Lecture 10: Gene Therapy

Question 1

What are 5 common genetic disorders?

Answer 1

1. Cystic fibrosis
2. Sickle cell anaemia
3. Thalassaemia
4. Duchenne muscular dystrophy
5. Familial hypercholesterolaemia
6. Haemophilia A
7. Phenylketonuria
8. Tay-Sachs disease

Question 2

What is gene therapy?

Answer 2

The introduction of recombinant DNA, typically via a viral vector delivery system, to alter the expression of a particular gene within the cells of an individual

Question 3

What are the four main targets of gene therapy and how gene therapy might be used in these scenarios?

Answer 3

1. Single gene recessive loss of function disorder = gene therapy allows the addition or replacement of the faulty gene to promote protein production (E.g. CF, haemophilia A)
2. Single gene, haploinsufficiency = gene therapy allows for addition of gene to give sufficient protein to overcome the haploinsufficiency (E.g. Dyschromatosis Symmetrica Hereditaria (DSH))
3. Single gene, dominant negative (toxic copy destroys function of normal copy) = gene therapy allows the silencing or replacing of mutated allele (E.g. HD)
4. Multi-gene or acquired = gene therapy allows addition of a therapeutic gene, CAR-T in cancer treatment (E.g. cancer, heart disease, rheumatoid arthritis)

Question 4

Which types of disorders are more attractive to drug companies for gene therapy treatment development and why?

Answer 4

Acquired diseases because they are much more common so there is more money to be made in this area

Question 5

What type of disease are the majority of gene therapies aimed at?

Answer 5

Cancer

Question 6

What is the name of the gene therapy aimed at cancer?

Answer 6

CAR-T (modification of T-cells to recognise cancer cells and restore immune function against cancer cells)

Question 7

Of the genetic diseases, what are a large amount of gene therapies aimed at?

Answer 7

Metabolic disorders

Question 8

What are the two approaches of gene therapy?

Answer 8

In vivo and ex vivo

Question 9

Describe the in vivo gene therapy approach?

Answer 9

• single step process (new gene delivered straight to patient via recombinant viral vector)
• vector administered directly to patient via blood stream or inhalation)
• the therapy can be targeted to specific organ or tissue by route of administration or specificity of vector in terms of cell specific entry

Question 10

Describe the ex vivo gene therapy approach?

Answer 10

• two step process (patient cells - usually blood stem cells - are harvested, modified by addition of gene in vitro via viral vector, cultured, and engineered cells re-introduced to patient)

Question 11

What types of cells are targeted by both in vivo and ex vivo gene therapy?

Answer 11

Somatic cells

Question 12

What are some barriers to gene therapy?

Answer 12

1. Viral vector entry - vector may encounter neutralising antibodies (especially if adenovirus vector used - common cold virus - many people have antibodies)
2. Uptake - the vector needs to be able to bind to the target cells so the cells need to possess cell surface receptors
3. Integration - the genetic material needs to be replicated (either incorporate into host genome or exist as an episome that replicated independent of host genome due to viral replication origin)
4. Transcription and processing - the gene needs to be transcribed and spliced to give protein
5. Immune response to new protein - the immune system may not have ever seen the protein before so recognises it as foreign and destroys it
6. Epigenetic effects - there is no control over where the gene inserts into the host genome - it may insert into a region of heterochromatin, in which it will be automatically silenced

Question 13

How can epigenetic effects influence the outcomes of gene therapy?

Answer 13

There is no control over where the gene inserts into the host genome - it may insert into a region of heterochromatin, in which it will be automatically silenced

Question 14

List some of the vectors for gene therapy? State which are used mostly for in vivo or ex vivo approaches

Answer 14

in vivo approaches typically use adenovirus, adeno-associated virus (AAV) and lentivirus vectors, some use retroviral vectors

ex vivo approaches typically use gamma-retroviruses

Other vectors: plasmids, mini-circles, transposons

Question 15

What is the main drawback of in vivo gene therapy approaches?

