Mutation Specific Therapies & Pharmacogenomics

Question 1

What are antisense oligonucleotides?

Answer 1

Small ssDNA or ssRNA molecules that affect translation by binding mRNA and blocking translational machinery

Question 2

How do ASO’s enter cells?

Answer 2

Endocytosis so must be stable

Question 3

3 concerns r.e. ASOs

Answer 3

Delivery to target tissue

Repeat treatments required to sustain effect

Complete inhibition not possible if level of ASO decreased compared to mRNA levels

Question 4

3 mechanisms for ASO therapies

Answer 4

Reading frame correction

Enhanced splicing

Downregulation of transcription

Question 5

1 example of reading frame correction therapy

Answer 5

Eteplirsen (Exondys51) for DMD patients with deletions where exon 50 skipping will restore the reading frame (14%) e.g. 49-50

Question 6

Where is reading frame correction therapy for DMD approved?

Answer 6

US only

Refused approval in Europe as main studies showed minimal effect and no long term studies showing lasting benefits to pts

$300k per year

Question 7

Example of enhanced splicing ASO therapy

Answer 7

Nusinersen (Spinraza) in SMA

Question 8

How does Nusinersen work?

Answer 8

Blocks SMN2 ISS-N1 site in exon 7, impairing access to negative splicing factors, stabilises U1snRNP machinery and promotes exon 7 inclusion. Increases full length SMN protein expression

Question 9

Trial outcomes of Nusinersen

Answer 9

Original trial in 2017 showed outstanding response with cohorts meeting normal motor milestones and no ventilation support

Approved in 2019 for Types 1-3 SMA

Question 10

2 other SMA therapies and brief mechanisms

Answer 10

Risdiplam - small molecule oral drug (pyridazine derivative) that modifies splicing
81% alive wo ventilation after 12m
(25% in untreated cohorts)

Zolgensma - viral vector delivered SMN1 transgene and synthetic promoter

Question 11

2 examples of downregulation of transcription ASOs

Answer 11

IONIS-HTTRx (Tominersen) for HD

Baliforsen for DM1

Question 12

What is the mechanism for Baliforsen?

Answer 12

Targets DMPK for degradation (through RNA-DNA heteroduplexes) and frees MBNL1 protein for alternative splicing function

Question 13

How has delivery to muscle been improved for Baliforsen ?

Answer 13

Combine with antibody for transferrin receptor (highly expressed on muscle cells due to high iron levels)

Ab+ASO are endocytosed into cell and improves targeted delivery to muscle

Question 14

What is readthrough therapy?

Answer 14

Aminoglycoside antibiotics allow ribosomes to readthrough premature termination codons

Interact with aminoacyl tRNA site = altered rRNA conformation = decreased accuracy

Question 15

When is readthrough therapy most effective?

Answer 15

When low levels of functional protein can ameliorate disease phenotypes

Question 16

Example readthrough therapy

effect on protein?

Answer 16

Ataluren/Translarna for DMD pts with nonsense mutations (15%)

10-20% increase in dystrophin expression which protects against some contractional damage

Question 17

What do CFTR modulators promote?

2 examples

Answer 17

correct folding and transport

Ivacaftor, Lumacafter (together = Orkambi)

Question 18

What CF variants are targeted by potentiator therapy?

Answer 18

Class 3 - mutant CFTr cannot transport Cl- through channel (decreased gating efficiency)

e.g. G551D

Ivacaftor binds CFTR to induce non-conventional gating

Question 19

What CF variants are targeted by corrector therapy?

Answer 19

Class 2 - CFTR is incorrectly folded/processed in ER and targeted for degredation

e.g. Phe508del

Lumacaftor faciliates proper maturation of CFTR and delivery to membrane. increases Cl- transport by ~14% (10% required for clinical response)

Question 20

What do pharmacogenomic tests look for?

Answer 20

Likelihood of altered responsiveness to drugs

Question 21

2 types of genes looked at in pharmacogenomics

Answer 21

Enzymes that activate drugs. Variants = decreased activation and sensitivity

Enzymes that breakdown drugs. Variants = accumulation

Question 22

What is the clinical utility of pharmacogenomics

Answer 22

Improve patient outcomes through personalised drug therapies and decrease healthcare costs. Improved drug safety and efficiency.

ADRs cause 2000 deaths & cost NHS £2billion annually

Question 23

5-fluorouracil mechanism

Answer 23

Antimetabolite chemo that affects with DNA synthesis by blocking thymidine formation, affects rapidly dividing cells

Question 24

Enzyme involved in 5FU inactivation

Answer 24

Dihydropyrimidine dehydrogenase DPYD

Question 25

ADR associated with DPYD deficiency

Answer 25

Diarrhoea, vomiting, cardiac/CNS issues

Question 26

DPYD testing

Answer 26

4 variants (3 SNV and 1 haplotype of 3 var in cis)

DPYD level depends on hetz, homoz or comp hetz status

Question 27

Levels of DPYD that confers intermediate activity

Answer 27

1-1.5

reduce dose 50%

Question 28

What is clopidogrel used for?

Answer 28

Anticlotting drug that prevents blood clot formation by making platelets less likely to bind

Used in pts at risk of clotting events e.g. strokes

Question 29

enzyme that activates clopidogrel?

Answer 29

CYP2C19

Question 30

Significance of CYP2C19 variants?

Answer 30

Decreased activity = decreased conversion of clopidogrel to active form = increased risk of clotting events

Question 31

Clinical utility for testing CYP2C19 in stroke patients?

How to test?

Answer 31

Identified pts that require alternative clotting medications

2023 NICE draft guidelines recommend POCT test (more expensive but does not require sample transit to genetics lab and results quicker in acute settings)

Question 32

Example of a mitochondrial pharmacogenomic variant

Answer 32

m.1555A>G in MT-RNR1

1 in 500 have this variant

Question 33

What does m.1555A>G cause?

Answer 33

Predisposition to aminoglycoside induced ototoxity (hearing loss post antibiotic treatment - implications in neonatal sepsis)

Question 34

Example of POCT pharmacogenomics

Answer 34

Development of rapid POCT for m.1555A>G in Manchester study. Device is able to genotype in ~26 min and does not disrupt normal clinical practice i.e. no delay in lifesaving treatment

Question 35

What did the PREPARE study conclude?

Answer 35

Pharmacogenomic panel testing is feasible in diverse European healthcare systems