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ATU 1 > Gene Therapy > Flashcards

Gene Therapy Flashcards

1
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”.

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2
Q

What types of inherited diseases could gene therapy cure?

A
  • Severe combined immunodeficiency disease
  • Cystic fibrosis
  • Ornithine transcarbamylase
  • Haemophilia
  • Duchenne muscular dystrophy
  • Cancer: Delivery of tumour suppressor genes and delivery of suicide genes for tumour destruction.
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3
Q

What hurdles have to be overcome for successful gene therapy?

A
  1. Target correct cell/ tissue type
  2. Express corrected gene as correct therapeutic dose
  3. Express gene for long term period (genetic diseases)
  4. Avoid immune system.
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4
Q

What is an ideal vector for gene therapy?

A
  • Safe: non-toxic/no unwanted effects
  • Easily produced: high titres required, reasonable shelf life
  • Infect dividing/ non-dividing cells
  • Size capacity: no size limit and including regulatory sequences
  • Sustained expression: long time period at the correct levels
  • Integration or episomal: allow for site specific integration or be retained as an episome in the nucleus.
  • Tissue targeted: delivery to the desired cell type
  • Immunologically inert: should not elicit an immune response.
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5
Q

What are the pros of non-viral vectors?

A
  • Non toxic or inflammatory
  • Non immunogenic
  • No chance of endogenous virus recombination
  • Ease of large scale production
  • No limitation of size of DNA.
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6
Q

What are the cons of non-viral vectors?

A
  • Low efficiency of cell transduction (especially in vivo)

- Lack of long term gene expression.

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7
Q

What subtypes of non-viral vectors are there?

A

Naked DNA, Lipoplexes, Polyplexes, Lipolyplexes.

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8
Q

Describe lipoplexes?

A

Cationic lipids formulated into liposomes and complexed with DNA
Composed of:
- hydrophobic tail (fatty acid, alkyl or cholesterol moiety), self assembly into bilayer vesicles in aqueous media
- amine group – DNA binding moiety
A positively charged lipoplex is necessary for cell binding prior to internalisation by endocytosis.

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9
Q

What methods of Naked DNA delivery are there?

A

Direct injections, systemic injections, electroporation, ultrasound, gene gun, hydrodynamics (high-pressure dynamics).

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10
Q

Describe electroporation/ultrasound as a method of Naked DNA delivery?

A
  • Controlled electrical field to facilitate cell permeabilisation
  • Enhances uptake of DNA after injection
  • Skin and muscles good candidates
  • Ultrasound also increases permeability of cell membrane.
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11
Q

Describe a gene gun as a method of Naked DNA delivery?

A

Shoot gold particles wrapped in DNA directly into tissues. DNA passes through the cell membrane into cytoplasm, even nucleus. However, expression is very short term (few days at most).

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12
Q

What are the barriers of non-viral gene delivery?

A

Vectors, which are taken up by the cells via endocytosis, are required to be released from the endosome before the DNA is destroyed. Therefore, endosomal disrupting peptides are incorporated into the lipoprotein complex.

Once released, the DNA is required to be targeted to the nucleus. The DNA contains NLS which interact with the nuclear import pathways and DNA is complexed with adenovirus Mu proteins which has a role in the virus.

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13
Q

Describe CF?

A
  • Most common lethal inherited recessive disease in Caucasians
  • Reduced life expectancy: 28 to 30 years of age.
  • Affects 1 in 2500 new-borns

Genetic mutations cause a build up salt in the cells that line the glands of the respiratory passages, sweat glands, pancreas and small intestine. This build up of salt causes the normally thin and slippery secretions of these glands to become thick and sticky. These secretions, which normally act as a lubricant, then build up in the organs. Thick secretions allow repeated cycles of infection which lead to continuous inflammation, damage to the lungs, respiratory failure. Continuous inflammation makes non-immunogenic non-viral vectors ideal for gene therapy of CF, as viral vectors may lead to further inflammation.

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14
Q

What causes CF?

A
  • Mutations in the CF transmembrane conductance regulator (CFTR)
  • Mutations CFTR protein results in the lack of cilary clearance of mucusin
  • Bacteria not effectively cleared which leads to the damaged cycles of inflammation.
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15
Q

What is CF disease severity dependent on?

A

CFTR activity.
• CF severity depends on the amount of CFTR expression
• If

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16
Q

What are the physical barriers of gene delivery for CF?

A
  • Airway epithelium have evolved complex series of barriers to prevent penetration
  • Mucus
  • Glycocalyx, may bind vectors and prevent binding to cell receptors
  • Apical cell membrane devoid of viral and growth/tropic receptors.
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17
Q

How can the physical barriers of gene delivery for CF be overcome?

