Gene Therapy

Question 1

What is gene therapy?

Answer 1

Use of nucleic acid polymers as a drug to treat disease by therapeutic delivery into the cells of a patient

Question 2

When does reverse transcription occur?

Answer 2

Used by virus
RNA virus
Specialised enzymes convert back to DNA

Question 3

What are the gene therapy approaches?

Answer 3

Replace non-functional gene
Swap abnormal gene
Selective reverse mutation
Gene regulation

Question 4

What are the methods of vector delivery?

Answer 4

In vivo
Ex vivo

Question 5

What is in vivo?

Answer 5

Directly into the body

Question 6

What is ex vivo?

Answer 6

Cell type taken out + modified then put back into the body

Question 7

What are the choices of gene delivery vectors?

Answer 7

Viral
Non-viral

Question 8

What are examples of viral vectors?

Answer 8

Retrovirus
Adenovirus
Adeno-associated virus
Herpes simplex virus

Question 9

What are examples of non-viral vectors?

Answer 9

Liposome
DNA-polymer conjugates
Naked DNA

Question 10

What characteristics would an ideal vector system have?

Answer 10

Undetectable by immune system
Non-inflammatory
Safe
Efficiency sufficient
Long duration of expression

Question 11

What is the objective of non-viral gene therapy?

Answer 11

Insertion of normal gene to replace abnormal

Question 12

Describe the non-viral approach: direct delivery

Answer 12

Direct introduction of DNA into target cells
Restriction to certain tissues
Large amount of DNA required

Question 13

How is non-viral direct approach done?

Answer 13

Electroporation = electric charge across membranes = open up
Sonoporation = sound waves
Magnetofection = magnets change the charge

Question 14

What are the pro of non-viral gene therapy?

Answer 14

Possible to work with large pieces of DNA
Non-toxic
No immune response

Question 15

What are the cons of non-viral gene therapy?

Answer 15

Inefficient
Limited to accessible cells

Question 16

What is a liposome?
Non-viral vectors

Answer 16

Artificial lipid sphere with aqueous core, carrying therapeutic DNA
= can pass through membrane + deliver gene

Question 17

What is receptor-mediated endocytosis?
Non-viral vectors

Answer 17

DNA molecule encapsulated in delivery device
Then endocytosed into cell

Question 18

Describe the viral approach of gene therapy

Answer 18

Carrier molecule (vector) used to deliver DNA
Viruses

Question 19

What happens to the virus in viral approach of gene therapy?

Answer 19

Virus genome modified to deliver therapeutic gene

Question 20

What are the requirements for viral vector?

Answer 20

Cell-specific
Immune evasion
Non-immunogenic
Safe
Express for long as required

Question 21

Describe retroviral vectors

Answer 21

Creates DNA via reverse transcription
DNA integrates into host chromosome via integrase

Question 22

What are the advantages of retroviral vectors?

Answer 22

Long term expression
Low toxicity
High capacity
Low immunity allowing repeat administration

Question 23

What are the problems of retroviral vectors?

Answer 23

Lack of cell specificity
Only infect dividing cells

Question 24

What is an example of retroviral vectors?

Answer 24

Treat CF by injections directly into bronchial lining

Question 25

What are X-SCID patients?

Answer 25

Immuno deficiency

Question 26

What happens to patients with X-SCID?

Answer 26

Cannot make T lymphocytes their B lymphocytes fail to make essential Abs for fighting infections

Question 27

What do X-SCID patients need?

Answer 27

Bone marrow transfer

Question 28

What is X-SCID caused by?

Answer 28

Mutations in X-linked gene

Question 29

Describe gene therapy of X-SCID patients

Answer 29

Stem cells taken out Genetically manipulated Put back into patient = express T + B cells Gene therapy via injection of retrovirally transduced stem cells

Question 30

Describe adenoviral vectors

Answer 30

Viruses with dsDNA genome = double stranded Remains in episome in cells DNA transcribed

Question 31

Why are adenoviral vectors widely used in gene therapy?

Answer 31

High transfection efficiencies Large quantities possible

Question 32

What are the cons of adenoviral vectors?

Answer 32

More likely to be attacked by immune system Trigger inflammatory response = NO repeat administration

Question 33

Describe adeno-associated vectors (AAVs)

Answer 33

ssDNA = single stranded DNA integrates into single site Infect many cell types

Question 34

What are the pros of AAVs?

Answer 34

Non-toxic Don't trigger immune response NO inflammation

Question 35

What are the disadvantages of AVVs?

Answer 35

Small = carry only 2 genes Difficult to make in large quantities

Question 36

Describe herpes simplex viral (HSV) vectors

Answer 36

Gene transfer in nervous system

Question 37

What are the advantages of HSV vectors?

Answer 37

Large genome = larger genes Infect wide range of tissues

Question 38

What are the disadvantages of HSV vectors?

Answer 38

Relatively untested Stigma due to "herpes"

Question 39

What are the disadvantages of gene therapy?

Answer 39

Short-lived nature Immune response Viral vectors = toxicity, immune response Multi-gene disorders = difficult to treat Insertional mutagenesis

Question 40

What is insertional mutagenesis?

Answer 40

Virus may target wrong cells

Question 41

What is Glybera?

Answer 41

Treatment for lipoprotein lipase deficiency Introduce normal, healthy LPL gene

Question 42

What is CF?

Answer 42

Autosomal recessive disease

Question 43

Why is CF an ideal candidate for gene therapy?

Answer 43

Single gene defect Recessive condition Main pathology = lung = accessible Progressive = at birth normal phenotype

Question 44

What does gene delivery for CF involve?

Answer 44

Large vol of vector-containing fluid to lung via the nose

Question 45

What is the problem with gene delivery for CF?

Answer 45

Safety problems = aspiration Large vol = enhanced alveolar exposure

Question 46

What is another way for gene delivery for CF?

Answer 46

Oral inhalation of corticosteroid

Question 47

What is the problem with oral inhalation for gene delivery for CF?

Answer 47

Small particles enhance delivery to peripheral airways = undesirable for gene vector delivery = adverse effects

Question 48

What is success of CF gene delivery restricted by?

Answer 48

Airways well defended

Question 49

How does the covid-19 vaccine work?

Answer 49

Raise immune response to spike protein mRNA in liposome then injected into patient = encode for spike protein mRNA degraded = NOT long lasting in genome