Gene Therapy

Question 1

What is gene therapy?

Answer 1

The application of genetic principles in the treatment of human disease in order to counteract the effect of a diseased gene or introduce new function.

Question 2

What does gene therapy correct?

Answer 2

Deficient phenotype so that sufficient amounts of normal gene product are synthesized to improve disorder

Question 3

Monogenic gene therapy

Answer 3

Provides genes to encode for the production of a specific protein

Question 4

Suicide gene therapy

Answer 4

Provides suicide genes to target cancer cells for destruction

Question 5

Antisense gene therapy

Answer 5

Provides a single stranded gene in an “antisense” orientation to block the production of harmful proteins

Question 6

Why does ex vivo delivery of gene therapy work better than in vivo?

Answer 6

If genetic information is delivered in vivo, it is unlikely to last long in the body and will be degraded quickly. As such, it is better to remove cells from the patient, use those cells to produce multiple copies of the genetic information through replication and cell division, and then reintroduce the mateiral back into the patient.

Question 7

In Vivo delivery of Viral Vectors for Gene Therapy

• What must be done to the virus to use it in this manner?
• Why are viral vectors used?
• What are some of the problems with using viral vectors to deliver genetic information for gene therapy?

Answer 7

• Viruses must be “crippled” (made so they can’t replicate) to disable their ability to cause disease
• They are used b/c they can stably integrate the desired gene into the target cell’s genome
• Viruses that have their replication capacity removed cannot spread the genes in the body, so many copies of the virus must be introduced, which makes this technique reliant on diffusion of the viral particles. Additionally, viruses often have a limited capacity to store genetic information.

Question 8

Retroviruses as a Vehicle for Gene Therapy

What are the benefits of using this virus for GT?

What are the downfalls?

Lentivirus is a subset of retrovirus. How does it overcome some of the limitations?

Answer 8

Benefits

• Non toxic to cell
• High efficiency transduction of appropriate target cells
• Integrated DNA is stable

Limitations

• Can only accomodate smaller transfer genes
• Target cell must undergo division for the transferred DNA to be integrated
• Integration is random - can lead to mutagenesis

Lentivirus

• Capable of DNA integration in non-dividing cells
• Does not show preferential integration into any specific gene locus –> reduces the changes of activating oncogene

Question 9

Adenoviruses as a Vehicle for Gene Therapy

• What are some of the advantages of this virus for GT?
• What are some of the limitations?

Answer 9

Advantages

• High transduction efficiency
• Will infect a wide variety of dividing or nondividing cells
• Accomodates a larger piece of genetic information than retrovirus
• Low risk of insertional mutagenesis

Limitation

• Transient expression and episomal inheritance
• Associated with at least 1 death in gene therapy trial due to elicitation of strong immune response

Question 10

Adeno-Associated Virus as a Vehicle for Gene Therapy

• What are some of the advantages of this virus for GT?
• What are some of the limitations?

Answer 10

Advantages

• Does not require cell division
• Site specific integration (Chromo 19)
• Do not illicit strong immune respones

Disadvantages

• Smallest amount of genetic information able to be incorporated of all viral types discussed in class

Question 11

Herpes Simplex Virus as a Vehicle for Gene Therapy

What are the advantages and disadvantages of HSV?

Answer 11

Advantages

• Does not require cell division
• HSV can accomodate a large therapeutic gene
• Can be produced at high titers

Disadvantages

• Transient expression (episome)
• Low transduction efficiency (episome)
• Very specific in the cells that it infects, so it has a very narrow application in gene therapy

Question 12

Describe the process of engineering a virus into a viral vector.

Answer 12

Question 13

What is a disadvantage of “crippling” the viral cell for gene therapy?

Answer 13

Loss of ability to self-replicate means that vectors are needed in very large numbers to achieve successful delivery of new genes into patient’s cells

Question 14

Why are the advantages and disadvantages of using viruses that insert their DNA into the host cell in the form of an episome?

Answer 14

Advantages - episome is stable, get long term expression and effect in infected cell

Disadvantage - when cell divides, the episome will not be replicated so one of the cells will not inherit the episome and will return to being a mutant cell –> requires readministration of vector over time

Question 15

Describe the process of electroporation.