A
  • Remove mucus and glycocalyx barriers
  • Pretreat patient with mucolytic agents (Nacystelyn) and neuraminidase which
  • Breakdowns glycoproteins
  • Endosomal escape
  • Endosomal disrupting peptides are incorporated into lipoprotein complex
  • Endosomolytic transmembrane domain of diphtheria.

However, still poor transduction efficiencies for both non viral and viral vectors for the treatment of CF.

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18
Q

Describe retroviruses?

A

Enveloped viruses containing a single stranded RNA molecule as the genome. Genome is approx. 8500 bps.

5’ cap, packaging signal, gag (encodes structural proteins), pol (encodes enzymes) and env (glycoproteins) and 3’ Poly A tail.

Maximum size is 8500bps –gag and pol= 7.5-8Kbp.

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19
Q

What is currently the most widely utilised vector system?

A

Retroviruses.

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20
Q

What is the life-cycle of a retrovirus?

A
  1. Associates and binds with one of the multiple retrovirus receptors (can be tailored)
  2. Once infected and entered the viral RNA is released
  3. Reverse transcription forms ds DNA (occurs in the cytoplasm)
  4. Integrated into the host genome, anywhere, randomly (required to occur in the nuclear membrane so it has to cross the nuclear membrane when the nuclear membrane has broken down as it has no other method way to cross, only occurs in dividing cell population whilst the membrane is broken down)
  5. Once expressed through translation to give a new viral genomic RNA
  6. RNAs exported from the nucleus and translated in the cytoplasm
    Assembly of viron, budding and release.
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21
Q

What are the pros of retroviruses?

A
  • Transduce a wide range of cells
  • Integrate into genome, therefore giving long-term persistence
  • Express transgene at high levels
  • Relatively high titre virus.
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22
Q

What are the cons of retroviruses?

A
  • Limited packaging capability (single-gene delivery systems)
  • Integration is random, can lead to insertional mutagenesis
  • Only integrate in dividing cells
  • Production of replication competent viruses from packaging cell lines
  • Silencing of promoters results in lack of long term expression.
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23
Q

How are recombinant retroviruses produced?

A

In packaging cell lines:

  • Used to make virus based vectors
  • Aka. Helper cell lines
  • Express gag, pol and env in trans using molecular contacts.
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24
Q

What is the most commonly used retrovirus?

A

Murine leukaemia virus.

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25
Q

What are the safety issues associated with retroviruses?

A

Retrovirus-based vectors have to be produced by packaging cell lines, recombination can lead to the production of replication competent wt viruses. In vivo application of retroviral vectors is still problematic and requires significant improvements.

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26
Q

What further developments of retroviruses are required?

A

In vivo application of retroviruses results in their rapid inactivation by the classical pathway of the complement system. Human/ primate cells are required as packaging cell lines to produce vectors resistant to human serum. Vectors express decay acceleration factors, protects vectors from the complement mediates lysis. Modification of vector tropism:

  • Pseudotyping: a retrovirus can non-specifically incorporate heterologous cell surface proteins into their lipid envelope. Improves viral infectivity towards a desired cell type. Good to use is gibbon ape leukaemia virus glycoprotein
  • Chemical modification of virus binding properties. Virus particles incubated with lactose (few other examples). Allows the interaction with specific receptor on hepatocytes.
  • Providing tissue specific transgene expression: Ensures expression of transgene in only the desired cell type by using transcription control elements of tissue specific promoter. Must be small due to the limited packaging capability, also may have implications of virus titre.
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27
Q

What are the types of retroviral gene therapy?

A

In vivo: make a retrovirus in the lab and directly inject

Ex vivo: better and more effective but more expensive for wide applications, take a patient and isolate the cells which need to be infected, grown in a laboratory, infect with the retrovirus and the put back into the patient. More specific and persoonalised. Commercially less viable.

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28
Q

How can retroviral gene therapy be clinically applied?

A

Severe combined immunodeficiency (SCID) syndrome, brain tumour therapy.

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29
Q

Describe SCID?

A
  • No immune system
  • Fetal disorder characterised by an early block in T and Natural killer cell differentiation
  • Near complete failure
  • Eg. Rhys Evans: First proof that gene therapy can cure a life-threatening disease
  • Extended clinical trials
  • In 2002 someone in the first trial was found to be developing T cell leukaemia-like condition, this was due to the retrovirus base vector integrated upstream of the LMO-2 oncogene, overexpression and up regulation
  • More patients in the SCID trial developed leukaemia, as the viral integration was in the LMO-2 locus but it should be random so why was it targeting this locus?
  • Still unknown.
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30
Q

Describe how retroviral gene therapy can be used for brain tumour therapy?

