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SOM > Viral Gene Delivery > Flashcards

Viral Gene Delivery Flashcards

1
Q

What is gene therapy?

A

Treatment of medical conditions by delivery nucleic acids into cells (transfection).

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2
Q

What is the difference between transfection and transduction?

A

Transfection is the process of introducing nucleic acids into cells via non - viral methods.

Transduction is the process of introducing nucleic acids into cells using viral methods.

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3
Q

What is transduction?

A

The use of a viral vector to delivery nucleic acids/ genetic material into cells.

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4
Q

Are viral vectors pathogenic?

A

❌NO their wild type viral genes are removed + replaced with 🌟therapeutic genes⭐️

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5
Q

What are the general benefits of using viral vectors as gene therapy delivery systems?

A
  • ✅ they are extensively studied
  • ✅ safe to use in humans
  • ✅ can target specific cells
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6
Q

Name 3 widely used types of viral vectors?

A
  • 📻 retrovirus vector
  • 🦠 adenovirus vector
  • 🦠 🦠 adeno - associated virus (AAV) vectors
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7
Q

What are retrovirus vectors?

A

They are a large family of viruses that use RNA as genomic material.

They transform the ssRNA genome into dsDNA and thus integrate the genetic material into the target cell genome.

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8
Q

What type of genetic material is enveloped in RETROVIRUS vectors 📻 ?

A

SsRNA

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9
Q

Are retrovirus vectors integrating?

A

✅ YES
They transform the ssRNA genome into dsDNA allowing for integration into the target cell genome.

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10
Q

What is encapsulated in retrovirus vectors?

A

SsRNA + reverse transcriptase enzyme

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11
Q

What is the function of reverse transcriptase enzymes in the retrovirus vectors?

A

They transform ssRNA into dsDNA

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12
Q

What is the avg size of retrovirus vectors?

A

100nm

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13
Q

Are retrovirus vectors enveloped?

A

Yes - as well as the capsid coat there is also a lipid bilayer that surrounds the viral capsid.

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14
Q

Give 2 examples of retrovirus vectors?

A
  • ✅ lentiviruses
  • ✅ gamma - retriviruses.
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15
Q

What are the advantages of retrovirus vectors?

A

They are integrating - fuse genetic material into the host cell genome and so when the cell divides it will be present in the daughter cells.
✅will allow for stable long term expression of the new gene.
✅ the therapeutic gene is copied into all new cells after the cell divides
✅ good for targeting dividing cells ie T cells, stem cells, etc those that involve ex vivo treatment.

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16
Q

Give 2 examples of commercially available medications that use retrovirus vectors?

A

💊 Libmeldy —> treat metachromatic leukodystropy , which causes severe organ damage
💊 Kymriah

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17
Q

What is Libmeldy indicated to treat?

A

✅ children with early onset MLD - a rare inherited disorder causing severe damage to the nervous system ansd organs.

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18
Q

What type of gene therapy is Libmeldy and what type of vector/ DDs does it use?

A
  • ✅ Autologous haemotopoetic stem cell gene therapy
  • ✅ lentivirus - a type of retrovirus.
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19
Q

Describe the structure of the Libmeldy lentivirus?

A

✉️ enveloped
🧬 ssRNA
✅ will integrate into the DNA

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20
Q

What causes metachromatic leukodystrophy?

A

A deficiency of the ARSA enzyme due to mutations in the ARSA gene.

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21
Q

What is the function of the ARSA enzyme ?

A

ARSA enzyme usually breaks down sulfatides (which are a major component of the myelin membrane in neuronal cells), in lysosomes to stop them from accumulating ==> this is bc we dont want TOO many sulphatides at any point.

Mutations to ARSA GENE means that there is a deficiency in the ARSA enzyme - thus less sulphatides are broken down.

Intra- lysosomal sulfatides accumulate leading to demyelination and neurodegeneration.

  • hence damage to nervous system and organs
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22
Q

How can we use Libmeldy to treat MLD?

A

Libmeldy is a ex vivo, autologous HSC therapy.

This means that we remove stem cells from the patients blood 🩸(ex vivo) - thus therapy is autologous.

The HSC are genetically modified ex vivo where we add the functional ARSA gene in a lentivirus into them via viral transduction. ✅

Cells are then cryopreseved and then px gets chemo to make space for GM HSC and then we administer the GM cells containing the functional ARSA gene in the lentivirus back into the cells.

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23
Q

What is the benefit + con of using Libmeldy to treat MDL?

A

✅ there was a statistical and clincal improvement in px after 24 months however long term safety and efficacy are not known.
- rmb, lentiviruses / retroviruses are integrating so as cells divide - will carry the functional ARSA gene.

❌ most expensive drug in the world - list price £2.8m - however has been reduced via negotiation with NHS

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24
Q

What type of gene therapy is Libmeldy and what is it used to treat?

A

It is an autologous haemotpoetic stem cell therapy that uses RETROVIRUSES (specifically lentiviruses) to treat rare inherited condition in children called Metachromatic Leuko - Dystrophy.

