Gene Therapy

Question 1

Describe the concepts of gene therapy?

Answer 1

• Definition - “The deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic purposes”
This is highly regulated

Aims of gene therapy:
Correct a gene defect  (cystic fibrosis)
Eradicate tumour cells (leukaemia)
Stimulate the immune system (cancer) 
Control an autoimmune disease (rheumatoid arthritis)
Gene editing?
Vaccination

Question 2

What justifies gene therapy?

Answer 2

It is a matter of last resort - normally a single gene mutation
No pharmacological therapy
Surgery is insufficient
Conventional treatments fail

Question 3

What diseases are addressed by gene therapy clinical trails?

Answer 3

Cancer diseases - 64.6%
Monogenic diseases - 10.5%
Infectious diseases - 7.4%
Cardiovascular diseases - 7.4%

Question 4

What are some (non-viral) vectors?

Answer 4

Vectors used in gene therapy trials:
Adenovirus - 21.2%
Retrovirus - 18.3%
Naked/Plasmid DNA - 17.1%
Lipofection - 4.6%

Question 5

Give a comparison of viral vectors and a liposome vector?

Answer 5

Viral vector with insert DNA
They interact with receptors on the surface, they uncoat and produce viral proteins - targeting to the nucleus (where transcription will take place)
Viral proteins may cause inflammation

Liposome with plasmid DNA
No physiological way of getting into cells - they can fuse with the plasma membrane
However, some of the nucleic acid will be destroyed in the cytoplasm and not make it to the nucleus
Less immunogenic - no antibodies will be raised against the lipids

Question 6

Describe the types of liposome?

Answer 6

Cationic - make sheets and have positive charges on the surface
DNA is held by charge interactions, remaining outside to the positively charged liposomes

Anionic - Make spheres and have negative charge on the surface
DNA is entrapped in negatively charged liposome
There could be some repulsion - so used less often

Question 7

Give a conclusion of chemical (non-viral) vectors?

Answer 7

Chemical (non-viral) vectors easy to produce, non-toxic, easy to formulate with nucleic acids
Problems with specificity and targeting
Possible recent success with Moderna and Pfizer/BioNTech mRNA-based SARS-Cov2 vaccine

Question 8

What are the categories of viral vectors?

Answer 8

Non-integrating, transient expression e.g. Adenovirus

Non-integrating, sustained expression e.g. Herpes virus

Integrating, sustained expression e.g. Adeno-associated virus, Retroviruses, Lentiviruses

Question 9

What are the advantages of viral vectors?

Answer 9

Efficient entry into cells
Many cell surface receptors
Often tissue-specific entry (adenoviruses and respiratory tract)
Can integrate or persist (only one administration needed; retroviruses, herpesviruses)
We can create disabled viruses that are replication-deficient for gene therapy

Question 10

What are the disadvantages of viral vectors?

Answer 10

Inflammation due to viral proteins
Pre-existing immunity against virus
Repeat dosing required if expression not sustained – leads to immune response against vector
Integrating viruses (e.g. retroviruses) may insert into critical genes in host DNA, altering gene function
Expensive to produce

Question 11

What is the life cycle of a retrovirus?

Answer 11

1. The virus fuses into the cell
2. Reverse transcription - transcribes the RNA of the virus into DNA
3. This viral DNA is integrated into the host-cell chromosomal DNA (pro-virus)
4. The host cell is transcribed forming retrovirus proteins
   They then bud off and enter other cells

Question 12

Describe the retrovirus genomes?

Answer 12

3 basic proteins - gag, pol and env - they encode for structural proteins and proteins present in the viral capsid
PSI - Signals for packaging (if mutated can’t make viable retroviruses)
Pol - encodes reverse transcriptase, protease and integrase proteins

Question 13

Describe the construction of a retroviral packaging cell line?

Answer 13

Mutant PSI -> cells -> Packaging cells
Normal PSI + therapeutic gene -> Packaging cells
The mutant/normal PSI and therapeutic gene forms viral proteins but no viable viruses
= Viable retrovirus (therapeutic gene)

Question 14

Describe some alternative approaches for constructing cell lines?

Answer 14

Transfect a mixture of plasmids, each encoding gag, pol or env along with a plasmid containing the therapeutic gene into human cells (293T)

The therapeutic retrovirus is secreted into and harvested from the cell culture medium

Question 15

Describe the Adeno-associated virus (AAV)?