A

Neoplastic cells are the only replicatin population of cell lines in brain.
As retroviral vectors can only infect, integrate and therefore express therepautic gene in dividing cells, it’s a specific targeted therapy. Purified vector are only injected into the tumour mass to express a cytotoxic suicide gene. Expression of these suicide genes (HSV-TK, cytosine deaminase). Action of the pro-drug acyclovir (a guanosine analogue), phosphorylated and incorporated into the cellular DNA and prevents elongation leading to cell death.

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31
Q

How do you overcome the problems of insertional mutagenesis in retroviral gene delivery?

A

Site specific recombinases:

  • Encoded by phage DNA to integration of into bacterial chromosomes
  • Enzymes ussualy recognise relatively short (-30-300BP) DNA sequences and mediates precise recombination between them.

Intgrase from phage PhiC31:
- Integrase recognises two different sequences, the phage attP and the bacterial host attB.

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32
Q

What are lentiviruses?

A

Complex retroviruses.

  • Include HIV, SIV, FIV
  • Infect non-dividing, terminally differentiated cells
  • Still randomly integrate into genome
  • Still have gag, pol and env with additional accessory proteins
  • Develop them exactly the same by expressing gag, pol and env and then the accessory proteins included are integrated into the genome.
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33
Q

How are lentivirus different to retroviruses?

A

Can effect non-dividing cells, as they don’t wait for the nuclear membrane to break they can traverse the nuclear pore to integrate into the host genome, and terminally differentiated cells.

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34
Q

What are the safety concerns of lentiviruses?

A

Obvious safety concerns with using HIV-1 as a vector
• HIV-2 developed as less pathogenic, safer to manipulate during design and production
• Vector biosafety can be investigated in primates as susceptible to HIV-2 infection
• Other animal lentiviruses being developed: SIV, FIV and EAIV. Pseudotyped with VSV-G and have been shown to infect a range of human cells.

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35
Q

What promoting results have come from using lentiviruses?

A

Neurological disorders:

  • No toxic effect but extremely high infectivity in the brain
  • Can be delivered in vivo via a stereotactic injection
  • VSVG-pseudotypes vectors exclusively infect neurons
  • Promising results for Parkinson’s disease

Multipotent haematopoietic stem cells:

  • Immune deficiencies
  • Lysosomal storage diseases
  • Haemoglobinopathies
  • Cancer.
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36
Q

Describe adeno-asscoated viruses (AAV)?

A

Small non-enveloped ssDNA viruses. Viral particle is an icosahedron of about 20 nm in diameter. Viral genome is approximately 4700 bps (4.7kbp), very small capacity for a unique and specific treatment. Belong to the family of Parvoviridae. Classified in the genus Dependovirus. Propagation is dependent on the co-infection of an unrelated virus (adeno or herpesviruses), for essential helper functions.

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37
Q

Describe AAV life-cycle?

A

2: Latent or productive infection.
Allows infection on a wide variety of cells.
1. AAV binds and enters a host cell via the primary receptor, heparin sulphate.
2. Virus particle then migrates to nucleus un-coats and releases its DNA
• In a latent infection, the DNA remains as a free episome or integrates in the host chromosome
• If co-infection with helper virus, rep and cap activated leading to virus production
• Similarly, upon superinfection with a helper virus the latent provirus is activated.

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38
Q

Describe the 5 gene products that have essential functions for a fully permissive production of AAV?

A

The helper functions:
• E1A – trans-activator of adenovirus gene expression and also activates AAV gene expression
• E1B and E4 – stabilise and facilitate the transportation of AAVs mRNAs and promote host cell to entry S phase (DNA synthesis phase of cell cycle)
• E2A – essential for efficient AAV RNA splicing and translation
• VA RNAs – stimulates efficient translation of AAV mRNAs.

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39
Q

How is AVV integrated into the host chromosome?

A

Via its inverted terminal repeats at either end of the genome. AAV DNA integrates in a site-specific manner. The cellular junction is on Chr 19 (19q13.3-qtr). This region contains a sequence with homology to consensus Rep-binding site in the AAV ITR. Rep 78 and 68 proteins are essentially required for the site specific integration, if removed then they can insert randomly which could cause insertional mutagenesis.

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40
Q

What are the pros of AVV?

A
  • Non pathogenic and non replicating virus
  • Not toxic to host cells as all genes can be removed, therefore no viral gene expression in host cells
  • Transduce a wide range of cells, dividing and non-dividing Integrate into genome, therefore giving long term persistence
  • Minimal immune response, non-pathogenic.
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41
Q

What are the cons of AVV?

