeutic strategies: a modified virus vector is used to carry a therapeutic gene for a protein to replace a defective or absent protein in the host

• Used in experimental treatment of cystic fibrosis

2) Cytolytic strategies: vector carries some gene that produces a protein to kill targeted cells (often used in cancer)

Question 3

discuss the advantages and disadvantages of adenovirus vectors

Answer 3

• Episomal
• High transduction efficiency
• Infects replicating & non-replicating cells
• Insert capacity: 8 - 36kb - Elicits immune response

Question 4

Simply, what is in vivo gene therapy?

Answer 4

a virus vector is modified to carry a therapeutic gene that will be inserted into specific cells of the patient’s tissue

Question 6

What is germline gene therapy?

Answer 6

genetic modification of germ cells that will pass the selected change on to the next generation

Germline intervention is strictly limited to animal model systems, and there is no intent to pursue this type of approach in humans at any time in the near future

Question 7

discuss the advantages and disadvantages of retrovirus vectors

Answer 7

• Inserts genome into host cell’s DNA
• non-pathogenic to humans
• Long term expression
• Insert capacity of 8kb
• Stably transduces replicating cells but not non-replicating
• Inactivated by human complement

Question 8

How does adeno-associated virus vector differ from other gene therapy vectors?

Answer 8

Does NOT stimulate inflammation in host.

Does NOT elicit antibodies against itself

Can enter non-dividing cells and dividing cells Integrates successfully into one spot in the genome of its host.

Chromosome 19 but can exist extrachromasomally.

Question 9

Explain the mechanism of adenovirus vectors for gene therapy. What is the disadvantage of this vector?

Answer 9

Non-enveloped with large dsDNA genomes; can accommodate large gene inserts up to 36kb; - Binds receptor, endocytosed, uncoats and inserts genome into nucleus - **Remains detached from host genome and replicates episomally. (NOT INTEGRATED) = extrachromasomally - Like retrovirus, have genes for structural and regulatory proteins - Disadvantage: These viruses are commonly seen by typical human, hence these can cause inflammation and immune response due to antigen commonalities.

Question 9

What are 4 problems that need to be solved before gene therapy can become an available tool for physicians?

Answer 9

1) avoiding immune response in patient —Particularly problematic with adenovirus vectors —Vector components can elicit Ab’s to destroy vectors when administered again —AAV circumvents this issue 2) Getting genes into non-dividing cells like liver, muscle and neurons. —E.g. retrovirus needs dividing cell —Lentivirus does not need dividing cell 3) Targeting specific cells successfully 3) Getting gene integrated so that it will be replicated and expressed indefinitely

Question 10

Explain the simple mechanism of liposome vectors for gene therapy. What is an advantage and a disadvantage of this vector?

Answer 10

Formulation of synthetic lipids that bind and encapsulate plasmid DNA; Fuse with cell membranes and deliver genome to cell - Do not bind to specific cell receptors - Advantage: Can deliver many different genes into all types of cells - Disadvantage: Efficiency of delivery is very LOW—So amount of delivery to patient has to be much higher.

Question 12

What is somatic gene therapy?

Answer 12

manipulation of gene expression in cells so as to be corrective for the patient, but this correction is not inherited by the next generation

Question 13

What is the example explained regarding an ex vivo somatic cell gene therapy case? HINT: LDL receptor

Answer 13

1. A piece of the patient’s liver was removed, and the cells were treated with a retrovirus carrying the low-density lipoprotein (LDL) receptor gene. 2. Liver cells failing to take up corrective genes were discarded; cells incorporating the corrective gene were re-implanted into the patients’ liver.

Question 13

What are 4 requirements and challenges in developing gene therapy?

