1.) Gene Therapy: Background & Delivery

Question 1

What is gene therapy?

Answer 1

Delivery of genetic material into a patient’s cells as a drug:

• treat diseases associated with genetic mutation/changes in gene expression
• Promising in theory but difficult in practice
• Ethical considerations

Question 2

What are the possible ways that gene mutation can lead to disease?

Answer 2

• Mutation in coding sequence
• Mutation in promoter
• Mutation in splice site
• Mutation in regulatory elements of UTR (untranslated region)

Question 3

Describe how mutation in coding sequence can lead to disease? What types of this mutation are there?

Answer 3

Mutated coding sequences of DNA results in the production of a mutant protein:
- AA sequence of protein is altered
- production/function is changd
- mRNA transcribed from mutated dsDNA is wrong, thus mutated code is translated to form mutant protein
»> Deletion/substitution/insertion variants alter AA sequence

Question 4

Name a disease that arises as a result of a mutated coding sequence, and the common mutations involved.

Answer 4

Cystic fibrosis:

• Dominant negative disease cause by loss of both copies of functional CFTR membrane transporter (autosomal recessive)
• Many mutations responsible: most commonly F508 deletion (3 base pair deletion)
• Loss of phenylalanine leads to misfolding of protein and subsequent degradation = transporter protein never makes it to the membrane to perform function

Question 5

Name possible damaging effects of coding sequence mutation.

Answer 5

• Premature termination colon: leading to production of truncated protein (e.g. missing C-terminal; protein is prematurely short/cut-off)
• Frameshift mutation leading to incorrect sequence downstream of mutation (insertion/deletion)

Question 6

Describe how mutation in promoter elements can lead to disease?

Answer 6

Can lead to change in expression level:

• Promoter required for transcription factors = influence how much mRNA is transcribed/made
• E.g. deletion in DNA sequence (of promoter element) removes binding site for essential transcription factor resulting in decreased protein expression

Question 7

Describe how mutation in regulatory elements of UTR/splice sites can lead to disease?

Answer 7

Mutation in control elements (splice sites, 5’ or 3’ UTR) leads to changes in regulation of expression by splicing or trans-acting factors:
- Splice sites: production of incorrectly spliced mRNA due to mutation (Intron 2 not removed e.g.)
- UTRs: mutation could lead to up/downregulation of protein production
»> E.g. Mutation in miRNA binding site prevents miRNA binding, leading to increase in protein production (miRNA involved in regulating expression)

Question 8

What diseases could be treated with gene therapy?

Answer 8

Normally diseases associated with genetic mutation/changes in gene expression:

Single-gene disorders:
- Rare inherited disease (chronic)
E.g. cystic fibrosis, sick cell

Infectious diseases:
- Persistent viral infections
E.g. HIV

Cancer:

• Attributed to reduced activity of tumour suppressor gene products/increased activity of oncogene products
• Multiple genetic changes therefore required
• Some inherited, most acquired

Immunotherapy:
- Gene therapy can alter host response to disease and make immune system more effective

Question 9

What are the different strategies for gene therapy?

Answer 9

• Gene therapy (OG approach): introduce a gene (dsRNA or mRNA) into cell to compensate for mutation/reduced expression of host gene
• Genome modification (editing): change sequence of genomic DNA to correct mutation
• Antisense: using oligonucleotides that bind to mRNA e.g. modifying splicing pattern of disease-associated gene
• RNA interference: using short interfering RNAs to knock down expression of gene
• microRNA therapeutics: using oligonucleotides to increase/decrease levels of disease-associated miRNAs

Question 10

What are the traditional challenges associated with OG gene therapy?

Answer 10

Challenges associated with introducing genes into cells:

• Delivery to correct target cell (genetic material = large molecule required)
• Maintenance of delivered gene in target cell (for chronic conditions)
• High cost of drug development

Question 11

What limitations surround OG gene therapy?

Answer 11

Limitations associated with introducing genes into cells:

• Only suitable for diseases caused by reduced proliferation of a protein product (dsDNA/mRNA gene inest compensates for mutation/reduced expression)
• Can’t achieve precise level of expression of delivered gene: only practicable if expression level does not matter too much (NOT a narrow window…)

Question 12

How are genes delivered into the patient (OG gene therapy)? Compare the two.

