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GH Coarsework 2023 > Animal Models +gene Therapy+ Ethics > Flashcards

Animal Models +gene Therapy+ Ethics Flashcards

1
Q

When would an animal model be used?

A
  1. Animal models are similar to humans in terms of genetic, anatomy, physiology.
  2. Gene mapping
  3. Trial of drug safely Andy efficacy
  4. Trial drug dosage
  5. Animal models are often preferable for experimental disease research because of their unlimited supply and ease of manipulation.
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2
Q

Majority of genetic studies, especially those involving disease, have employed mice, why?

A
  1. Genomes very similar to humans (99% of coding region)
  2. Availability
  3. Ease of handling
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3
Q

What are synteny groups.

A

Groups or blocks of genes occurring together across species or the conditions of two or more genes being close on the same chromosome.

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4
Q

Shared synteny describes

A

preserved co-localisation of genes on chromosomes of related species.

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5
Q

Synteny can reflect important functional relationships between genes how?

A

If the same genes are remaining close over time, they may well function as a unit.

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6
Q

What are some animal model with spontaneous animal mutants.

A
  1. Nod mouse- diabetes, insulin-dependent
  2. Max mouse- duchenne muscular dystrophy
  3. Hemophiliac dog-haemophilia
  4. Watanabe heritable hyperlilidemic rabbit-Familial hypercholesterolemia
  5. Splotch mouse -waardenburg syndrome
  6. Nf damselfish-neurofibfomatosis type 1
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7
Q

Two main approaches to induce human disease states.

A
  1. Non-directed and mutation driven
  2. Directed and disease driven
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8
Q

Explain Non-directed and mutation driven induced human disease.

A
  1. The Nondirected, mutation-driven method uses radiation and chemicals to cause mutations.
  2. X-ray often cause large deletion and translocation mutations that involve multiple genes.
  3. Treatment with the chemical N-ethyl-N-nitrosourea is linked to mutation within single genes, usually point mutations.
  4. The animal are screened in an attempt to determine which ones show phenotypes that are similar to human disease.
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9
Q

Explain directed and mutation driven induced human disease.

A
  1. The disease is defined and the causative gene known
  2. The directed, disease-driven approach can employ one of a number of techniques, depending on the exact type of mutation involved in the disease under study.
  3. Gain-of-function or loss-of -function? The same kind of mutation must be introduced into the homologous mouse gene.
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10
Q

What type of mutation that needs to be introduced into a animal model for a loss of functional mutation.

A
  1. Gene targeting to abolish normal functioning
  2. Inactivation of normal gene
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11
Q

What type of mutation that needs to be introduced into a animal model for a gain of functional mutation.

A
  1. Expression of dominant muation genes.
  2. Introduction of mutant gene
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12
Q

Common techniques used in direct mutation creation:

A
  1. Transgenesis
  2. Single-gene knock-out
  3. Single-gene knock-ins
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13
Q

What is transgenesis

A
  1. Process of introducin an exogenous gene- a transgene- into a living organism.
  2. The transgene integrates the host genome, will be expressed and transmitted to offspring.
  3. Can be facilitated by liposomes, plasmid vectors, viral vectors, pro nuclear injection.
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14
Q

Characterise Transgenic mice

A

Typically transgenic mice are used to show how the over-expression of a gene product affects physiology, behaviour, etc.

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15
Q

Considerations for transgenic mice

A
  1. Integration of transgene is random
  2. It may disrupt the function of another gene.
  3. May integrate into a part of the genome where gene expression is suppressed.
  4. May integrate into a part of the genome under the control of a locus control region.
  5. Number of copies cannot bee controlled
  6. Over time, the transgene is frequently silenced.
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16
Q

Characterise knock-in mice

A
  1. Insertion of a protein coding cDNA sequence at a particular locus.
  2. Difference between knock-in technology and transgenic technology is that a knock-in involves a gene inserted into a specific locus, and is a “targeted” insertion.
  3. Usually used to study the function of the regulatory machinery that governs the expression of the natural gene being replaced.
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17
Q

Modelling chromosome disorders

A
  1. Difficult (very difficult) due to rearrangement of genetic material across species.
  2. Use knowledge of synteny groups
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18
Q

What are different approaches in gene therapy.

