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Gene Therapy > Revision > Flashcards

Revision Flashcards

1
Q

What type of genome do Retroviruses have?

A

Single stranded RNA genome - around 8500 base pairs]

Enveloped

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2
Q

What genes does the Retrovirus genome encode?

A

Gag - structural (capsid, matrix and nucleoproteins)
Env - Glycoproteins
Pol - enzymes (RT and integrase)

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3
Q

Describe the life cycle of a Retrovirus.

A

Receptor-mediated infection
Viral core with the reintegration sequence was transported to the nucleus
RNA to DNA via RT
Integration into the most genome - more transcription and translation of RNA
Viral proteins produced - assembly of new virus particles and budding

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4
Q

What are the pro’s of Retroviruses?

A

High titre
Long term expression (integrated into the genome) and at a high level
Transduce a wide range of cells

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5
Q

What are the con’s of retroviruses?

A

Limited packaging capability
Insertional mutagenesis due to random insertion
Silencing of promoters - removes the long term expression
Only integrates into dividing cells

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6
Q

What is the most commonly used retrovirus?

A

Murine Leukaemia Virus

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7
Q

How are gag, pol and env supplied in retrovirus packaging cell lines?

A

In trans using a molecular construct

- Only the vector expressing the therapeutic gene is packaged into new formed particles

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8
Q

How have retroviruses been developed to overcome serum inactivation?

A

Use human cells in packaging cell lines - vectors express decay accelerating factor and therefore protects the virus from the complement system

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9
Q

What is pseudo typing?

A

The non-specific incorporation of heterologous cell surface proteins into their lipid envelope (their being viral) - improves viral infectivity to a specific cell type

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10
Q

Give an example of chemical modification on virus budding properties.

A

Incubation of virus with lactose - causes specific targeting of a specific cell type.

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11
Q

How do tissue specific transgene expression help develop a retrovirus?

A

Through transcriptional control elements or tissue specific promoters the transgene is only expressed in the desired cells type - however must be small due to the limited packaging capability.

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12
Q

What are the two types of retrovirus gene therapy are there?

A

In vivo (make in the lab and then put into the patient) and ex vivo

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13
Q

What is the cause of SCID?

A

An early block in and natural killer cell differentiation - near complete failure of the immune system to develop and function - low or absent T cells, NK cells and non-functional B cells

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14
Q

What are the two single gene defects linked to SCID?

A

X-linked SCID

Deficiency of the adenosine deaminase (ADA) enzyme

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15
Q

When did gene therapy trials in France and England begin?

A

2001-2002

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16
Q

What are the methods for overcoming insertional mutagenesis?

A

Site-specific recombinases: recognise 30-300bp DNA sequences and mediate precise recombination between them
Integrate from phage PhiC31: recognised and recombinases phage http and bacterial host attB
Site-Specific integration with PhiC31 integrate

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17
Q

Describe adeno-associated viruses.

A

Small non-developed ssDNA virus
Icosahedral - 20nm in diameter
4700 bps genome
Belong to the family of parvoviridae

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18
Q

Describe the AAV genome.

A

Rep 4 - non-structural genes

  • 2 promoters control Rep 4 (p5 and p19)
  • p5 = unspliced rep78 and spliced Rep68
  • p19 = transcribes larger rep52 and spliced rep40
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19
Q

What does Cap encode (AAV)?

A

Encodes 3 overlapping viral structural proteins

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20
Q

What promoter controls Cap? (AAV)

A

p40

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21
Q

What are the two AAV life cycles?

A

Latent and productive infection

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22
Q

What primary receptor does AAV bind and enter by?

A

Heparin sulphate

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23
Q

How does the DNA remain in the latent infection?

A

As a free episome or integrates into the host chromosome

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24
Q

What is needed in order to activate Rep and Cap?

A

Infection with a helper virus (Adeno or Herpes)

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25
Q

What are the 5 gene products essential for fully permissive production of AAV?

A

E1A - transactivator of adenovirus gene expression and activates AAV gene expression
E1B and E4 - stabilise and facilitate the transportation of AAVs mRNAs and promote host cell entry to S phase
E2A - Essential for efficient AAV RNA splicing and translation
VA RNAs - stimulates efficient translation of AAV mRNAs

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26
Q

How does AAV integrate into the genome?

A

Via its inverted terminal repeats at either end of the genome

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27
Q

Can insertional mutagenesis occur with AAVs?

A

No - site-specific integration at chromosome 19 (19q13.3-qter)
- Contains a sequence with homology to consensus Rep-binding site in the AAV ITR

28
Q

What Rep proteins are required for site specific integration?

A

Rep78 and 68

29
Q

What are the advantages of AAV?

A
  • Non-pathogenic and non-replicating virus
  • Not toxic to host cells - all genes can be removed resulting in no viral gene expression in host cells
  • Transduce a wide range of cells
  • Integrate into the genome - long term persistence
  • Minimal immune response
30
Q

What are the disadvantages of AAV?

A
  • Very limited packaging capability (4.7kb)
  • Rep-minus integration is random- adding rep decreases the packaging capability
  • Helper virus required and can leave contaminants
  • Fairly low titre vectors produced as they are quite hard to produce
31
Q

Describe the production of AAV-based vectors.

