gene Therapy

Question 1

Examples of various methods for treating genetic disease

Answer 1

1. Enzyme induction by drugs
2. Replacement of deficient enzyme/ protein
3. Enzyme/ protein preparations
4. Replacement of deficient vitamins or coenzymes
5. Replacement of deficient product
6. Substrate restriction in diet
7. Drug therapy
8. Replacement of diseased tissue
9. Removal of diseased tissue

Question 2

Define gene therapy

Answer 2

Genetic alteration of the cells of a patient with a genetic disease to achieve a therapeutic effect.

Question 3

Gene therapy can be divided into which two type

Answer 3

1. Modification of germ-line cells
2. Modification of somatic cells

Question 4

What modifications can be done to disease cells to alleviate disease.

Answer 4

1. Gene augmentation
2. Gene silencing
3. Repairing the mutant gene

Question 5

Explain gene augmentation therapy

Answer 5

1. Involves the replacement (supplementation) of a missing gene product by inserting a normal gene into a somatic cell
2. Best suited to correction of loss-of -function mutations that result in nonfunctional or missing gene product.
3. Suitable for recessive disorder, such as cystic fibrosis.
4. Not suitable for loss-of-function conditions where irreversible damage has occurred.

Question 6

Explain gene blocking/gene silencing therapies.

Answer 6

1. Especially relevant in infectious diseases, where essential functions of pathogen is targeted.
2. Could be used to silence activated oncogenes in cancer
3. Could be used to silence a gain-of-function mutant allele in inherited disease

Question 7

Explain what the delivery problem

Answer 7

Finding optimal and safe strategies to insert and express gene constructs.
- which cells/organ
- in vivo or ex vivo method
- viral or non-viral mediated

Question 8

Gene therapy is usually directed or limited to a particular target cells or organs an different strategies for different target cells. What are some aspects to consider when deciding.

Answer 8

1. Accessibility of the target cells (blood, skin, muscles, eyes vs liver and brain)
2. Life-span of the target cells:
   2.1 long lived: aim for efficient delivery and high levels of constant trans gene expression.
   2.2 short lived: important that the trans gene be transferred to daughter cells during mitosis.
3. The therapy should be safe and efficient

Question 9

Explain therapy procedure in vivo

Answer 9

In vivo: cells are modified within the patients body, success in difficult to monitor.

Question 10

Explain therapy ex vivo

Answer 10

Ex vivo: cells are removed from patient, genetically modified in cell culture, multiplied and returned too patient.
Cells can be analysed in depth before resting patient.

Question 11

Explain Non-viral delivery using liposomes

Answer 11

1. Lipid coat allows DNA to survive in vivo, it can bind to cells and allows DNA entry.
2. Fat body that can accept large DNA inserts.
3. Does it elicit immune response,
4. however efficiency of transfer is low.

Question 12

Explain what liposomes are:

Answer 12

Synthetic vesicles that form spontaneously when certain lipids are mixed in aqueous solution.

Question 13

Are non-viral deliveries safe

Answer 13

All non-viral delivery systems are safe, but have a relatively low efficiency

Question 14

List the viral vector classes

Answer 14

1. Retroviral vectors
2. Lentiviruses
3. Adenovirus vectors
4. Adeno-associated viruses

Question 15

Explain how retroviral vectors are used as a viral vector.

Answer 15

RNA viruses are modified to not replicate in host cells, most of the viral genome is deleted , replaced with normal copy of human gene (insert size -8Kb)

Question 16

Explain Packaging of therapeutic recombinant retrovirus

Answer 16

1. The retroviral vectors are Propagated in “packaging cells” that compensate for missing replication machinery.
2. Thus there is a production of multiple copies of retroviruses that cannot replicate
3. They are incubated with patients somatic cells (e.g bone marrow stem cells)
4. This is ex vivo therapy where retroviral genome inserts transgene into patient DNA

Question 17

Explain Gene therapy for severe combined immunodeficiency

Answer 17

1. Metabolic disease, total deficiency of the enzyme adenosine deaminase (ADA)
2. Delivered functional AD gene using retrovirus vector and ex vivo procedure to treat bone marrow cells.

Question 18

Explain what ADA deficiency is?