Answer 12

In vivo gene delivery:
- Introduce gene directly into patient (via method appropriate to target issue e.g. aerosol to lung)

Ex vivo gene delivery:

• Take target cells out of patient, introduce gene, return to patiebt
• Good approach for cells of haemtopoietic system (bone marrow, spleen, thymus and lymph nodes etc involved in the production of cellular blood components)
• Allows precise targeting and increased efficiency (currently more straightforward)

Question 13

What does firal delivery of gene therapy entail?

Answer 13

• Viral infection involves delivery of genetic material into host cell
• Viral genome modified to include gene of interest
• Host cell takes up virus
• Most common method of delivery currently

Question 14

What are the pros and cons associated with viral delivery of OG gene therapy?

Answer 14

Pros:

• Efficient uptake
• Can persist in cells
• Viral DNA can be modified to avoid problems of pathogenicity/tumorigenesis

Cons:

• Immune response
• Potential pathogenicity
• Potential for tumorigenesis if it integrates into host genome
• Limitation on size of gene able to be incorporated into viral vector

Question 15

What are some examples for non-viral delivery of OG gene therapy?

Answer 15

• Naked dsDNA

- Nanoparticles

Question 16

What are the pros and cons associated with non-viral delivery of OG gene therapy?

Answer 16

Pros:

• Low immunogenicity
• Large scale production
• Can package larger DNA molecules

Cons:

• Less efficient than viral delviery
• Can be difficult to target specific tissues
• Difficult to achieve sustained gene expression over time (more likely need to repeat dosing)

Question 17

What are the requirements that viral vectors must meet for gene delivery?

Answer 17

• Ability to attach to and enter target cell
• Transport of genetic material to nucleus and maintenance as DNA
• Sustained expression of genetic material over time
• Lack of toxicity/immunogenicity

Question 18

What viruses are used as viral vectors for gene therapy?

Answer 18

• Adeno-associated virus (AAV - > 50% of viral vectors)
• Other DNA viruses
• Retroviruses
• Lentiviruses

Question 19

How do retroviruses normally operate? Name an example.

Answer 19

Retroviruses:
• Encodes reverse transcriptase, which copies viral RNA to DNA, which is then integrated into host genome
• E.g. Murine moloney retrovirus

Question 20

How are retroviruses manipulated to deliver gene therapy? Issues with its use as a vector?

Answer 20

• Incorporation of delivered gene into host genome, therefore maintenance during cell division = inserted gene is not lost over time

Issues:
- Can’t control site of integration, which may lead to problems with insertional mutagenesis

Question 21

Define: insertional mutagenesis. Why might this be an issue?

Answer 21

Viral integrase inserting genome (gene therapy) wherever it wants:
- Could integrate within an important gene (e.g. tumour suppressor) and disrupt function
- Or strong viral promoter may be introduced next to a host oncogene, leading to disregulation of expression
»> Gene therapy could end up inducing cancer

Question 22

What are Lentiviruses? What advantages do they convey as viral vectors? Drawbacks?

Answer 22

• Highly modified versions of viruses, retrovirus subtype
• Integrates into host genome similar to retroviruses
• E.g. HIV

Advantages:
• Can integrate in non-dividing cells (unlike retroviral vectors)
• Current generation of lentiviral vectors better than retroviral vectors due to:
- Lower immunogenicity

Disadvantages:

• Insertional mutagenesis is still potentially a problem
• Small virus size; limits size of gene that can be delivered

Question 23

Describe the properties of AAVs.

Answer 23

Adeno-associated viruses:
• Non-pathogenic
• Low immunogenicity
• Infects dividing AND non-dividing cells
• Does not replicate without a helper virus

Question 24

Does AAV virus integrate into host genome?

Answer 24

Viral DNA is retained in cell nucleus (as episomal DNA):
• Native AAV virus integrates at specific site in host genome
• Gene therapy vectors have rep and cap genes deleted, so DO NOT integrate
• Gene therapy is just retained in cell nucleus as episomal concatemer for lifetime of non-dividing cell, or until lost through cell division

Question 25

Describe how gene therapy can treat SCID.