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25
Q

❌ What are the disadvantages of using retroviruses

A
  • ❌ small carrying capacity for gene insert- approx 8kb and thus the gene we are inserting needs to be very small …
    ❌ - integrating viral vectors - potential for genotoxicity ie (introduction of somatic mutation - damages host cell DNA)
    ❌ integrating viral vectors - potential for activation of oncogensis where cell may become cancerous…
26
Q

What are adenovirus vectors?

A

They are a family of NON - enveloped viruses.

27
Q

What type of genetic material does the wild type adenovirus have?

A

Double stranded DNA

28
Q

What type of wild type genetic material did retroviruses have?

A

SsRNA or ssDNA

29
Q

Are adenoviruses enveloped ?

A

🙅‍♀️ NO - they thus ONLY have a capsid layer and NO lipid bilayer coat.

30
Q

Are adenoviruses episomal/ genome integrating ?

A

🙅‍♀️ NO

31
Q

What is the size of the capsid diameter of adenovirus vectors?

A

90 - 100nm

32
Q

Describe the structure of the capsid layer of the 🦠 adenovirus?

A

It has 3 major protiens present (fibre, penton and hexon) - they all have a function in initial stages of infection of cells

33
Q

Can 🦠 adenoviruses replicate as vectors?

A

❌ no they have been engineered so that they cannot replicate.

34
Q

Give an example of a commercially available mediation that uses adenoviruses as the vector?

A
  • ✅ Vaxzevria (Covid - 19 vaccine).
  • ✅ Gendicine used in cancer treatment
35
Q

What are the advantages of using 🦠 adenovirus vector?

A
  • ✅ high packaging capacity - can accommodate gene inserts up to approx 39kB
  • ✅ can efficiently invade dividing and non dividing cells - thus can be used to deliver gene therapy to a broad range of cells and tissue tubes
    ✅ NON INTEGRATING - remains episomal - thus suitable for therapeutics where high but temporary gene expression is needed.
36
Q

When might we want to use retrovirus vectors? 📻

A

When we want to target dividing cells for long term trans gene expression.

37
Q

When might we want to use an adenovirus vector? 🦠

A

When we want high but temporary gene expression (ie vaccination) as they are non integrating (and thus transient) and can carry a lot of genetic material.

38
Q

❌ What are some disadvantages of using adenovirus vectors for gene therapy? 🦠

A
  • ❌ they are common human pathogens - WT adenoviruses cause respiratory infections such as the common cold and thus a lot ppl may already have pre existing immunity to them!!!!- and so the immune system may attack the adenovirus vector and thus we get ** TREATMENT FAILURE**.
  • ❌ host immunogenicity and cellular toxicity is a problem
39
Q

How can we counter and over come the pre - existing immunity associated with adenovirus vectors gene therapy? 🦠

A

💡 we can use rare human stereotypes, non - human adenovirus vectors - less likely to have been exposed to them in the past and this less likely to be immune
💡 we can shield the surface of the adenovirus vector with PEG to avoid immunogenicity
💡 we said it doesn’t have an envelope and so we could further encapsulate it.

40
Q

What are adeno - associated virus vector?

A

They are non - integrating viral vectors that are made up of dependoparvovirus which rely on adenoviruses to replicate.

41
Q

Give an example of an adeno - associated virus that we can use as a vector and explain why it’s *adeno - associated**?

A

Dependoparvovirus - it depends on adenovirus co - infection for replication.

42
Q

What is the size of AAV vectors?

A

Very small - approx 25nm in size

43
Q

What type of genetic material is encapsulated in AAV viral vectors?

A

SsDNA - think its an off brand adenovirus which uses dsDNA (like normal DNA)

44
Q

Are AAV genome integrating.

A

NO - they are largely non - integrating and thus are episomal.

45
Q

AAV are also high prevalent in humans, like adenoviruses, are they pathogenic?

A

NO - they are non pathogenic in their wild type.

46
Q

Give an example of a commercial preparation that uses Adenovirus vectors for gene therapy?

A

Vaxzevria - the AZ/Oxf Covid - 19 vaccine.

47
Q

What type of gene therapy is Vaxzevria?

A

It is a vaccine that contains dsDNA within a non - enveloped , non - integrating chimpanzee adenovirus.

48
Q

What is the gene therapy present in the adenovirus vaccine Vaxzevria?

A

SARS - cov2 - spike glycoprotein “ChAdOx1-S” present as a double stranded DNA

49
Q

Why do we use a chimpanzee adenovirus vector for the sdDNA sars cov - 2 spike glycoprotein in the Vaxzevria vaccine?

A

-adenoviruses are common human pathogens and so there is a risk of pre - existing immunity where the immune system will recognise the adenovirus and attack it resulting in treatment failure.

If we use an animal virus vector, there is a reduced chance of the immune system recognising it as pathogenic and eliciting an immune response against it.

50
Q

Describe the structure of the Vaxzevria Covid - 19 vaccine?

A

It is made up of a modified chimpanzee adenovirus vector which has been made non pathogenic and a double stranded gene coding for the SARS - COV2 - 2 glycoprotein spike protein.
The adenovirus is lacks an envelope and is non - integrating.