Answer 18

1. Autosomal recessive disorder
2. Adenosine deaminase (ADA) deficiency leading to a build up of deoxy ATP
3. Symptoms:
   3.1 destroyed T cells
   3.2 B cells not activated
   3.3 ( severe combined immune deficiency )

Question 19

Disadvantages of retroviral vectors

Answer 19

1. Could integrate randomly into hosts DNA, where it could possibly locate near a proto-oncogene + tumour formation which can be very severe combined with the immunodeficiency
2. Can only enter nucleus of a dividing cell (because nuclear membrane is dissolved) which means its ineffective in non dividing cells or slowly dividing cells

Question 20

What is a lentiviruse

Answer 20

Subclass of retroviruses, more complex genome structure

Question 21

How do Lentivirus vectors work?

Answer 21

1. vectors designed for greater safety, still high levels of transcription.
2. Also packaged to produce recombinant lentiviruses, also integrates into host genome
3. Used for ex vivo therapy
   4 are self-inactivating i.e enhancers in viral LTRs have been deleted.
4. Transgene expression driven by mammalian promoter

Question 22

Why may lentivirus be a better vector than normal retroviruses vectors

Answer 22

1. Because transgene expression is drive by mammalian promoter, Abnormal activation of a host gene is very rarely triggered Therefore it is Less likely to insert close to transcriptional start sites of host genes.
2. Lentivirus vector scan enter non-dividing or slowly dividing cells, including neurons through pores in nuclear membrane allowing it to integrate stably into the genome.

Question 23

Lentiviruses vectors have applications in?

Answer 23

They can deliver genes to neurons for treatment for Parkinson’s

Question 24

Retrovirus vectors have applications in?

Answer 24

Only want to target dividing cells e.g. cancer cells

Question 25

Characterise adenoviruse vectors

Answer 25

1. Double stranded DNA virus, large genome.
2. Engineered to accept inserts as large as 35 Kb
3. Packaged to produce recombinant adenoviruses, do not integrate into host genome, vector DNA exists as linear episomal molecule.
4. Can infect dividing and non-dividing cells, used for in vivo therapy.
5. High levels of transgene expression, but often transient

Question 26

Advantage of adenovirus vectors

Answer 26

They will not activate a proto-oncogene

Question 27

Disadvantage of adenoviruses

Answer 27

1. Adenoviruses are eventually inactivated, thus necessity for reintroduction of vector.
2. As only part of virus genome is removed, often an immune response is provoked

Question 28

What is the OTC-deficiency X-linked recessive

Answer 28

Insufficient transcription of the enzyme Ornithine transcarbamylase leads to a build up of Ammonia leading to mental deterioration.

Question 29

Characterise adeno-associated viral vectors

Answer 29

1. Type of parvovirus, small single stranded DNA genome of 4.7 Kb , non-pathogenic.
2. Recombinant vector similarly packaged as adenoviruses and used for vivo therapy
3. Remains mostly as non-integrated episomal molecules, but can sometimes integrate into genome.
4. Are non-pathogenic, elicits little adverse host immune responses

Question 30

advantages of adeno-associated vectors

Answer 30

1. Elicit little to no immune responses or inflammation
2. Can enter dividing and non dividing cells, some have narrow tissue tropism (liver)

Question 31

Disadvantage of adeno-associated vectors.

Answer 31

They can only have a max insert around 4,5Kb

Question 32

Factor that govern the choice of virus vector.

Answer 32

1. Capacity of vector: how big a piece of genetic material (transgene) can be inserted.
2. Tropism of virus: some viruses preferentially infected certain types of cells
3. Ability to infect dividing or non-dividing cells
4. Integrating or non integrating vector
5. Safety to patient

Question 33

Explain how uptake, transport and uncoating is a barrier to successful gene therapy.

Answer 33

Vectors bind to cellular membrane and are internalised by various processes. Most uptake steps involve a ligand-receptor interaction. Most vectors enter the endosome and undergo a complex set of reactions that can result in their full or partial degradation . If escape from the endosome is successful, transport to the nucleus is required.

Question 34

Explain how vector genome persistence is a barrier to successful gene therapy

Answer 34

The vector DNA can exist as an episomal molecule (and associate with the nuclear matrix) or it can be integrated into the host chromosome.

Question 35

Explain how sustained transcriptional activity is a barrier to successful gene therapy.

Answer 35

Transgene expression can be lost by Epigenetic modifications to the vector

Question 36

Explain how host immune response is a barrier to successful gene therapy

Answer 36

The immune response can limit the viability of the transduced cells and/ or the expression of the transgene product.

Question 37

List the barriers to successful gene therapy

Answer 37

1. Uptake, transport and uncoating
2. Vector genome persistence
3. Sustained transcriptional activity
4. Host immune response