Answer 15

Small DNA virus, non-pathogenic
Single-stranded DNA genome of approx. 4.6kb
Needs co-infection with adenovirus (or herpesvirus) for growth
Can enter human cells in absence of adenovirus and establish latent infection
Integrates into specific region of human chromosome 19
Multiple types (mainly AAV2 currently used for gene therapy)

Question 16

Describe the AAV genome?

Answer 16

There are two genes present - the REP/CAP (replicase and capsid)
They can be removed to add the DNA we want:
Need complementing (packaging) cell line expressing Rep and Cap
Or co-infection with adenovirus
Or triple transfection with plasmids encoding Rep/Cap, Adenovirus helper functions (E1A,E1B, E2A, E4, VA-RNA) and the therapeutic gene flanked by terminal repeats

Question 17

What are some recent devlopments in AAV as a vector?

Answer 17

Use of multiple types of AAV that have different tissue specificity
Engineering recombination between different AAVs co-infected into cells to produce larger genes within cells that can encode larger proteins

Notable success include:
Improvement in inherited degenerative eye disease (retinitis pigmentosum)
Licencing of Glybera - AAV1 encoding lipoprotein lipase (LPL) for inherited LPL deficiency
Zolgensma an AAV9 encoding SMN-1 (defective in Spinal Muscular Atrophy) – targets motor neurones

Question 18

Describe the Herpesvirus as a vector?

Answer 18

Large DNA viruses, approx. 150kb
Many non-essential genes provide targets for replacement with therapeutic genes
Infect non-dividing cells
Do not integrate into the host cell genome
Can establish latent infections (Herpes simplex virus)
Some herpesviruses (such as Epstein-Barr virus or monkey Herpesvirus saimiri) can be maintained as extra-chromosomal episomes in latently-infected cells
Recent development of T-VEC, passes phase III trial in head and neck cancer

Question 19

Give an overview of the adenovirus vector?

Answer 19

Have linear, double-stranded DNA genomes of approx. 36kb (=medium size virus)
Infect epithelial cells that line respiratory and alimentary tracts, conjunctiva, bladder and kidneys
Infect non-dividing cells
Contain 30-40 genes
Some non-essential genes can be replaced with therapeutic genes

Question 20

Describe the structure of the adenovirus?

Answer 20

They have an icosahedron structure (triangles)
Penton protein - the base for a fibre structure sticking out, that ends in a knob
Contains a RGD motif

Question 21

Describe infection of the host by an adenovirus?

Answer 21

1. Attaches to the cell
   The fibre knob attaches to the CAR (cell surface receptor)
   The penton base interacts with cell surface integrins - involved in adhesion of cells
2. Uptake via endosome and acidification - uses receptor mediated endocytosis
3. Release from endosome and breakdown of capsid
4. Transported to the NPC - along microtubules
5. Viral genome and core proteins enter via the NPC

Question 22

Describe the adenovirus genome structure?

Answer 22

Early genes
E1a, E1b - replaced by transgene (they are essential genes)
E3 - non-essential for virus growth so can be deleted

Late genes - L1-5
They don’t get destroyed in these vectors produced - so they can make virus particles

Question 23

What are some advantages of using adenoviruses in gene therapy?

Answer 23

Relatively safe
Used as vaccines in US military
Non-pathogenic (except in the immunosuppressed or very young)
Capacity for large inserts
Can infect a variety of human and mammalian cells (depending on the serotype)

Question 24

What are some drawbacks to adenovirus gene therapy?

Answer 24

Wide distribution of CAR receptor
Loss of CAR receptor in cancer tissues
Immune response to adenovirus proteins
Inflammatory response
Difficulty of re-administration

Question 25

Give an overview to using viruses as vectors?

Answer 25

Viruses form very promising systems for high-efficiency delivery of transgenes Problems exist with immune response and repeat dosing May be difficult to use in the general population (low temp storage etc) Need to be careful about possible toxicity

Question 26

Give an overview of SCID?

Answer 26

Serve combined immunodeficiency Normally it is supposed to recruit tyrosine kinases 19 types of SCID - some categories are: X-linked SCID - IL2RG, T(-), B(non-functional), NK(+) ADA SCID - ADA, T(-), B(-), NK(-) Jak3 mutation - JAK3, T(-), B(+), NK(-) David Vetter - bubble boy (1971-84)

Question 27

Describe ADA deficiency?

Answer 27

Adenosine deaminase deficiency This is an autosomal recessive disorder 1.5 kb cDNA and 1 in 100,000 Without treatment, these babies usually don't survive past the age of 2

Question 28

Describe the partial success and ex vivo gene delivery within SCID?