A

• Very limited packaging capability (

42
Q

What must AAV vectors contain and why?

A

Rep.
AAV-based vectors without Rep genes caused abnormally high number of mice with HCCs in preclinical trials. AV genome integrating in chromosome 12. Disrupting several maternally and paternally imprinted genes, over expression of adjacent transcripts telomeric to the proviruses, including the Rian and Mirg genes, encode multiple snoRNA and microRNAs - implicated in cancer.

All AAV vectors now used in clinical trials must have Rep gene – implications for packaging size.

43
Q

How can AAV be improved?

A

Specific tropism, evading the immune response, enhancing the packaging capacity and making higher titre AAV vector preperations.

Once learnt extend.

44
Q

What is haemophilia?

A

X-linked recessive bleeding disorder. Haemophilia A and B are due to the lack of the clotting factors VIII and IX, respectively, critical components of the middle stages of the blood coagulation cascade.
Haemophiliacs endure prolonged bleeding following an injury or wound, and in severe cases spontaneous bleeding into muscles and joints.
Incidence of haemophilia is 20 cases per 100,000 males Haemophilia A accounts for about 85% of these cases. No difference in incidence between races and geographical areas.

45
Q

Why is haemophilia a good target for gene therapy?

A

The disease is well understood at basic science and clinical perspectives

  • known gene products involved
  • a therapeutic level of factor is about 2% or greater of normal levels to convert a patient from frequent spontaneous bleeds to a mildly affected person
  • excellent animal model based on haemophilic dogs.
46
Q

Discuss clotting factor VIII?

A

One of the largest human genes known, consists of 26 exons spanning 186 Kb, cDNA of 9Kb. Adenoviruses and herpes viruses could package this amount of heterologous DNA but there is no way this can be treaed by a AAV as it is far too large.

47
Q

Discuss clotting factor IX?

A

Gene consists of 8 exons spanning 34 Kb, cDNA of 1.25 Kb. Suitable for AAV-based gene therapy. You would only have to increase factor by 2% for a dramatic effect for the patient.

48
Q

How has canine factor IX been used to assess gene therapy possibility of haemophilia?

A

AAV-2 vector expressing cFIX under the control of a CMV promoter provided via an intramuscular injection in a factor IX deficient dog gave rise to reduced blood clotting time, a circulating plasma concentration of IX which was sustained for 3 years.

49
Q

What problems arose when looking at gene therapy treatments with canine factor IX?

A

AAV-2 neutralising antibody persist preventing re-administration and the issues begin again. Requires immunologically distinct AAVs for effective administration. Even other AAV-1, 3, 4, 5, 6 serotypes are being neutralised upon re-administration.

50
Q

How could the problems with canine factor IX be overcome?

A

Recently identified 2 novel AAV from rhesus monkeys: AAV-7 and AAV-8.
Both have a divergent cap proteins with respect to AAV-1-6. Chimeric vectors based on AAV-2 containing the cap gene of AAV-7 or AAV-8.
- In vivo analysis suggests that these chimeric vector behave similar to wild type vectors
- Serological data suggests they are NOT neutralised after immunisation with other serotypes.

51
Q

Why is the eye suitable for gene delivery?

A
  • Easily accessible allowing local application of therapeutic agents with reduced risk of systemic effects
  • Non-invasive procedures for the determination of ocular structures and functions
  • Lack of inflammation by AAV-based vectors allows the transduction of very sensitive cells.
52
Q

What ocular cell types can be transfected with AAV?

A

A wide variety, including; photoreceptors, retinal pigment epithelial cells and retinal ganglion cells (RGCs).

53
Q

Discuss the clinical trials for retinal degeneration?

A

Leber congenital amaurosis (LCA) is a form of retinitis pigmentosa characterized by increasing loss of vision from birth into infancy – particularly in dim light
• In 2008, three independent research groups reported that patients with Leber’s Congenital Amaurosis had been successfully treated using AAV based vectors
• In all three studies, an AAV vector was used to deliver a functional copy of the RPE65 gene, which restored vision in children and adults suffering from LCA.

54
Q

Discuss the currently available AAV-based gene therapies?

A

UniQure : Glybera – First EU Approved Gene Therapy application. Glybera : AAV vector expressing human LPL gene under control of tissue-specific promoter, allows expression in muscle cells. • Restores the LPL enzyme activity required to enable the processing, or clearance, of fat-carrying particles formed in the intestine after a fat-containing meal.
In October 2012, the European Commission granted marketing authorization for Glybera® under exceptional circumstances as a treatment for adult patients diagnosed with familial lipoprotein lipase deficiency (LPLD) confirmed by genetic testing, and suffering from severe or multiple pancreatitis attacks despite dietary fat restrictions.

