SMA Flashcards
explain the mechanism of ASO therapeutics and give one example of its use in disease.
Antisense oligomers are small stretchers of RNA that bind RNA to prevent splicing.
This method has been applied in spinal muscular atrophy. An ASO now trialed in humans developed by Biogen and Ionis workds by binding to the intronic element ISN-1 on the SMN2 RNA preventing the splicing out of exon 7 allowing it to form a complete SMN protein.
.outline the main pathology of SMA, include findings from mouse studies.
SMA involves the selective loss of secondary motor neurons in the anterior horn of the spine. (ventral horn in mice). The central primary neurons are sparred (unlike in ALS). This reslts in symmertrical muscle weakness.
Mouse models have provided other insight. they showed that there was significant loss in the ventral horn. surviving motor neurons are swollen and chromatolytic, this often occurs along sided increased phosphorylated neurofilaments and autophagic vesicles.
these models have also shown wallerrian degeneration of intramuscular nerves.
muscle fibre is denervated and in severe cases cannot be reinervated and adapt an immature structure like myotubes, in less severe cases the fibres show grouping of fibres types and undergo cycles of innervation and dennervation.
They have very low SMN levels.
.What are the underlying genetics of SMA?
SMA is caused by mutations in the SURVIVAL MUSCULAR NEURON 1 (SMN1) gene. This is a RECESSIVE condition.
the gene is found at 5q and a inverted replication has been identified (5q11.2-5q13.2)
in 95% of cases then mutation drives a loss of function due to a mutation of 5q. in other cases mutations alter the reading frame.
on the same arm there is the SMN2 gene, this has a 1 nucleotide difference which drives the alteration the splicing modulator and the splicing out off exon 7 froming an incomplete protein (this occurs MOST of the time). All patients have atleast 1 copy of the SMN2 gene, variations in its copy number can alter the risk of disease.
what are the main diferences between the different types of SMA?
Type 1: This is the most severe form seen in 50% of cases.
This has a onset before 6 months usually at birth. The children will never walk and will die before 2.
They require both respiratory help and nutritional support (gastrotomy tube)
They have symmertical muscle weakness. this can be seen in the respiratory muscles in partuclular with a relative sparring of the diphram. This results in a bulb shaped stomack as they breath solely with the diaphram. also causes : WEAK CRY/COUGH, DIFFICULTY CLEARING SECRETIONS,
bulbar dennervation which can be visualised by visble fasciculation in the tongue and swallowing issues.
There are 3 subtypes- A presents at birth with joint contracture and repiratory compromise, B- has an onset before 3 months and C- has a onset after 3 months.
TYPE 2: this is the intermediate form. onset is seen after six months with patients often dying after 2 (Now with impoved awareness and care care survive till adulthood.). often DO NOT require ventilation support also respiratory weakness is still present.
These are able to sit but will never stand. This means that they develop bad scoliosis requring surgery and have frequent hip disclocations.
They also have Oro-bulbar dysfunction and malanutrition.
Type 3: this is the least sevevere from with an onset after 18 months and often after they have developed walking and so can stand.
they oten shown issues getting up from the sitting position called the GOWERS SIGN.
Best signs are a wobbling gate walk and msucular fasciculations.
Note: there is a very rare type 4 whcih can have adult onset.
How can varying SMN2 copy numbers alter the risk of SMA?
The SMN2 gene doesnt profuce a functional protein most of the time but it does sometimes.
It is a POLYMOROPHIC gene in the population with varied copy number.
Assesments of the risk of type 1 SMA shows are large shift from 97% at 1 copy to only 8% with 3 copies. by 5 copies the risk is 0%.
What is the main fucntional domain of SMN, what is SMNs main functional role? provide eveidence where possible
SMN has a Gems domain- This is a nuclear domain implicatesd in the modification of snRNPs (Small nuclear ribo-nuclear proteins). through this it is involved in RNA regulation.
SMN forms the complex with other Gemins and mediated the biogenesis of snRNP. it does this though loading the heptameric ring f splicesome fibres onto the snRNPs and this aids the formation of splicesomes for pre-mRNA intronic splicing splicing.
The SMN also has fucntions in altering the cytoskeletal elements of dendrites and axons. this is clear in axons where it is key to axonal transport and growth.
KO study showed reduced mature mRNA levels in the distal end of the axon, this was seen with reduced axonal growth and reduced levels of beta actin.
What is thought to underly the selectively neuronal death in SMA , provide evidence for a motor circuit specific SMN function.
One factor is the selctive role of SMN in the function of the U12 splicesome which meadites a minority of splicing.
Lottie et al- Studdies in mammalian (mouse) cell cultures and drosophilla with KD of SMN1 showed that SMN defficiency effected the u12-splicing and mRNA expression of a novel portein linked to the motor circuit Stansimon. they also showed that Stansimmon KO mimicked SMN KO phenotype.
Zhang et al- showed genes a;ltered in SMN KO involved many related to the NMJ maintencae like Agrins exon skipping (kEY TO ACHr cumulation in NMJ due to interaction with a synaptic complex of proteins consisting of LRP4 and MuSK which act to autophosphorylate themselves and down stream targts, DOK7, rapsyn and the delta subunit of AChrs to drive clustering at NMJ). Also upregulated the expression of synaptic pruning promoter compelment factor C1q.
effect on afferents- Mantis et al reported reduction in primary afferent inputs and porpprioceptive inputs into the Ventral horn prior to MN deficits. This is aslo shown in prior reductions in the Afferent provoked responses seen in the first 2 post natal weeks.