• Explain treatment
• Gene introduced/targeted
• Role in disease
• Method of delivery (and why)

Answer 25

Severe Combined Immunodeficiency Disorder:
- ‘Bubble babies’ - children with inherited immune defect v susceptible to infection (kept in sterile unit)
- V severe disease
• Single gene involved (different in different forms of SCID) = good target for gene therapy
• Latest treatment based on lentiviral vector (better than retroviral)
• Vector that allows genome integration necessary as immune cells turned over rapidly - can’t use AAV

Question 26

How were lentiviral vectors shown to be advantageous over retroviral vectors in SCID gene therapy?

Answer 26

• 2002 gene therapy trial suspended after x4 children developed leukaemia
  »> Insertional mutagenesis of retroviral vector

• 2013 confirmed x5 children were doing well with treatment delivered via lentiviral vector

Question 27

What is CAR-T immunotherapy, and what is it indicated for?

• Explain treatment
• Gene introduced/targeted
• Role in disease
• Method of delivery (and why)

Answer 27

Form of adoptive T-cell transfer; patient’s T-cells are engineered and then reintroduced:
• Kymriah (Novartis) approved by FDA for ALL
• T-cells taken from patient (ex vivo), genome engineered to produce chimeric antigen receptors (CARs) which recognise antigens on cancer cells
• Return cells to patients, where they multiply, attack and kill cancer cells
• Usually via retroviral vector
»> V effective in acute lymphoblastic leukaemia (ALL)
> Indicated for cancers with specific CAR for specific tumour antigen

Question 28

What drawbacks are there with CAR-T immunotherapy?

Answer 28

Serious side effects can occur:

• E.g. cytokine release syndrome - cytokine storm, uncontrolled immune activation

Question 29

Describe the applications of gene therapy in the retina.

Answer 29

Gene therapy can be delivered subretinal/intravitreal (in vivo):
- Rare inherited disease affecting the retina (potentially causing blindness)
E.g. Choroidermia
E.g. Retinal dystrophy (biallelic RPE65 mutation-associated)

Question 30

What is choroidermia, and how can it be treated via gene therapy?

Answer 30

X-linked disease causing progressive vision loss, caused by mutation in CHM gene:
• Wildtype CHM gene delivered in AAV viral vector
• Via injection in vicinity of retina
• One eye treated and one untreated as control
• Vision in treated eye much improved 3.5 years after treatment

> > > The CHM gene encodes a special protein called Rab-escort protein 1 (REP1) which plays a key role in the metabolism of the cells making up the retina, which is the light-sensitive layer (like a camera film) that lines the back of the eye. The absence of REP1 in the retinal cells causes them to die over time, resulting in a progressive degeneration of the retina and consequent loss of vision.

Question 31

Describe the applications of gene therapy in the treatment of haemophilia.

Answer 31

Haemophilias are blood clotting disorders due to lack of clotting factors:

• X-linked recessive inheritance
• Haemophilia A and B caused by lack of factor VIII and IX respectively

Treatment:
PI/II trial results in December 2017 for haemophilia B were promising:
• Hepatocyte-targeted AAV used to introduce factor IX into liver
• Single dose = 9/10 patients had no bleeds for +18 months

Question 32

Describe the hurdles presented with haemophilia gene therapy and the corresponding solutions.

Answer 32

Expression of sufficient clotting factor:
• Used gain-of-function over wild-type mutation yielding 7-fold greater activity

Pre-existing immunity to AAV in 30-50% of people:
• Modifications to AAV capsid, but requires more work to be suitable for all patients

Transient (short-term) liver cell damage (hepatocyte) in 25% of treated patients:
• Short term corticosteroid treatment

Question 33

Why would delivering mRNA as gene therapy be advantageous over dsDNA?

Answer 33

• No danger of integration into genome

* Can be rapidly developed

Question 34

What barriers need to be dealt with in developing mRNA therapeutics as gene therapy?

Answer 34

• Avoid immune response
• Avoid degradation
• Be efficiently translated

Delivery:

• Lipid nanoparticles
• Non-lipid polymers

Question 35

What indications are mRNA gene therapeutics being developed towards?

Answer 35

Vaccines:

• Prophylactic e.g. Zika, influenza A (intramuscular)
• Personalise anti-tumour vaccines (direct injection to tissue)

CVD:
- VEGF mRNA for wound healing