A

AAV vector - contains the transgene and ITR - all viral genes are removed.
Need the packaging plasmid - Rep and Cap as well as helper functions - Ad genes of plasmid also provided in 293 cell line

32
Q

When Rep and Cap are expressed what do they recognise?

A

ITR - replication of DNA then occurs and is packaged into AAV particles

33
Q

What happens in mice if Rep is not incorporated?

A

A high number of mice with HCCs in pre-clinical trials

- Get a range of small to large tumours

34
Q

What happens if the AAV incorporates into chromosome 12 opposed to chromosome 19?

A

Disrupt several eternally and paternally imprinted genes

Oveqexpressoin of adjacent transcripts telomeric to the pro-viruses - Rian and Mirg genes - implicated in cancer

35
Q

What are the improvements to AAV based vectors?

A

AAV infectivity of specific cell types
Evading the immune response
Increasing the packaging capacity of AAV vectors: heterodimerisation
Producing higher titre AAV vectors

36
Q

What is the basic concept of gene therapy?

A

Introduce the correct gene, and its product should cure or slow down the progression of a disease

37
Q

Give examples of two diseases gene therapy could cure?

A

Cystic fibrosis

SCID

38
Q

How could gene therapy treat cancers?

A

Delivery of tumour suppressor genes

Delivery of suicide genes for tumour destruction

39
Q

What are the major hurdles to overcome in gene therapy?

A

Target the correct cell/tissue type
Express corrected gene at correct therapeutic dose
Express gene for long term period
Avoid immune system

40
Q

Describe the ideal vector system

A 
Safe - not toxic to target cells 
Easily produced - at high titres
Infect dividing/non-dividng cells
Size capacity - no size limit 
Gives sustained expression
Integration or episomal 
Tissue targeted
Immunological inert - not elicit an immune reponse
41
Q

What are the non-viral vector systems?

A

Naked DNA

Chemical carriers

42
Q

What are the viral vector systems?

A

Retrovirus
Aden-associated virus
Adenovirus
Herpesvirus

43
Q

What are the advantages of non viral vectors?

A 
Non toxic of inflammatory
Non immunogenic 
No change of endogenous virus recombination
Easy to produce at large scale
No limitation of size of DNA
44
Q

What are the disadvantages of non-viral vectors?

A

Low efficiency of cell transduction

Lack of long term gene expression

45
Q

What are the types of non viral vectors?

A

Naked DNA
Lipoplexes
Polyplexes
Lipopolyplexes

46
Q

Describe lipoplexes

A

Cationic lipids formulated into liposomes and complexed with DNA

47
Q

Describe polyplexes

A

Cationic polymers complexed with DNA

48
Q

Describe lipopolyplexes

A

Combination of both lips and polyplexes

49
Q

What are the ways of delivering naked DNA?

A

Direct injections
Electroporation/ultrasound
Gene gun
Hydrodynamics

50
Q

How does a gene gun work?

A

Shoot gold particles wrapped in DNA directly into tissues

DNA passes through the membrane into cytoplasm/nucleus

51
Q

What is the disadvantage of a gene gun?

A

Expression is very short term

52
Q

Describe electroporation

A

Controlled electrical field to facilitate cell permeabilisation - enhances uptake of DNA after injection

53
Q

What are good targets of electroporation?

A

Skin and muscles

54
Q

What are lipoplexes composed of?

A

Hydrophobic tail

Amine group - DNA binding moiety

55
Q

Why does the lipid need to be positively charged?

A

Necessary for cell binding prior to internalisation by endocytosis

56
Q

How can lipoplexes be delivered?

A

Systemically as it is stable in the bloodstream

57
Q

What are the extracellular barriers for non viral gene deliver?

A

DNA degradation in plasma
Inability to target to specific organs
Largely ineffective via the oral route

58
Q

What are the intracellular barriers?

A

Eadosomal escape of DNA

Lysosomal degradation of DNA

59
Q

What is a major development of non viral gene delivery?

A

Non viral vectors are taken up by cells via endocytosis

  • Vectors must be released from the endosome before the DNA is destroyed
  • Eadosomal disrupting peptides are incorporated into the lipoprotein complex
60
Q

How is the DNA targeted to the nucleus?

A

Contain NLS OR complex with adenovirus Mu protein - role in the life cycle of the protein to get into the nucleus

61
Q

Why can viral vectors not be used for cystic fibrosis?

A

Continous inflammation makes non-immunogenic non-viral vectors ideal for gene therapy - viral vectors may lead to further inflammation

62
Q

Why is CF a good gene therapy potential?

A
63
Q

What was found in clinical trials of CF?

A

Viruses had low transduction rate
Enhance inflammation
Produce neutralising Abs preventing repeats administration

64
Q

What are the physical barriers to gene delivery in the CF lung?

A

Airway epithelium has:

  • mucus
  • glycocalyx - may bind vectors and prevent binding to cell receptors
  • Apical cell membrane devoid of viral and growth/tropic receptors
65
Q

How can you overcome mucus and glycocalyx barriers?

A

Pre-treat patient with mucolytic agents and neuraminidase which breakdowns glycoproteins

66
Q

What has the latest clinical trial found?

A

Monthly application of the pCM169/GL67A gene therapy formulation was associated with a significant benefit in stabilisation of lung function compared with placebo at 1 year

Gene Therapy (1 decks)

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