Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

2. BIOC 2306 S2 > Gene Therapy > Flashcards

Gene Therapy Flashcards

1
Q

Describe the concepts of gene therapy?

A

• 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What justifies gene therapy?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What diseases are addressed by gene therapy clinical trails?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are some (non-viral) vectors?

A 
Vectors used in gene therapy trials:
Adenovirus - 21.2%
Retrovirus - 18.3%
Naked/Plasmid DNA - 17.1%
Lipofection - 4.6%
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Give a comparison of viral vectors and a liposome vector?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Describe the types of liposome?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What are the categories of viral vectors?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are the advantages of viral vectors?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the disadvantages of viral vectors?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is the life cycle of a retrovirus?

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Describe the retrovirus genomes?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Describe the construction of a retroviral packaging cell line?

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Describe some alternative approaches for constructing cell lines?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Describe the Adeno-associated virus (AAV)?

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Describe the AAV genome?

A

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

17
Q

What are some recent devlopments in AAV as a vector?

A

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

18
Q

Describe the Herpesvirus as a vector?

A

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

19
Q

Give an overview of the adenovirus vector?

A

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

20
Q

Describe the structure of the adenovirus?

A

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

21
Q

Describe infection of the host by an adenovirus?

A
  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
22
Q

Describe the adenovirus genome structure?

A

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

23
Q

What are some advantages of using adenoviruses in gene therapy?

A

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)

24
Q

What are some drawbacks to adenovirus gene therapy?

A 
Wide distribution of CAR receptor
Loss of CAR receptor in cancer tissues
Immune response to adenovirus proteins
Inflammatory response
Difficulty of re-administration
25
Q

Give an overview to using viruses as vectors?

A

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

26
Q

Give an overview of SCID?

A

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)

27
Q

Describe ADA deficiency?

A

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

28
Q

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

A

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

29
Q

What are problems with the SCID gene therapy?

A

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

30
Q

Describe adenoviruses within clinical applications of gene therapy?

A

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

31
Q

Describe the recorded death due to adenovirus gene therapy?

A

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

32
Q

What is T-VEC?

A 
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

33
Q

Describe the mechanism of action of T-VEC?

A

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)

34
Q

How can gene therapy be used within cystic fibrosis?

A

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

35
Q

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

A

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

36
Q

What are ATMPs?

A

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

37
Q

What are the challenges of gene therapy?

A 
Safe vectors (low immune response, hepatotoxicity)
Ability to re-administer
Selective targeting of cells 
Effective delivery 
Sustained expression of transgene
38
Q

Give an overview of gene therapy?

A

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

2. BIOC 2306 S2 (15 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions