Emerging Treatments

Question 1

How do errors in metabolism lead to disease

Answer 1

Lacks enzyme
Can’t make product
Leads to excess substrate
Leads to alternative product which may cause disease

Question 2

Examples of metabolic error diseases

Answer 2

PKU ( Protein Ketone Urea)
MCAD Deficiency
Maple Syrup urine disease
Homocystinuria

Question 3

PKU ( phenylketonuria) cause

Answer 3

Caused by a lack of phenylalanine hydroxylase ( enzyme) and this leads to the creation of phenylketones which are neurotoxic and kills brain cells.

Question 4

Symptoms of untreated PKU

Answer 4

Major cognitive impairment 
Behavioural difficulties
Fairer skin , hair and eyes than siblings
Lack of melanin 
Recurrent vomiting

Question 5

Treatment of PKU

Answer 5

Treatment with low protein diet - tyrosine supplements

Question 6

Haemophilia symptoms

Answer 6

Uncontrolled bleeding 
Bleeding into joints
Excruciating pain 
Bleeding into brain 
Internal bleeding

Can be fatal if untreated

Question 7

Haemophilia treatment

Answer 7

Fresh Frozen Plasma

Question 8

Solutions to contaminated blood scandals for FFP

Answer 8

Heat treat products kills virus
Factor 8 can be cloned
Recombinant factor 7 treatment

Question 9

Pompe disease treatment

Answer 9

Injection of alpha glucosidase

Question 10

Lysol also storage disease

Answer 10

Injection recombinant alpha galactosidase A/ agalsisase beta ( faradenzyme)

Question 11

Do therapies targeting proteins cure the disease

Answer 11

No they are treatments not cures as they try to normalise the function of mutant proteins - they treat the condition not the symptoms so need to take these treatments for as long as you live

Question 12

What are pharmacological chaperones

Answer 12

Protein folding is complex sometime fails
System in endoplasmic reticulum degrades misfolded proteins
Some mutations prevent proteins folding properly > so they are subject degradation pathway
If folded correctly would be active
Chaperones can stabilise the shape of the protein so it folds properly and does not get degraded. (It’s acts as a competitor to the original defective enzyme)

Question 13

Example of a pharmacological chaperone

Answer 13

Fairy disease - deficiency of alpha galactosidase A
Build up of glonotriaosylceramide
Some mutations cause misfolding of proteins
Migalastat small molecule chaperone
Stabilises enzyme in correct shape

Question 14

Pharmacological modulators

Answer 14

Pharmacological modulators are commonly used drugs
Receptor agonists/antagonist
Ion channel activators/blockers
Can design one that has these effects on mutant receptor or channel
Bcl-abl Kinase inhibitors (Philadelphia chromosome)

Question 15

Cystic fibrosis

Answer 15

Defective chloride channel
Mutations (33) cause channel not to open
Design a drug which causes activation - Ivacaftor
Is mutation specific

Question 16

Example of combination therapy

Answer 16

Cystic fibrosis
Defective chloride channel + One mutation (f508del) misfolded, inactive channel
Treat with combination chaperone and activator (turns it on in situations when it wouldn’t be on)
Orkambi (Ivacaftor/lumacaftor) – NICE approved Oct ’19
Not cure but does improve lung function

Question 17

Diseases caused by stop codon read through

Answer 17

Some diseases are caused by non sense mutations where there is a premature stop codon
This prevents protein production

Question 18

Example of a drug which treats non sense mutation

Answer 18

Mutation causes release factors to bind prematurely and so there is a shorter protein formed. Aminoglycoside antibiotics bind to the ribosome and causes mistranslation . Drugs based on these prevent the release factors from binding and so a full length DNA is produced

Aminoglycoside Abs is an example as it binds to ribosome causing mistranslation so skip the nonsense in a loss of function

Question 19

Duchenne muscular dystrophy

Answer 19

DMD premature stop codon and Becker Muscular dystrophy has a missing section

If read through premature stop codon DMD-> BMD
Atalurenis an example
But it is non sense mutation specific and so doesn’t cure but reduces the symptoms

Question 20

In vivo

Answer 20

In living

Question 21

Ex vivo

Answer 21

Out of the living

Question 22

In vitro

Answer 22

In glass ( foetus)

Question 23

How to fix a recessive diseases by gene therapy

Answer 23

Recessive disease - rePlace defective gene

Dominant disease - delete defective gene

Question 24

Why is in vivo hard

Answer 24

Very difficult to achieve in practice
	Achieving specificity
	Getting therapy to right place
	Maintaining expression
Much easier to achieve in vitro than in vivo

In vivogene therapy means that therapy is administered directly the patient. The targeted cells remain in the body of the patient. Withex vivogene/cell therapy the targeted cells are removed from the patient and gene therapy is administered to the cellsin vitrobefore they are returned to the patient’s body.

Question 25

How does mitochondrially inherited disease therapy work

Answer 25

It is ex vivo / in vitro

Only effective therapy
Requires IVF
Take DNA from fertilised patient egg
Transfer to donor egg normal mitochondria

Question 26

Virus gene therapy

Answer 26

Can Engineer virus to carry therapeutic gene
Wide variety of virus used
	AAV
	Adenovirus
	Lentivirus – HIV 
	Vaccinia

Virus choice depends on target tissue : virus tropism has affinity of injecting certain viral phenotypes
Amount of DNA limited depends on virus

Question 27

In vitro gene therapy SCID

Answer 27

X linked
Can be treated with bone marrow transplant as it is caused by adenosine dealings deficiency

But not possible for all children (90% ADa -SCID) no match
Has own risks

Question 28

Process of treating in vitro gene therapy ADa -SCID

Answer 28

Strimevelis
Autologous transplant
Isolate patients Haemopoietic stem cells 
Isolate and expand CD34+ 
Transferred with ADA -lentivirus 
Grow transformed cells
Treat patient with busulfan (kills HSC) 
Reinfused transformed cells into patients

Question 29

In vivo therapy supplement

Answer 29

Lever congenital amaurosis type 2
Recessive disease caused by mutation REP65
Progressive blindness - loss of rentinal cells
Luxturna rAAV2 expressing RPE65
Not cure ; greatly improves vision
Patients need sufficient remaining cells

Question 30

Anti sense oligonucleotides

Answer 30

Shortnmodifies nucleic acid complementary to target
Modification prevents degredation allow entry to cell
Binds to target
Block translation
Can also alter splicing
Relatively cheap

Question 31

In vivo therapy knockdown

Answer 31

Useful for disease caused by gain of function
Inotersen -
Transthyretin- related hereditary amyloidosis
Mutation in transthyretin ( TTH)
TTH cannot form tetramers , forms aggregates
Mutation specific

Question 32

Transthyretin related hereditary amyloidosis

Answer 32

Symptoms between 20-40
Pain muscle weakness and this eventually leads to heart and brain problems
Insotersen binds to wildtype mRNA and stop production /slows down overall rate of production

Question 33

Exon skipping

Answer 33

During pre-RNA processing
Oligonucleotides cause exon to be skipped
Can be used to skip disease causing exon
To put RNA back in reading-frame

Useful in limited circumstances
Exons skipped mustn’t be vital otherwise worse damage
Generally only large proteins - small proteins will lose too much

Question 34

Exon skipping in duchenne muscular dystrophy

Answer 34

Duchenne muscular dystrophy – premature stop (nonsense)
Becker muscular dystrophy – missing section
If prevent incorporation mutant exon DMD → BMD
It skips an exon and moves it back into frame.

Question 35

Drug used to treat exon skipping in duchenne muscular dystrophy

Answer 35

Eteplirsen – oligonucleotide cause skipping exon 51

Will result in production partially active dystrophin

Question 36

Gene editing CRISPR- Cas 9

Answer 36

Bacterial system disable bacteriophages

Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR – derived from previous bacteriophage
CAS9- Crispr associated protein 9- Endonuclease
Recognises and Cleaves DNA CRISPR hybrids

Gene editing correct relatively small errors
Cannot correct large changes
Large deletions OR triplet expansion

May have off target effects
Not currently used in humans (1 exception)
Same problem as other methods
	Targeting
	Getting into cell