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BMS3031 > PRECISION MEDICINE AND THERAPEUTIC GENE EDITING > Flashcards

PRECISION MEDICINE AND THERAPEUTIC GENE EDITING Flashcards

1
Q

What is the difference between precision medicine and personalised medicine?

A
  • Precision medicine is using MEDICAL INTERVENTIONS to alter molecular mechanisms that cause disease or INFLUENCE a patients response to certain treatments
  • Personalised medicine is working out which medical treatments work best for each patient.
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2
Q

What is Moore’s law?

A
  • Long term trend in computer hardware that involves the doubling of computer power every 2 years
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3
Q

What is the general term for Anti Sense Olgio nucleotides?

A
  • Small pieces of DNA that change the way DNA is expressed

- Can be shortened to ASO

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4
Q

Does it matter if introns are correctly or incorrectly splieced?

A
  • YES because we need to get the right reading frame

- IF not correctly spliced, can lead to non-functional proteins

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5
Q

What is a phase 0 intron?

A
  • When there is NO CODON shared between exons after splicing
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6
Q

What is a phase 1 and 2 intron?

A
  • When after splicing out of introns, there is a codon shared between two exons, e.g. C and G
  • phase 2 intron where there are 2 nucleotides on the first exon that are spliced with 1 nucleotide on the third intron to make one codon
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7
Q

What can happen if exons are incorrectly spliced together?

A
  • proteins can be our of reading frame and thus non functional
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8
Q

What is an approach used to work around non-functional (out of reading frame) proteins?

A
  • Therapeutic approaches using antisense oligonucleotides (ASOs)
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9
Q

What is the specific definition of antisense oligonucleotides. ( ASOs)?

A
  • Short single stranded sequence of DNA or RNA designed to target specific RNAs to modify gene expression.
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10
Q

What are three ways that ASOs exert their effects and what does this depend on?

A
  1. RNA degradation.
  2. preventing protein translocation.
  3. modifying RNA splicing.
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11
Q

What are two diseases that ASOs are used to treat?

A
  1. Spinal Muscular Atrophy

2. DMD (Duchennes Muscular Dystrohpy)

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12
Q

What is SMA caused by and characterised by?

A

Loss-of-function mutations in SINGLE gene –>SMN1 (Survival Motor Neuron 1)
- Characterized by progressive degeneration of the spinal cord motor neurons.

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13
Q

Is there a second SMN gene and how much of the normal protein is produced?

A

Yes SMN2

- But only 10-20% of the normal protein is produced

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14
Q

How do the SMN1 and SMN2 genes differ in terms of the strucutre?

A
  • Differ in 5 bases
  • C–> T transition in exon 7 means that SMN2 favors SKIPPING of exon 7 during splicing
  • This means that majority of SMN2 products are UNSTABLE and thus truncated –> SMN delta7
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15
Q

Can SMA patients with no SMN1 still produce a functional SMN protein at sufficient levels for function?

A
  • NO –> levels are insufficient for normal function so this results in degeneration of the spinal cord motor neurons
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16
Q

How can ASOs be used to treat SMA (Spinal Muscular Atrophy)?

A
  • Can try and shift the machinery so it recognises exon 7
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17
Q

How is the drug ‘Spiranza’ used to treat SMA?

A
  • Shifting machinery to recognise exon 7
  • Blocking the 15 nucleotide sequence in intron 7 SMN2 (this is crucial for splicing regulation) –> ISS-N1 (intronic splicing silencer N1)
  • ISS-N1 blocking ASOs lead to the production of exon 7 and INCREASED EXPRESSION of SMN2
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18
Q

What is the target for Spiranza to allow for a fix of SMA?

A
  • ISS-N1
  • This is the intronic splicing silencer N1 that is imprtant in splicing regulation
  • If it is blocked, then exon 7 will be recognised and integrated to produce a full length SMA2
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19
Q

What is dystrophin?

A
  • Parts of a protein complex and anchors the cytoskeleton in muscle cell with the ECM (linker between Actin and ECM).
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20
Q

What happens if we don’t have dystrophin?

A

The muscles degrade.

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21
Q

Is the dystrophin gene small or large?

A

It is the largest gene known at 2.4 mega bases and has 79 exons.

22
Q

What are the two main domains that the dystrophin gene has?

A
  • Actin Binding domain and the dystro glycan Binding domain.
23
Q

What lies between the actin binding domain and dystro glycan binding domain in the dystrophin gene and is it important for function?

A
  • A linker sequence

- It is not important for function

24
Q

What happens to the exon/s in muscular dystrophy?

