Spinal Muscular Atrophy

Question 1

what is SMA

Answer 1

a multisystem disorder

• primarily/prefers to attack MNs since main signs are muscle wasting and weakness following degeneration and death of MNs
• includes a wide spectrum of NMDs with a variation in onset and severity

Question 2

SMA genealogy

Answer 2

• autosomal recessive - both parents need to be carriers for children to have it
• it’s the 2nd more common autosomal recessive genetic disorder after CF
• most common cause of infant death resulting from a genetic defect

Question 3

SMA classification

Answer 3

• diagnosis based usually on genetic testing of WBCs
• SMA patients present with muscle wasting and many other symptoms
• there are 5 types of SMA numbered 0-4 with zero being the most severe

Question 4

type 0 SMA

Answer 4

is the most severe
affects only a small percentage of people
- we don’t usually talk about it and consider type 1 SMA to be most severe

Question 5

what happens as SMA type number increases?

elaborate

Answer 5

the severity of the disorder decreases

• type 1 has earlier onset and is more severe than the higher number types
• as SMA type number increases, live expectancy increases too and onset is often later

Question 6

SMN2 copy number and disease severity relationship?

also go write out the table of SMN copy numbers for each SMN type

Answer 6

as SMN2 copy number increases, disease severity decreases

Question 7

do healthy individuals need SMN2?

Answer 7

no
we have SMN1
- in fact, we often have lower copy numbers of SMN2 but it’s irrelevant bc we have SMN1

Question 8

what’s the number 1 genetic modifier for SMA?

Answer 8

SMN2 copy number for SMA patients

- it determines the severity of the disease

Question 9

can someone be missing both SMN1 and SMN2?

Answer 9

nope, they would not survive

Question 10

SMN1 and SMN2 copy number healthy individuals?

Answer 10

2 of each, one on each 5q

Question 11

what are the % SMA pts with SMN2 copy numbers for:
SMA 1
SMA 2
SMA 3

Answer 11

SMA 1: 80% have 1 or 2 SMN2 copies
SMA 2: 80% have 3 SMN2 copies
SMA 3: 95% have 3 or 4 SMN2 copies

Question 12

what’s the difference b/w SMN1 and SMN2?

Answer 12

• there’s a C to T transition on exon 7 from SMN1 to SMN2 - it’s a sense mutation so the same AA is produced, but the transition affects pre-mRNA splicing so that exon 7 is not included in mature mRNA from SMN2
• 80-90% of the time the premRNA code for a mature mRNA that produces dysfunctional protein
• 10-20% of the time the premRNA includes exon 7 which codes for full length functional SMN protein

Question 13

pre-mRNA splicing regulation of SMN1

Answer 13

• exonic splicing enhancer (ESE) on 5’ end of exon 7 facilitates splicing of exons 6 and 7 together (this is good)
• ESE attracts snRNPs and ASFs which aid in splcing

Question 14

pre-mRNA splicing regulation of SMN2

Answer 14

C to T transition in exon 7 changes splicing enhancer (ESE) to splicing silencer (ESS) which inactivates ASF and promotes exclusion of exon 7

• hnRNP1
• ISS N1

Question 15

hnRNP1

Answer 15

the main factor that bind to the ESS to suppress the splicing of exons which eliminates exon 7

Question 16

ISS N1

Answer 16

intronic splicing silences N1

- contains 2 hnRNP A1 binding sites that have a much stronger effect on exon 7 exclusion than the C to T transition

Question 17

roles of SMN protein

Answer 17

1. SMN complex - is important for snRNP assembly which are critical for splicing and processing mRNA
2. transport of mRNAs from cell body down the axon where it needs to go

Question 18

spinraza/nusinersen facts

Answer 18

• 1st drug approved for SMA in 2016
• improves and prolongs quality and quantity of life
• an ASO that increases the amount of SMN that can be produced by acting on SMN2, not SMN1

Question 19

what’s an ASO?