Answer 13

1) Gene identification and cloning: Identify the gene (or genes) responsible for a particular disease state, so when you replace it, the disease is gone 2) Delivery: Delivery of genetic material to the appropriate cells of the patient in a way that is specific, efficient and safe 3) Ensuring control of gene expression: Make the correct amount of therapeutic protein at the right time. Maintaining long-term expression of the gene in the correct cells or tissue. 4) Avoiding Host Immune Responses: Remember that we are using some foreign agent to deliver genetic material to host cells, which would often trigger an immune response. • Often if multiple doses are needed, we may have a problem because of acquisition of memory responses

Question 15

Simply, what is ex vivo gene therapy?

Answer 15

cells are taken out of a patient, modified with some treatment to alter gene expression, then successfully altered cells are implanted back into the person

Question 17

discuss the advantages and disadvantages of liposome (naked DNA) vectors

Answer 17

• No limit to size of gene to be delivered - Low immunogenicity - Poor levels of gene transfer - Requires high dose level

Question 18

What are 5 factors that will influence the development of gene therapy in the future?

Answer 18

A. Optimizing vector safety to reduce adverse event risk B. Overcomingthetechnologicalobstacles C. Additional analysis of the human genome, including understanding of microRNAs D. Expansion of the diagnostic industry E. Reducing risk to patients which is essential for public acceptance of gene therapy

Question 19

What is gene therapy?

Answer 19

Gene therapy is a novel approach to treating diseases based on modifying the expression of a person’s genes toward a therapeutic goal. Gene therapy remains a highly experimental collection of technologies whose fullpotential is yet to be realized

Question 21

What are the vehicles of delivery, when delivering gene therapy to targets?

Answer 21

1) Attenuated or modified viruses – Must remove disease-causing components of virus and insert therapeutic genes 2) Non-viral vectors – Liposome transfection (DNA inside lipid) and “gene gun” (delivery of DNA on gold particles)

Question 22

discuss the advantages and disadvantages of herpesvirus vectors

Answer 22

• Large insert capacity - Broad host range - Infects replicating and non-replicating cells

Question 23

How are retroviruses produced in the lab for gene therapy? (Strategy)

Answer 23

How Retroviruses are produced for therapy:

• MAIN POINT: End game is to remove structural proteins because this removes potential effect that the vector will self-replicate in vivo using these structural genes. So, how is this done?
• 1st, viral structural genes are deleted from viral genome and (MOVED) cloned into a plasmid expression vector (separate from viral genome)
• This plasmid is transfected into a cell genome so it can produce structural proteins but no viral genome = called packaging cell line
• To create gene therapy vector itself: Sequences involved in packaging of genome into virus particles are called “packaging signals” are removed from the viral genome and cloned into a plasmids with the therapy gene of interest.
• Therapy gene of interest is then cloned between these packaging signals
• These plasmids with packaging signals and therapy gene are introduced into the packaging cell line
• Structural proteins assemble the therapeutic gene into virus particles => gene therapy vehicle = vector
• This is called a producer cell line
• These are released and can infect nearby cell in ex vivo
• Or, can be introduced to patients in vivo but since the therapy gene lacks the genes for structural proteins, there is no risk of self-replication and/or spread.
• so, no new infection of virus is made

Question 24

The death of Jesse Gelsinger was the first directly due to gene therapy. Evaluation before and after autopsy found tha this bone marrow was completely depleted of precursor cells and tha this cytokine levels were abnormal. Cause of death = ARDS and MOF due to anoxia. It’s believed that the vector caused SIRS. What two cytokines were in dangerous proportoin to each other?

Answer 24

Increased IL-6

No change in IL-10

Question 25

Which cytokine coud best stimulate the immune system to recognize and eliminate a tumor in a patient?

One modification for this strategy is to irradiate the tumor cells to prevent them from dividing prior to transduction with gene therapy vector. Based on this information, which viral vector could not be used?

Answer 25

GM-CSF

Retrovirus

Question 26

Name a monogenic disease that has been succesfully treated with gene therapy.

Answer 26

lipoprotein lipase deficiency