Answer 28

Treated ex vivo with retrovirus expressing cytokine receptor subunit Injections of the ADA protein to try and offset the deficiency Six patients reported to be successfully treated in Paris Two patients successfully treated at Great Ormond Street However potential problem with leukaemia reported Ex vivo gene delivery Application in lymphoid and haematological disorders e.g. SCID 1. Bone marrow removed 2. Cells transduced by recombinant retrovirus 3. Transduced cells returned to patient

Question 29

What are problems with the SCID gene therapy?

Answer 29

In 2005, three children in the French group had been diagnosed with leukaemia Reasons not clear but could be due to insertion of retrovirus into a gene (LMO2) associated with leukaemia 4/9 subjects developed leukaemia, with one fatality - integrations into the LMO2 gene Additional integrations also observed in proto-oncogenes BMI1 and CCND2 1/10 subjects in London trial developed leukaemia following integration next to LMO2

Question 30

Describe adenoviruses within clinical applications of gene therapy?

Answer 30

Disappointing results in cystic fibrosis - quite transient Immune response against vector Inflammatory response Re-administration not effective Encouraging results in head and neck squamous cell cancer (ONYX-015 replication-competent or oncolytic virus) ONYX-015 licenced in China as H101

Question 31

Describe the recorded death due to adenovirus gene therapy?

Answer 31

Jesse Gelsinger from liver toxicity Suffered from inherited ornithine transcarbamoylase (OTCase) deficiency = High levels of urea Received adenovirus containing OTCase gene via hepatic artery Part of study involving 18 patients and died four days later Study stopped (proved controversial amongst other participants) and FDA investigation followed

Question 32

What is T-VEC?

Answer 32

``` Talimogene laherparepvec (Imlygic) - a licenced oncolytic herpesvirus for melanoma First live virus to be approved by the FDA for the treatment of cancer (melanoma) Engineered version of herpes simplex virus type 1 (HSV-1) ``` Genes were removed to prevent toxicity of the virus and enhancing the immune response against the cancer cells

Question 33

Describe the mechanism of action of T-VEC?

Answer 33

T-VEC is injected in a specific tumour It will infect both normal and cancer cells but it is attenuated so the virus doesn't replicate in healthy cells = undamaged T-VEC selectively replicates and synthesises GM-SCF in tumour cells The tumour cells lyse and release the replicated virions and GM-CSF and tumour specific antigens Replicated virions repeat cell lysis in neighbouring tumour cells GM-CSF recruits dendritic cells to the tumour - which present TSAs to mediate a tumour specific immune response (adaptive immunity will destroy the cancer throughout the body)

Question 34

How can gene therapy be used within cystic fibrosis?

Answer 34

Several trials performed in 1990’s using 1st generation recombinant adenoviruses expressing CFTR (USA) or Liposomes containing CFTR plasmid Transient CFTR expression and some evidence of restoration of chloride transport No sustained effect Patients receive the therapy by inhaling fat globules (liposome) contains DNA from a nebuliser This targeted the epithelial cells in the lungs

Question 35

What was involved in the clinical trials of CF using lipsome-based therapy?

Answer 35

Monthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo Limitations - although encouraging, the improvement in FEV1 was modest and was not accompanied by detectable improvement in patients’ quality of life. Future work will focus on attempts to increase efficacy by increasing dose or frequency, the co-administration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration

Question 36

What are ATMPs?

Answer 36

Advanced therapy medicinal products (ATMP, European medicines agency (EMA)) Includes gene, cell and tissue therapeutic products Can be high cost ($0.5-1 million dollars per patient for inherited diseases) Need to determine which patients will derive most benefit Challenge to healthcare systems ATMPs approved by the EMA Strimvelis - ADA-SCID - April 2016 Imlygic - Melanoma - October 2015 Glybera - Lipoprotein lipase deficiency (LPLD) - November 2012

Question 37

What are the challenges of gene therapy?

Answer 37

``` Safe vectors (low immune response, hepatotoxicity) Ability to re-administer Selective targeting of cells Effective delivery Sustained expression of transgene ```

Question 38

Give an overview of gene therapy?

Answer 38

Viruses form a key vector in human gene therapy for cancer as well as inherited diseases Direct in vivo, intra-tumoral use (onyx-015) – also h101 and Gendicine Used ex vivo to create cell therapy products (Strimvelis – t-cells modified by retrovirus expressing ADA) Cost implications – balanced against cost of care Ethical and safety implications