EXPENSIVE- £1million a shot.

55
Q

What question have recently arose about AAVs?

A

Safety concerns regarding AAV without Rep gene.

56
Q

Discuss adenoviruses?

A

Medium sized DNA viruses isolated from avian and mammalian species.

Human adenovirus consist of over 50 serotypes:

  • Infect and replicate a wide range of organs; resprittory tract, eye, bladder, GI tract abd liver
  • Most serotypes cause respiratory infections

Icosahedral capsid consisting of 3 main proteins; hexons, fibre, penton base and the minor cement proteins.

Genome is linear ds DNA, wrapped in proteins VII and Mu.

57
Q

Discuss the life-cycle of adenoviruses?

A
  1. Absorption of virus to target cell receptors via high affinity binding of the knob portion of the fibre to primary coxsachie/adenovirus receptor
  2. Internalisation of the virus by receptor mediated endocytosis is via the interaction of the penton with secondary receptors, interns alphabeta3 and alphavbeta5
  3. Virion escapes from the endosome through the lysis of the endosomal membrane
  4. Virion translocates to the nuclear membrane via the microtubules
  5. Viral genome is then released into the nucleus
  6. Remains in nucleus as non-integrating episome- different to two previously, no integration remains as a non-integrated linear episome.
58
Q

How are genes expressed in adenoviruses?

A

Harder to use as it needs more engineering due to size.
Two phase event, early and late
- Before and after DNA replication

Initially, transcription and complex splicing events produces 4 early cassettes, E1, E2, E3, E4 (E1 divided into E1A and E1B)
DNA replication
followed by late transcription, with 5 cassettes resulting from a series of complex splicing events (produced from the major late promoter)
- Late proteins encode structural components of the virus.

59
Q

What are the pros of adenoviruses?

A
  • Transduce a wide range of cells (expressed receptor on many cells)
  • Infection of both dividing and non-dividing cells (ubiquitous receptor)- no cancer cells
  • Relatively easy to produce high titre virus stocks
  • Third generation vectors can incorporate large amounts of DNA Episomally maintained – no integration problems
  • No insertional mutagenesis.
60
Q

What are the cons of adenoviruses?

A
  • Immune response (CTL and humoral)
  • Lack of persistence of viral episome in dividing cell population
  • Transient expression of therapeutic gene
  • Lack of long-term persistence within that episomes, only useful for short, sharp mechanisms (2-3 weeks).
61
Q

Why do adenoviruses require to be disabled before use as a vector?

A

As they are human pathogens.

62
Q

How are adenoviruses prepared to be used as vectors?

A
  • First generation: deletions in the E1 gene (major transcriptional activator gene).
    Problems:
    • Allow the incorporation of 6.5 kb of foreign DNA
    • Inhibited expression of E1 therefore in theory should not get any replication of virus DNA or production of capsid proteins. However, in vivo trials showed that transgene expression was transient and depressed.

Second generation: deletion in the E1, E2 and E3 genes
• With deletions in E2 these vectors have no capacity to replicate virus DNA or generate replication competent viruses (RCAs)
• RCAs minimised by expressing E1, E2 and E3 on different plasmids. However, long term transgene expression was not evident
• Still host immune response to virus proteins, especially on repeat administrations

Third generation:
• Much effort has been put into removing as many viral genes from the vector as possible
• The gutless vectors therefore offer the “ultimate prize”
• These vectors contain only the ITRs and a packaging signal
• These can therefore be filled with 36,000 bp of any DNA you want, if therapeutic genome doesn’t add up to this add stuffer.

More detail when learnt.

63
Q

What is an adenoviruses primary receptor?

A

CAR, which is widely distributed.

64
Q

How can specific targeting of CAR with adenoviruses be performed?

A
  • Retarget ad using bispecific conjugates
  • Usually knob domain of fiber interacts with CAR
  • Bispecific conjugate ablates normally binding, by preventing binding to CAR, and introduces a novel tropism, by directing vector to another receptor
  • Basic fibroblast growth factor (FGF2) has shown to enhance infectivity of vascular endothelial and smooth muscle cells
  • Pseudotyping of fibre protein.
65
Q

What do adenoviruses have potential to treat?

A

Cancer.

66
Q

Discuss the Jesse Gelsinger case?

A

First patient that died in a clinical trial for gene therapy, had ornithine transcarbamylase deficient. Genetic defect -> deficiency of OTC activity -> accumulation of ammonia ->ammonia is a neurotoxin affecting the central nervous system (CNS).