A
  1. A normal gene introduced to compensate for a nonfunctional gene.
  2. An abnormal gene replaced by a normal gene by homologous recombination
  3. Change the regulation of a gene
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19
Q

For any candidate disorder, you need to answer the following questions:

A
  1. Which genes are involved
    1.1 genetic cause must be known
    1.2 The best candidates for gene therapy are single-gene disorders
  2. which tissue are affected: can the tissue be accessed
  3. What do you know about the consequences of the mutation involved.
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20
Q

Hallmarks of successful gene delivery.

A
  1. Targeting the right cells
  2. Activating the gene
  3. Integrating the gene in cells the cells
  4. Avoiding harmful side effects
21
Q

Characterse Targeting the right cells

A
  1. You do not need to correct the gene in tissue that do not normally express that gene.
  2. How can you ensure that the gene gets into the correct cells?
22
Q

Characterise avoiding harmful side effects

A
  1. Anytime you introduce an unfamiliar biological substance into the body, there is a risk that it will be toxic or that the body will mount an immune response against it.
  2. If the body develops immunity against a specific gene delivery vehicle, this, and future rounds of the therapy, will be ineffective.
23
Q

Vectors should provide:

A
  1. High safety level with minimum side effects
  2. High efficiency and specificity
  3. Large packaging capacity
  4. Able to be regulated to control gene expression.
24
Q

Common viruses used:

A
  1. Adenovirus
  2. Retrovirus
  3. Adeno-associated virus
  4. Lentivirus
  5. Herpes simplex virus
25
Q

Characterise adenovirus

A
  1. Double stranded DNA genome, nonenveloped, no integration into host genome.
  2. Causes respiratory, intestinal, and eye infection in humans
26
Q

Advantages and disadvantages of adenoviruses

A
  1. Ad: Large packaging capacity-36kb
  2. Dis: non integrating thus not replicated, can induce immune response leading to removal of infected cells by macrophages.
27
Q

Characterise retrovirus

A
  1. ssRNA genome, envelope
  2. Packaging sizes of 8-10kb
  3. Widely used
  4. The retrovirus goes through reverse transcription
28
Q

Adv and dis of retrovirus

A

Adv: can lead to long term gene expression.
Disadvantage: inserts randomly into host genome so many cause insertional mutagenesis.

29
Q

Characterise adeno-associated viruses

A
  1. Packaging capacity up to 4.7Kb
  2. Integrates into the human chromosome 19 normally but viral integration genes removed when used as a vector (non-integrating)
  3. AAV-based gene therapy vectors form episomal concatamers in the host cell nucleus.
  4. Gene is always ‘on’ so the protein is always being expressed.
30
Q

Advantage of adeno-associated virus.

A

Causes no known disease in humans and doesn’t trigger patient immune response theoretically .

31
Q

Characterise herpes simplex virus.

A
  1. Ds DNA virus that infects neurons
  2. Like Ad and AAV, HSV is maintained episomally (non-integrated)
  3. Used for targeting neuronal tissue
  4. Still considered one of the most desirable vectors for cancer therapy.
32
Q

Are there non-viral options for gene therapy delivery.

A
  1. Liposomes, which are artificial lipid sphere with aqueous core can be used to carry therapeutic DNA through membrane.
  2. But it only works for certain tissue.
  3. Requires a lot of DNA
33
Q

Non viral vectors need to be….