Answer 15

there may be difficulty in delivery - for example some organs, such as liver, retina and brain are much more inaccessible for gene therapy.

Question 16

True or false: in vivo gene therapy can be used for the treatment of single gene disorders as well as acquired diseases?

Answer 16

True

Question 17

Give three advantages and three disadvantages of adenoviral vectors?

Answer 17

Advantages:
- large capacity
- broad specificity (infect broad range of cell types)
- efficient transduction
- easily purified
- potential vector for cancer treatment (expression of anti-cancer proteins)

Disadvantages:
- highly immunogenic capsid protein - common cold virus - neutralising antibodies
- potentially fatal systemic inflammatory response (toxic shock syndrome)
- transient expression of transgene (not permanent - but may be useful for cancer treatment as don’t need permanent expression)

Question 18

Give three advantages and a disadvantage of using adeno-associated viral (AAV) vectors in gene therapy

Answer 18

Advantages:
- non-pathogenic and minimal immune response
- can be maintained in non-dividing cells as episome
- shows tissue specificity depending on the serotype used

Disadvantage:
- small capacity (for instance, AAV would not be able to carry the dystrophin gene)

Question 19

Give an example of a disease that has been treated with AAV gene therapy

Answer 19

Leber’s congenital amaurosis (LCA)

Question 20

What is Leber’s congenital amaurosis (LCA)?

Answer 20

Autosomal recessive disorder caused by mutation in 14 different genes (including RPE65 that encodes for retinal pigment epithelium-specific 65kDa protein required for photoreceptor function)
- causes early onset blindness and lack of photoreception despite photoreceptors being present in the affected individuals

Question 21

How has AAV vector gene therapy been used to treat LCA?

Answer 21

• AAV2 serotype capsids are injected directly beneath the retina
• the virus is taken up by retinal epithelium
• RPE65 gene expressed from episomal vector
• light sensitivity was restored and was maintained for over 3 years (no full vision restoration though)

Question 22

Other than the retina, what are three other target organs of AVV vector gene therapies?

Answer 22

Liver
- for treatment of plasma protein deficiencies (fibrinogen, albumin, enzymes), metabolic disorders, Haemophilia B (factor IX)

Muscle
- for treatment of haemophilia B, alpha-1-antitrypsin deficiency, DMD

Brain
- AAV9 can cross BBB for treatment of Parkinson’s disease, Canavan’s disease, Batten’s disease

Question 23

What type of cancer has CAR-T shown success in the treatment of?

Answer 23

triple negative breast cancer

Question 24

Describe the process of ex vivo gene therapy for treatment of SCID

Answer 24

• HSPCs isolated from patient’s bone marrow
• the cells are transduced with IL2RG (for X-linked SCID) or ADA (for autosomal recessive SCID) gene via gamma-retroviral vector ex vivo
• the modified cells are then reintroduced to patient’s bone marrow and in theory should repopulate the bone marrow for long term production of B-cells, T-cells and NK cells

Question 25

Why might gamma-retroviruses lead to the development of cancer in gene therapy patients?

Answer 25

They have LTRs that have promoter enhancer activity so if integrate near promoter end of a host proto-oncogene, they can transform them into oncogenes

(GO OVER SLIDE 21/22 AGAIN ONCE LECTURE RECORDING UP)

Question 26

How can the safety of gamma-retroviral vectors be improved?

Answer 26

use self-inactivating vectors with LTRs deleted so don’t have such a strong enhancer effect.

Question 27

What are the (3) main problems with gamma-retroviral vectors?

Answer 27

• preference for insertion near promoters of active genes
• strong enhancer and promoter in LTRs that can activate nearby oncogenes
• splice donor site downstream of 5’ LTR and can therefore be spliced to exons of oncogenes

Question 28

What are alternatives to gamma-retroviral vectors and why might they be better?

Answer 28

Lentiviruses:
- LTRs lack strong enhancer
- self-inactivating vectors (delete LTRs for added safety)

DNA vectors (simple plasmids/mini-circles)
- no pre-existing immunity
- high capacity
disadvantage though is difficult to deliver in vivo