51
Q

How does the Vaxzevria Covid - 19 vaccine work?

A
  1. Genes coding for the sars cov 2 spike glycoprotein are taken from the virus and loaded into a modified chimpanzee adenovirus that has been rendered non - pathogenic forming the Chadox1 ncov - 19 vaccine.
  2. The vaccine is administered to the patient. Adenovirus vector transducers genetic material into the cells - endocytosis etc - cells now express the spike protein.
  3. The body produces antibodies against the spike protein
  4. If the patient is infected with the Covid - 19 virus , ie pathogenic virus, a secondary response is mounted where the antibodies will recognise the sars cov 2 spike protein and attack it.
52
Q

Is Vaxzevria used in the UK as part of the booster programme for Covid - 19?

A

NO

53
Q

What are the cons / side effects of using Vaxzevria ? (And so why are there pre cautions about using adenovirus vector based therapies? )

A
  • a few cases reported of blood clots in ** younger people** —> small no of cases of “vaccine induced immune thrombotic thrombocytopenia” with Vaxzevria and the Johnson and Johnson Covid - 19 vaccine which was also adenovirus vector based.
  • this decreased the public perception of the virus.
    And so not included in booster programme
54
Q

Briefly describe how the adenovirus vectors used in the Covid - 19 vaccines may have lead to blood clots?

A

Platelet factor 4 - involved in blood clotting.

It’s thought that the adenovirus vector may bind to PF4, forming a complex which the immune system now recognised as a foreign complex.
As a result, anti - PF4 antibodies were produced which bind to and activate platelets leading to blood clots.

55
Q

What are some advantages of using adenovirus associated vectors for gene therapy? (AAVs)

A

✅ good safety profile - rmb, they were non pathogenic : )
✅ they are able to introduce genetic material into dividing AND non - dividing cells and can persist for long periods (like Adenovirus vectors)
✅ there are lots of different types of AAV serotypes available and new additional ones can be created by changing the design of the capsid - good for research !
✅ can be transduced into a plethora of different cell types

56
Q

Give two examples of commercially available gene therapies that use AAV vectors as thier DDS?

A

Zolgensma - for spinal muscular atrophy in children
Hemgenix - haemophilia in adults

Rmb these are DNA therapies - go to the nucleus : )

57
Q

❌ what are some potential disadvantages of using AAV vectors for gene therapy delivery?

A
  • highly prevalent in human and so there is the risk of pre - existing immunity (could try to use rare or non human viruses instead).
  • ❌ severe size limitation of the gene insert - must be very very small abt 4.7 kb (bc very small abt 25nm).
  • *thus targeting of larger genes * - very difficult
  • targeting particular disease may be limited due to the limited size of gene we can insert into the AAV vector.
    ❌ small size brings a about challenges to manufacture - ie large (n) of AAvs needed for efficient transduction (thus not very potent) and there are also inefficiencies in vector production.
58
Q

Briefly describe how viral vectors are synthesised?

(Ie AAV vectors)

A
  1. A transgene, helper and packaging plasmid are produced, containing all AAV component genes - these genes code for the therapeutic genetic code, adenovirus to help with replication (rmb AAV), and the cap side proteins to form the virus itself.
  2. They are then co - transfection into packaging cells , often HEK293 (ie allows for the formation and development of the viral vectors.
  3. AAVs containing therapeutic gene insert are produced.
  4. The AAVs are then isolated from the HEK293 cells and are then purified.
59
Q

What is the function of the (a) transgene plasmid, (b) helper plasmid and (c) packaging plasmid in the formation of AAVs?

A

Transgene - contains the genetic code for our gene of interest. It’s our therapeutic gene

Helper - this is the adenovirus used to help AAV replicate - rmb we said AAVs cannot replicate on their own.

Packing virus - think packaging as in the viral capsid. The genes code for the actual bits of the virus itself ie the capsid etc.

60
Q

What is the function of the HEK293 cells in the formation of AAVs?

A

They are packaging cells and they are used to transpire the genetic material from the genes on the plasmids, allowing for the assembly of the AAV vectors

61
Q

What does the choice of viral vector selection depend on?

A
  • 📦 packaging capacity (ie if we want to pack larger / lots of genes adenovirus is the best, if very very small genes, or minimal amount AAV or retrovirus)
  • 🚪 transduction cell type - ie if we want to transduced dividing cells (retrovirus) or both dividing and non dividing ( AAV and AV)
  • 🧬 length of gene expression stable and long term (retro), transient ( AV) , either (AAV).
  • 💉 gene therapy strategy - ie ex vivo (retro - SCT), or in vivo / direct (AAV and AV).
62
Q

Overall what are some of the challenges ❌ 🧠 we need to consider in general when we want to use viral vector?

A
  • 🙅‍♂️ limitiations when it comes to the size of genetic martial we can deliver
  • 🥵 repeated dosing may be an issue essp with those that have risk of triggering an immune response
  • 🤢 risk of triggering an adverse immune response - ie Vaxzevria
  • 🏭 challenges with quality control, scalable and economic production.

This has lead to an increasing interest in non - viral methods.

SOM (7 decks)

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