A
  1. Be of biocompatible composition.
  2. Bind to cell surface and get internalised
  3. Traffic through the cytoplasm
  4. Deliver the gene to the nucleus
34
Q

Advantage of non viral vectors

A
  1. Less immunogenicity than viral vectors -safer and also allows for repeat administration.
  2. Increase genetic-carrying capacity- allowing for multiple gene delivery and more complicated treatment designs.
  3. Easier to mass-produce
35
Q

Disadvantage of non viral vectors

A

Far less efficient

36
Q

Characterise In vivo route of administration

A
  1. Systemic delivery- intravascular injection
  2. Muscular dystrophy - direct muscle injection
  3. Liver - direct hepatic artery injection
37
Q

Characterise ex vivo route of administration

A
  1. Especially when using retroviral vectors
  2. Cells are removed from patient, infected and then reintroduced.
38
Q

When to use integrating and non integrating vectors ?

A

Integrating vectors: especially useful in rapidly dividing cells like bone marrow
Nonintegrating: more useful in non dividing or slowly dividing cells eg. Muscle, liver, neurons

39
Q

Problem with gene therapy .

A
  1. The early integrating vectors carried the risk of insertional mutagenesis.
  2. For in vivo vector administration, the risk was related to deleterious immune response.
  3. Short lived: hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long term effects.
  4. Cost: expensive large-scale production of vectors
  5. Multi-gene disorders: trying to target all causative genes will be impossible. (Also environment)
  6. Expression pattern: How to get the gene to be expressed appropriately, protein produced where, when, and in the amount needed.
40
Q

What is a newer gene therapy approach used to counteract the harmful effects of genes within cells

A

Interfering RNA

41
Q

Characterise antisense RNA therapy.

A
  1. An antisense RNA designed to base pair with, and inhibit, a target RNA.
  2. SiRNA used to degrade RNA of particular sequence
  3. Abnormal protein wont be produced.
  4. Applicable for treating conditions where a gain of function mutation results in a novel, toxic protein.
42
Q

Strategy of gene therapy for recessive disease.

A

Need to provide the patient with a viable copy of gene for which they have no functional copies

43
Q

Strategy of gene therapy for dominant disease. (Gain of function)

A

Require knockdown of the mutant protein

44
Q

Strategy of gene therapy for dominant disease.(haploinsufficient)

A

Use of small activating RNAs (saRNAs) to modulate gene expression for such diseases.

45
Q

Methods for detecting off target at risk sequences

A
  1. Bioinformatics analysis- identify sequences with similarity to target sites.
  2. Identify sequences sites where cleavage has occurred
46
Q

Questions to ask when considering heritable genome editing.

A
  1. Have alternative reproductive technologies been considered?
    A- donor sperm or egg
    B- prenatal Testing
    C- preimplantation genetic testing
    D- mitochondrial Replacement therapy
  2. Is it known that the mutation causes the disease?
  3. Are there testing facilities .
47
Q

Factors that would affect the success of germline genome editing.

A
  1. Full understanding of the genetic basis for the disorders and the type of mutation.
  2. Quality and experience of the lab handling the processes
  3. Maternal age and response to ovum stimulation.
  4. In vitro fertilisation
  5. Editing approach
  6. Blastocyst available for biopsy and assessment
  7. Number and quality of embryos.
48
Q

Knowledge gaps in human genome editing

A
  1. Fully understanding the cause and effect of a mutation.
  2. The science of genome editing technologies (accuracy and safety)
  3. Effect on foetal development (Epigenetic and transcriptional effects)
  4. Effect on future generations
49
Q

Ethical issues to consider

A
  1. Who will set the standard for safety and efficacy.
  2. Who will indicate when the technology is advanced enough to be clinically implemented.
  3. How will it be decided which couples should have access to HHGE.
  4. Which disease will be prioritised.
  5. How will HHGE be regulated. Nationally and internationally
  6. Physiologically and psychological health need to be considered. Will long term follow-up be mandatory?

GH Coarsework 2023 (13 decks)

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