Answer 19

antisense oligonucleotide

• a sequence of synthetic nucleotides generated in labs by biotech companies
• drug is usually injected intrathecally (spinal canal into CSF)
• antisense bc is complementary to the RNA
• designed to be specific and stable

Question 20

nusinersen mechanism

Answer 20

binds to a site on intron 7 of SMN2 pre-mRNA:
- keeps splicing suppressor proteins hnRNP A1 and A2 from binding (ASO displaces them)
- allows U1 snRNPs to bind to 5 splicing sites
all in all, supports inclusion of exon 7 into SMN mRNA

Question 21

how well does nusinersen work to produce full length SMN protein? (%)

Answer 21

normally full length of an SMN protein produced by SMN2 is 20% but nusinersen increases this to 50%

Question 22

gel electrophoresis to measure SMN expression & length of SMN proteins technique

Answer 22

• tissue sample put in reaction buffer to give it a negative charge
• load sample into well with a thick gel matrix and then apply an electric field to the gel
• molecules move from + to - to stretch out the molecule (mRNA or protein)
• levels of mRNA or proteins in the sample were then identified using probes (oligonucleotide for mRNA and antibodies for proteins)
• gel electrophoresis separates molecules according to size - smaller molecules move further

Question 23

does ASO nusinersen increase full length SMN protein?

what was measured to confirm this?

Answer 23

yes - in many tissues

• measured % inclusion (the amount of full length SMN mRNA produced)
• as drug dosage increased, % inclusion increased

Question 24

is there a dose-dependant relationship for ASO nusinersen efficacy?
is re-dosing necessary?

Answer 24

dose-dependent? yes - mice who received greater doses lived significantly longer
re-dosing? yes - mice had ASO effects decrease after 30 and 180 days, suggesting re-injections would be good

Question 25

how does ASO nusinersen improve quality of life (mechanism)?

Answer 25

• the ASO increases the number of αMNs AND CSA of muscle fibres which makes them comparable to healthy individuals (mouse study)
• ie. the increase in SMN caused by the ASO corresponds to an increase in the survival of MNs

Question 26

ASO nusinersen administration methods

which is better?

Answer 26

2 options:

• ICV - intracerebroventricular
• SC - subcutaneous
• SC is better

Question 27

ICV vs SC administration of nusinersen

Answer 27

ICV

• injection directly into CSF where ASO has direct effect on the brain
• researchers found SMN increased in the CNS but not anywhere else (not muscle or liver)
• doubled animals lifespan

SC

• injection into peripheral NS, much less invasive
• mice saw increased SMN in muscle, heat and other tissues while also increasing lifespan 25 times!

Question 28

can ASOs cross the blood brain barrier?

Answer 28

no. so subjects must be dosed while they are young when the BBB hasn’t completely formed yet - in mice anyway (can’t rely on SC injections after P5)

Question 29

what happened in phase 2 clinical trial when nusinersen was given to SMA infants?

Answer 29

• drug found to be safe
• hammersmith test found functional improvements (stat. sig) for subjects (better score = better performance)
• nusinersen was able to move through CNS, along with other tissues of the body
• kids who received drug had greater amount of full length SMN in SC after multiple intrathecal injections

Question 30

what might be a next step for drug companies and SMA research

Answer 30

look for a drug or ASO that can block the ESS found just upstream of exon 7 so that kids with SMA see even better functional improvements

Question 31

zolgensma - what is it?

Answer 31

a viral mediated gene therapy for SMA approved May 2019

- a virus delivers a functional copy of the SMN1 gene which has nothing to do with SMN2

Question 32

zolgensma - does it work? in comparison to nusinersen?

Answer 32

yes
- in studies, nusinersen increased survival rates in subjects compared to placebo group
BUT - everyone who received zolgensma survived
- subjects also see a 22% increase in function after 1 injection (only one needed ever) so it seems to be more effective than nusinersen

Question 33

McCartney 1988 study: details, significance, results?

Answer 33

• @ Mcmaster
• 1st study done where we specifically identified exercise effects on SMA pts
• resistance training in type 3/4 SMA pts
• found subject’s % improvement increased for everyone
• patients got stronger and more fatigue resistant
• found resistance training to be safe for SMA patients

Question 34

Madsen 2015 study: details, results?

Answer 34

• aerobic training in SMA patients
• fitness measured by VO2 max and outcomes measured by ADL questionnaire pre and post training
• after training, SMA pts saw 25-30% increase in VO2max (2x that of healthy controls)
• ADL questionnaire found training wasn’t quite as positive, resulted in a lot of fatigue, need to sleep in SMA patients
• overall found exercise to be safe

Question 35

in mice with SMA, what are the effects of exercise? is running or swimming better? how was this identified?

Answer 35

• both types of exercise increase lifespan, mice were stronger and more fatigue resistant
• swimming was better than running for protecting medium and large diameter MNs (>600um) - ID’d by ChAT immunofluorescence
• running was better at protecting smaller diameter MNs (<600um) - ID’d by estrogen related receptor Beta (ERRB)
• all neuroprotective effects were independent from SMA expression - in the ventral horn of SC - there could be changes in SMA expression in other tissues