Trial:
Cohort 18 patients (as rare) all with partial OTC activity were given increased does of 2nd generation (E1/E4 deleted) adenovirus vector

Jesse received 6x1013 particles expressing OTC via intra-hepatic (hepatic artery direct to the liver, first time ever done) administration. Within 2 hours of administration the patient experienced severe complications and died 2 days later.

Adenovirus produced a massive innate immune response which caused multiple organs failure and Jesse died adult respiratory distress syndrome. Problems attributed to the systemic administration, direct into the liver, and now adenovirus is only injected directly into the tissue of interest.

Major errors:
1. Jesse was not eligible for the trial; liver function was abnormal (after a viral infection) The trial changed their criteria (without FDA approval) so Jesse could be included
2. Patients were not informed of the pre-clinical data. A number of primates had died after receiving similar treatment (albeit with a higher dose)
3. Other patients had experienced Grade III toxicity reactions at a lower dose, therefore the trial should have stopped there and then
4. Findings were not disclosed due to pressures of commercialisation of this technology
James Wilson and colleagues who ran the trial are banned from conducting trials using human subjects.

67
Q

How could adenoviruses be used to treat cancer?

A

50% of tumours having loss of p53 function, involved the induction of cell death pathways. Can you target this difference of p53 status in virus so that it can replicate in the p53 mutated cancer cell but is inhibited in WT p53 normal cells, ie: selectively replicate and lyse tumour cells?

In cancer therapy you want to develop a virus that can identify between normal and cancer cell. In a normal cell you don’t want the virus to replicate but in a cancer cell you want replication and destruction. This could then destroy tumours, and leave the healthy cell alone without damage. The treatment has to be specific. The genetic differences between the cancer cell and a normal cell can be exploited to create this.

More detail available.

68
Q

What are the subfamilies of Herpesvirus?

A

Gammaherpesvirinae: EBV, MHV68, KSHV, HVS

Alphaherpesvirina: HSV, VZV

Betaherpesvirinae: HHV7, HHV6, CMV

69
Q

Discuss the structure of the HPV?

A
  1. Core containing the large DNA genome (all different sizes) surrounded by a proteinaceous core.
  2. The complex isosahedral capsid surrounds the core.
  3. Outside the capsid is the tegument, a protein-filled region
  4. On the outside of the particle is the envelope, which contains numerous glycoproteins.
70
Q

Discuss HPV genome?

A
  • Genome is large consisting of 150,000 bp
  • HSV encodes for at least 80 gene products (40 of which unessential in vitro)
  • Composed of two covalently linked segments, long and short, both flanked at either end by inverted repeats
  • The repeats allow rearrangements of the unique regions and Herpesvirus genomes exist as a mixture of 4 isomers.
71
Q

What are the pros of HPV?

A
  • Large virus, ability to package and deliver large amounts of heterologous DNA
  • Genome remains in cell as non-integrated episome (no insertional mutagenesis, but no long-term persistence, except in the brain)
  • Infect a wide variety of dividing and non-dividing cells
  • HSV-1 can establish a long term latent infection in neutrons
  • High titre virus produced.
72
Q

What are the cons of HPV?

A
  • Complex genome, expression of genes induce cytotoxicity and immune responses
  • Direct injection of virus can lead to encephaltitis due to viral replication
  • Herpesvirus vectors need to be disabled by deleting essential genes for lytic virus replication
  • Disabled viruses require packaging cell lines, these have reduced virus titre.
73
Q

Discuss the life-cycle of HPV?

A

Latent:
• Reversible non productive infection of a neurone
• This can be a lifelong state
• Viral genome persists long term in the host cell
• To maintain latent state, the virus must evade host immune response, therefore limited virus gene expression
Lytic:
• Virus replicates in epithelial cells
• Replicate in the host cell nucleus
• Leads to cell destruction and infectious particle production.

74
Q

Discuss HPVs latent state?

A
  • Major site for HSV latency is the sensory neurons in the ganglion tissue
  1. After initial infection (oral/genital mucosal surface), the virus travels along the neuronal axon to the neuronal cell body
  2. Enters a quiescent state, genome remains in the nucleus non-integrated episome
  3. Virus is transcriptionally silent apart from LATs expression Role of LATs unknown, help keep genome silent and may prevent apoptosis
    - Allows the virus to avoid the host immune surveillance, remains in this state until signals reactivate virus
    - Signals poorly understood, but physical stresses such as illness and exposure to UV light increase reactivation.
75
Q

What is the point in using replication defective vectors?