Answer 15

B: short burst of electricity applied across cells to drive DNA into cell w/o damaging cell. Then, select for cells that took up the therapeutic gene. Then characterize and re-introduce into patient.

Question 16

What are some of the advantages and disadvantages to using a liposome for introducing genetic information into a person?

Answer 16

Advantages

• Not limited by size or # of genes they can accomodate
• They are safe
• They are easy to produce

Disadvantages

• They offer only short term expression

Question 17

What are 2 patient saftey concerns when using viruses as vectors for gene therapy?

Answer 17

1. Immune hyper-sensitivity - reactions directed against viral vector components or against transgenes expressed in treated cells
2. Integration is not controlled, so the gene could insert into the DNA and activate an oncogene or silence a tumor suppressor leading to cancer

Question 18

Answer 18

cDNA

Regualtory

enchances, promoters, etc.

Question 19

Answer 19

Promoters

Gene expression patterns

Question 20

Answer 20

Insertion

Insertion

Silence

Question 21

Why is gene therapy costly?

Answer 21

B/c of cell culturing needs involved in ex vivo techniques and the number of subjects involved in studies are very small so hard to make GT that is broadly applicable

Question 22

Briefly explain Severe Combined Immunodeficiency Disease (SCID).

Why wasn’t gene therapy successful in early trials with this disease?

Answer 22

• Gene for adenosine deamidase (ADA) is on chromo 22. Mutations at locus for ADA gene lead to deficiency of enzyme –> ADA deficiency leads to build up of deoxyadenine which is toxic to T cells –> failure to develop T lymphocytes –> failure to develop mature B cells –> patients cannot withstand infection of any kind –> death
• Lymphocytes are short lived so the patients needed to receive a lot of treatment over the course of their lives, if that was not kept up with they would die

Question 23

Briefly describe what Ornithine transcarbamylase deficiency is.

Relate this to the gene therapy case of Jesse Gelsinger.

Answer 23

Ornithine transcarbamylase = enzyme in urea cycle. Genetic defect –> decreased enzymatic function –> increase of ammonia –> travels to brain –> coma –> death

Jesse was being treated for OTC deficiency with drug that bound excess ammonia in blood and excreted it. He was tired of having to constantly take the drugs, so he signed up for clinical trial for gene therapy with adenovirus. He was given a dose of the virus that was too high and a massive inflammatory response was triggered in his body leading the production of inflammatory molecules and multiple organ failure. He was in a coma within 24 hours.

Question 24

What is the CRISPR Locus?

Answer 24

Part of genetic information of bacteria that is part of the bacterial immune system. Every time a bacterium is infected by a phage, the bacteria cuts the invading DNA using restriction enzymes and then takes a piece of the invading DNA and incorporates that DNA into its own geneome at the CRISPR locus.

Question 25

Describe how the CRISPR/Cas system works.

Answer 25

Phage infects bacterium. Bacterium digests phage DNA with restriction enzyme. Bacaterium takes part of phage DNA and incorporates into bacterial genome at CRISPR locus. Bacterium transcribes CRISPR locus to produce pre-crRNA that contains all the sequences of DNA that the bacterium recognizes as foreign DNA. Cas protein separates the different phage sequences from the pre-crRNA into mature cr-RNA, which then bind with Cas9 protein. Cas9 protein with bound cr-RNA exist in bacterium ready for next phage infection. Next time bacterium is infected by same phage, the crRNA in Cas9 will hybridize with foreign DNA as a recognition and Cas9 will cleave the foreign genetic material to degrade it.

Question 26

Explain how CRISPR/Cas could be used in organisms for genomic engineering.

Answer 26

1. Could be engineered to recognize specific DNA sequence, cleave DNA at specific programmable site, which would trigger DNA repair mechanisms. Could introduce healthy Donor DNA to promote homologous repair and restoration of normal chromosome function.
2. Could be engineered to recognize specific DNA sequence, cleave DNA at specific programmable site, which would trigger DNA repair mechanisms. Could introduce healthy Donor DNA to promote insertion of new donor DNA into chromosome.
3. Could be engineered to recognize specific DNA sequence and Cas protein could be conjugated with activator or repressor to affect gene expression of gene directly.