Modern approach to human disease
Lecture 1
Intro to evidence-based medicine and SMA
The bench to clinical practice
1. Initial dug discovery animal model analysis
cell line does the drug work in the lab
animal-based models
Therapeutic does doses it works how much do I have to give for it to work and the lethal
doses what does start to kill animals.
Are they close far apart if too close won’t get to clinical trial if fair apart has more
chance?
Side effects- modification of the genome
Renal effects
Looks at if the drug does what it is supposed to do
2. Clinal Trials (discovery and validation)
Does the drug work in humans?
Increase in beneficial outcomes
Put into human and can repeat those outcomes in multiple trials remove bias
NICE review
Controlled by a regulatory body of meta-analysis from multiple reviews
Compare drug offering to current therapies to show improvements in clinical management.
Reviews basic science to approve clinical trials NICE
Reviews trail data NSC
3.Clinical practice
Evidence-Based Medicine
Drug A: 25% mortality within 20 months
Drug B: 25% mortality within 80 months
Drug A has left beneficial effects than B, but does this mean that the drug is better
Evidence-based is when you to in with side effects long impacts collation of multiple pieces of
evidence
What is the evidence?
Authority - It is a clinical trial-based analysis gone through vigour’s peer review (clinical trial)
Anecdote - Less weight than authority evidence case studies no statistics more bias (case study)
Law - Legal requirement to give drug A instead of B this can be due to secondary complications
associated with drug B
Evidence Hierarchy
, 3. Systemic reviews – meta-analysis least biased
4. Randomized controlled trials
5. Non-randomised controlled trails
6. Case series
7. Case reports and expert anecdotes
8. None
9. Common knowledge, authority most biased
Basic Science Spinraza
SMA is the first FDA approved treatment for spinal muscular atrophy
Spinal Muscular Atrophy
Aetiology
2nd most common genetic cause of infant mortality
Carrier freq is 1 in 50
Incidence of 1 in 10,000 births
Pathophysiology and cell Biology
Caused by loss of anterior horn cells alpha motor neurons
Causes progressive atrophy of skeletal muscle
Prognosis is dependent on respiratory involvement -more involvement poorer prognosis
Genetics
Caused by a mutation in SMN which is Autosomal recessive
SMN gene is located at Chr 5q13 which is an unstable part of the genome
Mutations identified point mutations, frameshift mutations and large-scale deletions
Types of SMA
Type 1 SMA severe form
Onset:4-6 months floppy babies
Characteristics: high intercostal involvement
Prognosis: Poor (death usually occurs between 2-4 years)
SMN2 X2
Type 11 SMA intermediate
Onset: 1-2 years
Characteristics: intermediate interictal muscles.
Prognosis: Poor (death usually occurs between 8-16 years)
SMN2 x4
Type 111 SMA mild form
Onset: 2+ years
Characteristics: low intercostal involvement
Prognosis: good (normal life span)
SMN2 X6
,SMN gene
SMA is caused by a mutation in the SMA gene
SMN has 2 copies SMN1 and SMN2
SMN1 = the disease determining gene deleted in all SMA patients
SMN2+ = disease-modifying gene controls the severity level of protein SMN makes.
SMN1 makes 100% full-length protein
SMN2 makes 10% full-length protein
SMA severity is 100% due to SMN protein levels
What controls the severity?
SMN 2 copy number because of the 10% produced from the SMN2 gene
If you look at an asymptomatic carrier or controlled individual who has no mutated copies of SMN 1
and two copies of SMN2 compared to an asymptomatic carrier who has one copy of SMN1 and
another one deleted there is a reduction in protein expressed because they lack this copy of SMN2.
If you compare that to a type 1 patient who has no copies of SMN1 and only two copies of SMN2.
Both of which are only producing about 10% of protein there is a significant drop in SMN protein
levels between a normal control individual and a type 1 patient. This is what causes the severe
phenotype.
If you compare that to type 2 where there is no copy of SMN1 and 2 copies of SMN2 per
chromosome. There is a high level of protein expressed compared with the type one which is why
the severity is reduced but a significant low level compared to the normal level which is why you still
see disease.
If you compare that to a type 3 patient where there are no copies of SMN1 3 copies of SMN2 per
chromosome. There is 3 times as much protein than type 1.
Therapeutic Strategy
All patients have one copy of SMN2
RNA splicing modification small compounds that change SMN2 RNA splicing - Spinraza
RNA splicing
The splice sites are the cis elements splicing factors are the trans elements
5’ splice site GU which is the start of an intron
Branch point
Polypyrimidine tract
3’ splice site end of the intron
These dictate an intron
The u1 binds a recognises the 5’ splice this what dictates that this is the start of an intron. The
polypyrimidine tract and the 3’ splice site is recognised by U2AF. Both of these binding forms the
complex E. This is the rate limiting factor for the complex A formation. Once U2AF is bound it can
facilitate the binding of U2 onto the branch point. The formation of this is what controls the majority
, of alternative splicing events. If you can block that you can trigger an alternative splicing event if you
stimulate it, you can trigger correct splicing of the intron and the exon.