Fletcher et al 2017- The reduced MN firing has been related to a reduction in Kv2.1 on MNs. Pharmoclogical upregualtion of central activity resulted in an increase in the expression of Kv1.2 and relief of the phenotype.
(smae study to show evidence of entral activity in the correct wiring of sensory-motor connectivity)
what is the issue with drosophilla and zebra fish models of SMN KO.
Zebra fish and drosophilla only have the 1 SMN copy and thus KO of SMN1 has not SMN2 for partial recovery and this leads to embryonic death.
are there lifelong changes in SMN importance, what can evidence around this tell us about theapeutics.
Kariya et al 2014- They did age progressive KD in mouse models and identified a point at which KD did not reuslt in the prgressive developement of SMA, this COINCIDED WITH NMJ MATURATION.
This suggests that its significance is seen in adulthodd. this means that treatment maybe not needed thoughout life. and more acute action early on is most important.
does SMN dysfucntion soleley effect the spinal motor neurons? provide evidence
Hunter et al- they showed the impact of SMN LOF on schwann cell function. showing over expfression of SMN in schwann cells reduced the volume of demyelinated muscular neurons. hence, LOF is also linked to wallerian degeneration.
role and support for central motor drive in regulating the correct wiring an connectivity of the Motor neuron systems and its disruption in SMA (SMA)
Fletcher et al 2017- The reduced MN firing has been related to a reduction in Kv2.1 on MNs. Pharmoclogical upregualtion of central activity resulted in an increase in the expression of Kv1.2 and relief of the phenotype.
(smae study to show evidence of entral activity in the correct wiring of sensory-motor connectivity)
what underpins SMA Motor neruon death?
This is though to be a Cell automnomous event.
SMN loss of function results in the increased expression of P53 which shows selective accumulation in vulnerbale vs resistant MNs.
Phosphorylation of P53 on serine residues marks vulnerable MNS for cell death.
Later on this is ransferred to resistant MNs causing there death.
shRNA KD of p53 in models by Simoni et al 2017- this was able to rescue motor neurons and increase MN survival.
What are the main therapeutic methods in SMA.
there are both those effecting expresion of SMN (expression) and those effecting the symptoms without effect SMN (agnostic)
Agnostic- FDA have approved the use of FASUDIL. this acts as a ROCK/RHOA kinase inhibitor. this was reported to mitigate the SMA phenotype and greatly improve motor function. would be a good option alongside ASOs.
(Neuroprotective driugs like Gabapentin hav been traialed to limitted application.)
Expression
ASOs-antisense oligomers have been sed to prevent the splicing of SMN2 thus allowing it to form a full SMN1 protein and replace the lossed SMN1.
One developed by Ionis and biogen was traille din humans called NUSINERSEN. This targets the intonic element ISN-1 to prevent the splicing out of exon 7 to allow for a full protein.
-ve These cannot cross the BBB and so hav to be introduced intrathecally. +ve this means they have a long life span. after a LOADING set of 6 injections only 3 are required a year.
Early studies in mice in whic the SMA mice were alot smaller and has shrotened life spans. ASO injections into liver was able to reverse issues with size and significntly prolong life. Through this they identified a THERAPEUTIC WINDOW OF ACTION.
ENDEAR trial 0They trialled this type 1 SMA patients in comparision ot a vehicle. and found that it extremely effective. it improved mortality by 63% and reduced the likely hood of needing repiratiry intervention by 47%. long term trials recently eneded in may 2018.
small molecules- The main benefit f these is they can cross the blood brain barrier.
1 produced by Roche and PTC was shown to be extremely selctive affecting SMN2 and a few others showing very little side affects in mice.
small doses were able o induces A LIFE SPAN SHIFT FROM 20D TO 60D. in large doses this was shown to almost increase life span to normal lab life spans (1 year)
Pharmacological- The work by infleuncing th
prevention of alternative splicing or by transcriptional activation.
a drug developed by PTC RG3039 targets the DcpS enezymes and ha d dramtic effect on mouse life span. 14 days going to 6 months.
This is now being optimised fro humans.
Gene replacement therapy> the best example is being worked on by avexis. this uses a novel ADENO-ASSOCIATED VIRUS that can cross the BBB and infect many cell types. AAV9-SMN greatly increased the expression of SMN and counterers disease poehenotype.
outline in detail the efficacy of ASOs in SMA.
antisense oligomers have been sed to prevent the splicing of SMN2 thus allowing it to form a full SMN1 protein and replace the lossed SMN1.
One developed by Ionis and biogen was traille din humans called NUSINERSEN. This targets the intonic element ISN-1 to prevent the splicing out of exon 7 to allow for a full protein.
-ve These cannot cross the BBB and so hav to be introduced intrathecally. +ve this means they have a long life span. after a LOADING set of 6 injections only 3 are required a year.
Early studies in mice in whic the SMA mice were alot smaller and has shrotened life spans. ASO injections into liver was able to reverse issues with size and significntly prolong life. Through this they identified a THERAPEUTIC WINDOW OF ACTION.
ENDEAR trial 0They trialled this type 1 SMA patients in comparision ot a vehicle. and found that it extremely effective. it improved mortality by 63% and reduced the likely hood of needing repiratiry intervention by 47%. long term trials recently eneded in may 2018.
Outline the findings of DUQUE ET AL 2015, how does this support the role of SMN in SMA?
Duque et al 2015 used intrathecal injections to introduce scAAV9 linked SMN1 shRNA (short hair pin RNA) this KD of SMN1 caused proximal muscle weAkness, fibrillations and reduced expression of CMAP and MUNE in pigs.
