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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
What are the 5 gene products essential for fully permissive production of AAV?
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
26
How does AAV integrate into the genome?
Via its inverted terminal repeats at either end of the genome
27
Can insertional mutagenesis occur with AAVs?
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
What Rep proteins are required for site specific integration?
Rep78 and 68
29
What are the advantages of AAV?
- 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
What are the disadvantages of AAV?
- 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
Describe the production of AAV-based vectors.
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
When Rep and Cap are expressed what do they recognise?
ITR - replication of DNA then occurs and is packaged into AAV particles
33
What happens in mice if Rep is not incorporated?
A high number of mice with HCCs in pre-clinical trials | - Get a range of small to large tumours
34
What happens if the AAV incorporates into chromosome 12 opposed to chromosome 19?
Disrupt several eternally and paternally imprinted genes | Oveqexpressoin of adjacent transcripts telomeric to the pro-viruses - Rian and Mirg genes - implicated in cancer
35
What are the improvements to AAV based vectors?
AAV infectivity of specific cell types Evading the immune response Increasing the packaging capacity of AAV vectors: heterodimerisation Producing higher titre AAV vectors
36
What is the basic concept of gene therapy?
Introduce the correct gene, and its product should cure or slow down the progression of a disease
37
Give examples of two diseases gene therapy could cure?
Cystic fibrosis | SCID
38
How could gene therapy treat cancers?
Delivery of tumour suppressor genes | Delivery of suicide genes for tumour destruction
39
What are the major hurdles to overcome in gene therapy?
Target the correct cell/tissue type Express corrected gene at correct therapeutic dose Express gene for long term period Avoid immune system
40
Describe the ideal vector system
``` 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
What are the non-viral vector systems?
Naked DNA | Chemical carriers
42
What are the viral vector systems?
Retrovirus Aden-associated virus Adenovirus Herpesvirus
43
What are the advantages of non viral vectors?
``` 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
What are the disadvantages of non-viral vectors?
Low efficiency of cell transduction | Lack of long term gene expression
45
What are the types of non viral vectors?
Naked DNA Lipoplexes Polyplexes Lipopolyplexes
46
Describe lipoplexes
Cationic lipids formulated into liposomes and complexed with DNA
47
Describe polyplexes
Cationic polymers complexed with DNA
48
Describe lipopolyplexes
Combination of both lips and polyplexes
49
What are the ways of delivering naked DNA?
Direct injections Electroporation/ultrasound Gene gun Hydrodynamics
50
How does a gene gun work?
Shoot gold particles wrapped in DNA directly into tissues | DNA passes through the membrane into cytoplasm/nucleus
51
What is the disadvantage of a gene gun?
Expression is very short term
52
Describe electroporation
Controlled electrical field to facilitate cell permeabilisation - enhances uptake of DNA after injection
53
What are good targets of electroporation?
Skin and muscles
54
What are lipoplexes composed of?
Hydrophobic tail | Amine group - DNA binding moiety
55
Why does the lipid need to be positively charged?
Necessary for cell binding prior to internalisation by endocytosis
56
How can lipoplexes be delivered?
Systemically as it is stable in the bloodstream
57
What are the extracellular barriers for non viral gene deliver?
DNA degradation in plasma Inability to target to specific organs Largely ineffective via the oral route
58
What are the intracellular barriers?
Eadosomal escape of DNA | Lysosomal degradation of DNA
59
What is a major development of non viral gene delivery?
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
How is the DNA targeted to the nucleus?
Contain NLS OR complex with adenovirus Mu protein - role in the life cycle of the protein to get into the nucleus
61
Why can viral vectors not be used for cystic fibrosis?
Continous inflammation makes non-immunogenic non-viral vectors ideal for gene therapy - viral vectors may lead to further inflammation
62
Why is CF a good gene therapy potential?
63
What was found in clinical trials of CF?
Viruses had low transduction rate Enhance inflammation Produce neutralising Abs preventing repeats administration
64
What are the physical barriers to gene delivery in the CF lung?
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
How can you overcome mucus and glycocalyx barriers?
Pre-treat patient with mucolytic agents and neuraminidase which breakdowns glycoproteins
66
What has the latest clinical trial found?
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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