Alternative Splicing Controlled by Exon SPLICE sites and wPPTS
Strong PPT prevents alternative splicing events because PPT binds strongly to UTAF
Strong PPT is made up of 10-15 pyrimidine residues made up of U and C at the RNA level
Weak PPT is made up of 5-10 pyrimidine residues and 5-10 purine – has a lower affinity for
UTAF which causes alternative splicing
Wppt Promotes alternative splicing controlled by downstream cis elements, Exon splicing enhancers
and Silencers
SMN1 and SMN2
1. In SMN1 and 2 there is a Weak PPT between exon 6 and exon 7 so in intron 6. Which means
UTAF binds weakly so U2 loads weakly.
2. To overcome this in SMN1 there is an exon splicing enhancer in exon 7 a CA rich motif which
starts with the C at position 6 of exon 7. This exon splicing enhancer binds to the splicing
factor ASF/SF2.
3. Anchors U2AF onto the weak PPT
4. Anchors U2 onto branch point allows the formation of the correct complex on the intron
5. Exon 6 is correctly spliced to 7 as U1 can then bind to exon 7 U2
6. Same complex formed on intron 7 so 7 is spliced to 8
So, you get correct Splicing of the c-terminus of SMN1 RNA which means 100% because of the exon
splicing enhancer in Exon 7 you get full length inclusion because 7 is spliced correctly in SMN1. This
is why SMN1 produces 100% full length protein
SMN2:
1. The C at position 6 has a snip (silent nucleotide polymorphism) converted to a U RNA level.
2. The U disrupts the splicing enhancer and creates a splicing silencer
3. ASF/SF2 does not bind to exon 7 but hnRNPA1 which blocks the polypyrimidine tract and the
branch point
4. No U2AF binding or U2 which means exon 7 is not recognised as an exon so it is alternatively
spliced.
5. U1 looks for the nearest U2 which is in intron 7 recognises exon 6 and 8 thinks exon 7 is part
of an intron
6. Which means you get an alternative splicing event which splices 6 to 8 remove exon 7 from
RNA forming the product delta 7 non-functional
7. Intron 7 Intronic splicing silencer prevents U1 from binding here which means 7 cannot be
spliced to 8 U2 doesn’t bind so 6 can’t be spliced to 7
Doesn’t happen all the time
90% of the time product delta7 6-8
10% Full length SMN 6-7-8 processed correctly
Antisense Oligonucleotides
CIS elements - complementary RNA sequences that bind splicing factors (trans elements)
Lecture 1
Intro to evidence-based medicine and SMA
The bench to clinical practice
1. Initial dug discovery animal model analysis
cell line does the drug work in the lab
animal-based models
Therapeutic does doses it works how much do I have to give for it to work and the lethal
doses what does start to kill animals.
Are they close far apart if too close won’t get to clinical trial if fair apart has more
chance?
Side effects- modification of the genome
Renal effects
Looks at if the drug does what it is supposed to do
2. Clinal Trials (discovery and validation)
Does the drug work in humans?
Increase in beneficial outcomes
Put into human and can repeat those outcomes in multiple trials remove bias
NICE review
Controlled by a regulatory body of meta-analysis from multiple reviews
Compare drug offering to current therapies to show improvements in clinical management.
Reviews basic science to approve clinical trials NICE
Reviews trail data NSC
3.Clinical practice
Evidence-Based Medicine
Drug A: 25% mortality within 20 months
Drug B: 25% mortality within 80 months
Drug A has left beneficial effects than B, but does this mean that the drug is better
Evidence-based is when you to in with side effects long impacts collation of multiple pieces of
evidence
What is the evidence?
Authority - It is a clinical trial-based analysis gone through vigour’s peer review (clinical trial)
Anecdote - Less weight than authority evidence case studies no statistics more bias (case study)
Law - Legal requirement to give drug A instead of B this can be due to secondary complications
associated with drug B
Evidence Hierarchy
, 3. Systemic reviews – meta-analysis least biased
4. Randomized controlled trials
5. Non-randomised controlled trails
6. Case series
7. Case reports and expert anecdotes
8. None
9. Common knowledge, authority most biased
Basic Science Spinraza
SMA is the first FDA approved treatment for spinal muscular atrophy
Spinal Muscular Atrophy
Aetiology
2nd most common genetic cause of infant mortality
Carrier freq is 1 in 50
Incidence of 1 in 10,000 births
Pathophysiology and cell Biology
Caused by loss of anterior horn cells alpha motor neurons
Causes progressive atrophy of skeletal muscle
Prognosis is dependent on respiratory involvement -more involvement poorer prognosis
Genetics
Caused by a mutation in SMN which is Autosomal recessive
SMN gene is located at Chr 5q13 which is an unstable part of the genome
Mutations identified point mutations, frameshift mutations and large-scale deletions
Types of SMA
Type 1 SMA severe form
Onset:4-6 months floppy babies
Characteristics: high intercostal involvement
Prognosis: Poor (death usually occurs between 2-4 years)
SMN2 X2
Type 11 SMA intermediate
Onset: 1-2 years
Characteristics: intermediate interictal muscles.