A
  • They are deleted

- this interferes with the rest of the gene being pieced together

25
Q

In muscular dystrophy can any of the exons be deleted?

A
  • YES
26
Q

In the muscular dysrophy EXAMPLE, which of the exons are deleted and what is the effect of this?

A
  • Exon 52 is deleted
  • This means that Exon 51 and 53 join–> BUT the ORF will be disrupted from a codon mismatch (sticky ends joining)
  • this means the dystrophin protein will be non functional thus disease
27
Q

How can ASOs help with Muscular Dystrophy?

A
  • They can SKIP the exon and put the protein BACK into reading frame so exon 53 is removed and the two non-sticky ends can join
  • Some of the aas will be lost but protein will still be functional
28
Q

In dmd what are the specifics for how the EXON skipped by ASOS?

A
  • ASOs mask the splice site.

- Means the machinery moves over that exon to the splice site in the next exon

29
Q

What is an example of an FDA approved drug that works via the mechanism of ASO skipping for DMD and what is the function?

A
  • Exondys 51 (Eteplirsen)

- Skips exon 51

30
Q

Is there a clinical benefit for Exondys 51 for dmd?

A
  • NO –> increase of 0.1% after 48 weeks

- 30mg per kg twice weekly

31
Q

With the exon skipping for Exondys 51 treatment for DMD, is there functional dystrophin produced?

A
  • Somewhat

- Shortened functional dystrophin produced but not as functional as the full length

32
Q

Is there any benefit for the other drug to treat DMD (Vyondys 53)?

A
  • NO
  • This is for patients with a mutation that can be helped by exon 53 skipping
  • FDA approval not originally given BUT granted on appeal
33
Q

What does gene therapy involve?

A

Viral mediated introduction of defective cells with the corrective gene
- for example ADA (Adenosine Deaminase) in SCID was corrected by the retroviral gene therapy but stopped in 2002 (because of off-target effects causing leukaemia).

34
Q

What do we choose to express viruses in now?

A
  • AAV (recombinant adeno-associated virus).
35
Q

Is AAV very immunogenic?

A

No

36
Q

What type of virus is AAV?

A
  • A single stranded DNA virus.

- Icosahedral capsid (protein shell).

37
Q

What happens to the Genome of AAV for Biotechnology?

A

It is gutted so the proteins can be placed in and delivered.

38
Q

Can a AAV transduce both dividing and non dividing cells?

A
  • Yesq
39
Q

Can AAV be integrated into the Human Genome?

A
  • No (very rarely)
40
Q

What is a limiting Factor with the use of AAV for biotechnology?

A

-The size of insertion as you can only put a certain amount of DNA in there.

41
Q

How does the drug Zolgensma work for the treatment of SMA?

A
  • It delivers the NORMAL copy of the gene encoding the SMN protein to patients
  • AAV has a transgene encoding the SMN protein (under the control of cytomegalovirus enhancer and chicken-beta actin hybrid promoter)
42
Q

Are there multiple injections with the SMA treatment for Zolgensma or single?

A
  • A SINGLE ONE!!
  • But this is very expensive—> 2.1 million per patient
  • throughout 2020 they are giving 100 doses for free via a lottery
43
Q

In terms of germline therapy for mitochondrial disease, how many of the mtDNA genes are non mendelian?

A

-35/37 genes

44
Q

Can mitochondrial disease change throughout oocytes?

A
  • YES
  • you can have someone with a high level of mutation, or a lower level of mutation that might get disease later on in life.
45
Q

What is an option for preventing mitochondrial disease?

A
  • Mitochondrial donation or mitochondrial replcaement therapy
46
Q

What are the two forms of mitochondrial donation?

A
  • Maternal Spindle Transfer (MST)

- Pronuclear transfer (PNT)

47
Q

What occurs in MST?

A
  • Mitotic spindle from a woman with bad mtDNA transferred to a site with nucleus removed and healthy mtDNA
  • Oocyte site then fertilised with women’s partners sperm
  • Creates an oocyte with HEALTHY mitochondria
  • Occurs before fertilisation
48
Q

Does the mitochondrial manipulation in Maternal Spindle Trasnfer occur before or after fertilization?

A
  • BEFORE
49
Q

What occurs in Pronuclear Transfer (PNT)?

A
  • Affected woman has oocyte fertilised by partners sperm
  • Zygote is created
  • Pronuclei removed and placed into another zygote created using donor oocyte and partners sperm (with their pronuclei removed)
  • Embryo developed and inserted into woman
  • Mitochondrial manipulation occurs AFTER FERTILIZATION
50
Q

In PNT does the Mitochonrial manipulation occur before or after fertilization?

A

-After

BMS3031 (6 decks)

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