A

They reduce cytotoxicity.
• Latent genomes are not toxic because lytic gene expression is silenced
• Upon initial infection or reactivation viral gene expression from the vector genome may be detrimental to host cell
• As viral gene expression is regulated in temporal cascade, deleting IE (immediate early) genes should stop viral gene expression, as no early genes or structural proteins will be expressed.

76
Q

What are first generation HPV-based vectors?

A

HSV-1 has 5 IE genes which are mainly involved in activating lytic gene expression and providing the right environment of viral synthesis. Deletions in multiple IE genes and therefore are required to be grown on complementing cell lines.
~80 kbp packaging capability and no toxic effects.

77
Q

What are second generation HPV-based vectors?

A

No HPV genes.
Amplicons are infectious bacterial plasmids, only contain origin of replication and packaging sequence from the Herpesvirus. Contain therapeutic gene/agent, a viral origin of DNA replication and DNA cleavage/packaging sequence. In the presence of “helper” functions, amplicons are replicated and packaged into infectious visions. Reduced toxicity and immunogenicity attributed to virus-encoded regulatory and structural proteins. Totally disabled vector. Helper virus free systems currently being developed where all functions being provided in trans. Hopefully, in the future it will work very very well.

78
Q

What is a major obstacle for all viral vectors?

A

Providing long-term therapeutic gene expression. - Problem is that heterologous promoters (IE CMV) get silenced after few weeks in vivo. Ideal promoters would be active in the latent state (must be active to maintain latency). In HSV latency the viral genome is transcriptionally silent apart from LATs expression. LATP can provide long term gene expression in mouse dorsal root ganglion.

79
Q

Briefly describe Parkinson’s disease?

A

Loss of the pigmented neurons in the compact zone of the substantia nigra, which contain the chemical dopamine as their neurotransmitter/neuromodulator.
Dopamine acts as a neurotransmitter, or chemical messenger, between cells in the brain exerts an important influence on many aspects of brain functioning, from control of movement to higher cognitive processes.

Hard to treat as you have to try and inhibit the destruction of the remaining dopamine expressing cells and the ones that are still functioning you want to up-regulate them to dry and produce the normal amount of dopamine.
Not due to a single gene defect: Multiple genes need to be delivered to help in the treatment.

80
Q

What genes can be expressed using gene therapy to aid in the treatment of Parkinson’s?

A

Genes to increase expression of dopamine:
• Tyrosine dehydroxylase: helps by converting the precursor tyrosine
• GTP-cyclohydrolase: regulates tetrahydrobiopterin (required for optimal TD activity)
Genes to halt or slow death of dopaminergic
• Glial-derived neurotrophioc factor: neurone survival factor.

In theory advantageous to express all 3.
Proving difficult but has potential.

81
Q

What is ICP34.5?

A

A herpesvirus protein, neurovirulence factor.

Can infect a wide variety of cells in the brain- brain tumour treatment.

More info if required.

82
Q

What happens when HPV is without ICP34.5?

A
  • Cannot replicate in neurons and does not induce any cytopathology
  • Cannot go latent in neurons (as deletion overlaps the LATs)
  • Can replicate in tumour - dividing cells with aberrant PKR signalling.
83
Q

What is inherent tumour selectivity?

A

Some RNA viruses grow exclusively in cells with defective antiviral responses. Sensitive to interferon so can’t replicate in normal cells but replicate in cancer with defective interferon response.

84
Q

Give some examples of viruses with inherent tumour selectivity?

A

Newcastle disease virus (NDV) (Avian paramyxovirus): similar to mumps, preferentially eplicates in cancer cells due to the defective interferon response.

Vesicular stomatitis virus (VSV).

Horse/cattle rhadovirus (rabies)- sensitive to ds RNA activated protein-kinase. However, in cancer cells RAS overexpressed which inhibits PKR.

Reovirus.

85
Q

Describe Newcastle Disease Virus?

A
  • ss RNA genome
  • 16,000 nucleotides (16kb)
  • Replication takes place in the cytoplasm, classic RNA replication in the cytoplasm
  • NDV is a contagious and fatal viral disease affecting most species of birds.
  • NDV is so virulent that many birds die without showing any clinical signs.
  • Asymptomatic, no effect in humans
  • A death rate of almost 100 percent can occur in unvaccinated poultry flocks.
86
Q

Why does NDV replicate only in tumour cells?

A

In cancer cells interferon response is weaker and delayed giving the virus time to replicate producing infectious particles which go on to infect other cells. NDV replicate x10,000 better in human cancer cells than normal cells. NDV infect elicits a very strong interferon response upon surface contact with of normal cell. A timeline which allows the replication of the disease in the cancer cells.

87
Q

Describe Reovirus?