Prognosis: Poor (death usually occurs between 8-16 years)
SMN2 x4
Type 111 SMA mild form
Onset: 2+ years
Characteristics: low intercostal involvement
Prognosis: good (normal life span)
SMN2 X6
,SMN gene
SMA is caused by a mutation in the SMA gene
SMN has 2 copies SMN1 and SMN2
SMN1 = the disease determining gene deleted in all SMA patients
SMN2+ = disease-modifying gene controls the severity level of protein SMN makes.
SMN1 makes 100% full-length protein
SMN2 makes 10% full-length protein
SMA severity is 100% due to SMN protein levels
What controls the severity?
SMN 2 copy number because of the 10% produced from the SMN2 gene
If you look at an asymptomatic carrier or controlled individual who has no mutated copies of SMN 1
and two copies of SMN2 compared to an asymptomatic carrier who has one copy of SMN1 and
another one deleted there is a reduction in protein expressed because they lack this copy of SMN2.
If you compare that to a type 1 patient who has no copies of SMN1 and only two copies of SMN2.
Both of which are only producing about 10% of protein there is a significant drop in SMN protein
levels between a normal control individual and a type 1 patient. This is what causes the severe
phenotype.
If you compare that to type 2 where there is no copy of SMN1 and 2 copies of SMN2 per
chromosome. There is a high level of protein expressed compared with the type one which is why
the severity is reduced but a significant low level compared to the normal level which is why you still
see disease.
If you compare that to a type 3 patient where there are no copies of SMN1 3 copies of SMN2 per
chromosome. There is 3 times as much protein than type 1.
Therapeutic Strategy
All patients have one copy of SMN2
RNA splicing modification small compounds that change SMN2 RNA splicing - Spinraza
RNA splicing
The splice sites are the cis elements splicing factors are the trans elements
5’ splice site GU which is the start of an intron
Branch point
Polypyrimidine tract
3’ splice site end of the intron
These dictate an intron
The u1 binds a recognises the 5’ splice this what dictates that this is the start of an intron. The
polypyrimidine tract and the 3’ splice site is recognised by U2AF. Both of these binding forms the
complex E. This is the rate limiting factor for the complex A formation. Once U2AF is bound it can
facilitate the binding of U2 onto the branch point. The formation of this is what controls the majority
, of alternative splicing events. If you can block that you can trigger an alternative splicing event if you
stimulate it, you can trigger correct splicing of the intron and the exon.
Alternative Splicing Controlled by Exon SPLICE sites and wPPTS
Strong PPT prevents alternative splicing events because PPT binds strongly to UTAF
Strong PPT is made up of 10-15 pyrimidine residues made up of U and C at the RNA level
Weak PPT is made up of 5-10 pyrimidine residues and 5-10 purine – has a lower affinity for
UTAF which causes alternative splicing
Wppt Promotes alternative splicing controlled by downstream cis elements, Exon splicing enhancers
and Silencers
SMN1 and SMN2
1. In SMN1 and 2 there is a Weak PPT between exon 6 and exon 7 so in intron 6. Which means
UTAF binds weakly so U2 loads weakly.
2. To overcome this in SMN1 there is an exon splicing enhancer in exon 7 a CA rich motif which
starts with the C at position 6 of exon 7. This exon splicing enhancer binds to the splicing
factor ASF/SF2.
3. Anchors U2AF onto the weak PPT
4. Anchors U2 onto branch point allows the formation of the correct complex on the intron
5. Exon 6 is correctly spliced to 7 as U1 can then bind to exon 7 U2
6. Same complex formed on intron 7 so 7 is spliced to 8
So, you get correct Splicing of the c-terminus of SMN1 RNA which means 100% because of the exon
splicing enhancer in Exon 7 you get full length inclusion because 7 is spliced correctly in SMN1. This
is why SMN1 produces 100% full length protein
SMN2:
1. The C at position 6 has a snip (silent nucleotide polymorphism) converted to a U RNA level.
2. The U disrupts the splicing enhancer and creates a splicing silencer
3. ASF/SF2 does not bind to exon 7 but hnRNPA1 which blocks the polypyrimidine tract and the
branch point
4. No U2AF binding or U2 which means exon 7 is not recognised as an exon so it is alternatively
spliced.
5. U1 looks for the nearest U2 which is in intron 7 recognises exon 6 and 8 thinks exon 7 is part
of an intron
6. Which means you get an alternative splicing event which splices 6 to 8 remove exon 7 from
RNA forming the product delta 7 non-functional
7. Intron 7 Intronic splicing silencer prevents U1 from binding here which means 7 cannot be
spliced to 8 U2 doesn’t bind so 6 can’t be spliced to 7
Doesn’t happen all the time
90% of the time product delta7 6-8
10% Full length SMN 6-7-8 processed correctly
Antisense Oligonucleotides
CIS elements - complementary RNA sequences that bind splicing factors (trans elements)