A
  • Small, non-enveloped human virus
  • Asymptomatic- near none disease
  • Segmented RNA genome
  • Ubiquitously found in environment
  • Most humans infected, usually sub-clinical without symptoms
  • Reoviruses are naturally oncolytic
  • Seems to work on ras regulated tumours- may be onlymechanism but not certain definitely major
  • Replicate in transformed cells and cell derived from human carcinomas
  • Activation of Ras signalling renders cell susceptible to reovirus oncolysis
  • Activating mutations of Ras have been found in >30% of human cancers.
88
Q

What is ras?

A

A small G protein that active when bound to GTP and inactive upon hydrolysis of GTP to GDP.

89
Q

How is ras related to cancer?

A
  1. Epidermal growth factor receptor ligand binding leads to auto-phosphorylation of EGFR
  2. Leads to phosphorylation of effector molecules (Shc/Grb)
  3. Phos-Grb then recruits SOS, which leads to activation of Ras-GTP
  4. Ras-GTP promotes membrane recruitment / activation of 18 effectors
  5. Modulate activity of variety of signalling pathway
  6. Oncogenic transformation occurs when regulation is lost, through constitutive activation of Ras
  7. Leads to proliferation, anchorage dependent growth, enhanced survival.
90
Q

How does reovirus replicate to the point of cell lysis in ras-activated cells?

A

In normal cells reovirus replication is restricted as early viral transcripts activate dsRNA-activated protein kinase. In tumour cells, Ras counteracts this inhibition by activating a phosphatase that antagonises PKR effects.

91
Q

How are reoviruses thought to be able to treat metastasis?

A

During metastasic program, tumour cells select high RAS activity to go metastatic. Therefore, more favourable for reovirus replication.

92
Q

What are ribozymes?

A

Ribozymes are molecular scissors that cut RNA at a defined sequence. They are RNA molecules with a defined sequence and structure that recognises and hydrolyses a determined phosphate bond of a specific RNA sequence.

93
Q

How could ribozymes aid in the treatment of Ewing’s sarcoma?

A

Design ribozyme to specifically target EWS/FLI1 RNA, this is the gene which is mutated in the cancer due to a chromosomal breakage at a specific point and then rebinding. Hydrolyse the RNA and stop EWS/Fli1 protein being expressed, therefore restricting metastasis.

94
Q

How are viruses related to cancer?

A 
Widely implicated.
eg:
• Papillomavirus
• Herpesviruses (EBV, KSHV) – particularly in AIDs 
• Hepatitis C
• SV40
• Adenoviruses
• Human T cell leukaemia virus

If ways of inhibiting virus replication reduce possibility of virus related cancers.

95
Q

What is RNAi?

A

Long double-stranded RNAs used to silence the expression of target genes in a variety of organisms and cell types.

96
Q

Describe the RNAi pathway?

A
  1. The dsRNAs get processed into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) called Dicer (initiation step)
  2. The siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes
  3. The siRNA strands are then unwound to form activated RISCs
  4. The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step).
  5. Leads to effect gene silencing.
97
Q

How can siRNAs be used to prevent HIV?

A

siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by HIV-1 can specifically block Tat and Rev expression and function.

98
Q

Discuss cervical cancer and how this can be treated by siRNAs?

A

Over 90% of human cervical cancers are positive for papillomavirus and abnormal cell proliferation is driven by co-operative effects of viral E6 and E7 genes.

E6 and E7 genes prevent apoptosis of the cell by inhibiting the p53 mediated apoptosis pathway (p53 is a major apoptosis inducing agent).

If the viral proteins are destroyed their inhibitory effect of p53 is removed and p53 is up-regulated, causing apoptosis of the cells.

One caveat how are you going to deliver these to cervical cancer cells? Difficulties in delivery mechanism.

99
Q

What is the future of gene therapy?

A

Somatic: targets somatic cells or tissues

Germline: targets germ cells, gametes, zygotes, early stage embryos, testes or ovaries.

Germline gene therapy: transfer gene into germ cells in order to prevent a person being born with the disease

Target early stage embryos: In theory easier as only have to target single or clump of cells, and gene should be passed to all cells in the body.

100
Q

What concerns are there with the future of gene therapy?

A

Gene editing in embryos could have a bright future because it could eradicate devastating genetic diseases before a baby is born. However, others argue against. Crosses an ethical line because the genetic changes to embryos, known as germline modification, are heritable, they could have an unpredictable effect on future generations.
Researchers have also expressed concerns that any gene-editing research on human embryos could be a slippery slope towards unsafe or unethical uses of the technique (designer babies).

ATU 1 (4 decks)

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