The abundance of the SMN2 gene is increased by 1.5 times with the addition of SCM-3 compound compared to a fibroblast of a SMA patient and in vivo models the SCM-C3 has also shown to increase the amounts of SCM-C1 produced naturally by the mice. The mice have also produced a significantly increased amounts of the SMN 

protein in both the quadriceps and the brain, but it seems like the effect of the SCM-C3 has decreased over time. 

All of the above means that SCM-C3 increases the production of naturally occurring SCM-C1 which in turn upregulates the transcription of the full SCM gene that includes the exon 7 to produce a functioning protein. Therefore, this compound could potentially help the individuals with spinal muscular dystrophy as it helps them to produce a functional protein. 

Although Jones et al. also showed that SMN2 has no significant change in mRNA abundance, the abundance of 6 genes doubled and halved for 6 others. This begs some questions regarding the risks. What are these 12 genes, and does their mRNA abundance have any adverse side effects? What happens when the quantity of these genes is doubled? Halved? These are crucial points to consider before moving forward with studies on SMN-C1, C2 and C3. Nonetheless, Jones et al. showed optimistic findings regarding the possibility of increasing the level of SMN2 pharmacologically, which could ultimately prove to be significant in future research on SMA treatments and therapeutics.

 

To understand how SMN-C1, C2, and C3 molecules prevent the ∆7 form of alternative splicing, an experiment of mRNA SMN abundance was conducted in fibroblasts treated with SMN-C3. The results showed that SMN-C3 does affect alternative splicing in fibroblasts. However, changes in SMN gene expression is not the reason why. Therefore, it can be assumed that SMN-C3, and the others, alter splicing during the mRNA maturation process. Further experiments on mice indicated that the drug has a higher effect the longer it has been used. The starting level of SMN on the first day of SMN-C1, C2 and C3 oral dosage was 0, which was increased to 100% change from baseline on day 10.

 

This study has proven that SMN-C1, C2, and C3 are effective in reducing the levels of transcription of mutant SMN mRNA, especially SMN-C1 and SMN-C2. However, as the in vivo was performed on mice, before using it as treatment it would have to go through a clinical trial. 

Jonas et al then investigated the effect of SMN-C3 on gene expression. Figure 2 highlights that SMN-C3 can up and down regulate almost an equal number of genes, however, most of the genes have a Log2 fold change of 0. This suggests that SMNC-3 has no significant change in total mRNA abundance, especially for SMN2.

SMN-C3 is in higher concentrations in the brain compared to the plasma, and peaks before the first 6 hours. Continuous treatment is required to increase the SMN2 baseline, as after one day there is small peak which quickly decrease, but after 10 continuous days of treatment, there is a much larger peak and the protein decreases comparatively more gradually.

Overall, this study is significant as we found that SMN-C3 will help treat SMA as increases healthy SMn2 gene expression. Yet, it is important to investigate the significance of this treatment and its side effects, especially after daily dosage.

This study could have many potential applications and shows promise as a ‘magical bullet’ for this non-infectious disease. Further studies could be used to determine the best time in the infant development for these drugs should be introduced and where- in the germ line or the somatic cell line. 

In vitro, treatment with increasing SMN-C1, C2 and C3 was correlated with increased transcript of full length SMN2 and a decrease in delta7 SMN, the shortened unfunctional protein.

Data is missing for SMN-C1 and C2 but figure 2 shows that treatment with SMN-C3 does not affect significantly SMN ene expression. Meaning that the increase in the SMN2 protein aimed at would be due to the action of the treatment on the splicing of the mRNA and not gene expression.

Treating living mouse with SMN-C3 led to an increase in brain and muscle SMN2 for the approximate 40 next hours, before the treatment has been metabolized and the SMN levels go back to their original point. 

This treatment seems to be promising as it is a simple oral administration. However, data is missing to assess the efficiency of SMN-C1 and C2. Also, the time lapse of SMN-C3 efficiency means that the treatment should be administered every day or so. Finally, further research should be done to assess any potential secondary effects and changes in effects from mouse to humans. 

 

The efficacy of each tested compound were then compared. SMN-C3 was tested on an in vivo model (mouse) and SMN2 levels in the brain and quadriceps were shown to initially increase after administering a dose of SMN-C3. However, when measuring the difference in total transcript expression in fibroblast cells after treatment with SMN-C3, only 7 out of 11,714 genes appeared to be upregulated, and a further 7 were downregulated. Importantly, the SMN2 gene was not one of the 7 that were found to be significantly upregulated by the compound. This data shows that although positive changes in SMN2 levels were observed after treatment with SMN-C3, the changes may not be significant enough in comparison with SMN-C1 and SMN-C2.

 

The data indicates that SMN-C3 does have a measurable effect on increasing transcription of the full length SMN2 gene and decreasing transcription of the alternatively spliced form missing exon 7, suggesting that it could be a potential target for therapeutic interventions for SMA. However, SMN-C3 showed some off-target effects and had stronger absorbance in some tissues than others. This needs to be carefully studied to determine the other effects that the drug could have.

Inside SMA mouse models, the daily oral dose of SMN-C3 increased SMN protein in both muscle and brain tissues over time. A greater initial SMN protein level in both quadriceps and brain tissues on day 10 than day 1 after the dose was shown in figure 2B.

Another finding is that both plasma and brain SMN-C3 level seemed to plateau above its initial level 24 hours after the dose. Even though plasma and brain SMN-C3 concentration decreases quickly after the dose, it remains higher than before treatment. Hence, the impact of SMN-C3 is shown to be possibly long-lasting in SMA mice. Since mice are mammals, they are genetically comparable to humans. Scientists may be able to translate this finding to SMN-C3 application to SMA patients. 

 

Then, SMN-C3 was applied to patient fibroblasts and the gene transcription was measured. While the transcription level of most genes, including SMN2, did not show a significant change, 6 genes were upregulated, while 6 downregulated. 

 

Next, SMN-C1, C2 and C3 were given to SMA mouse orally. SMN-C3 reached peak concentrations in both the plasma and the brain about 3 hours after administration, while showing a major target site in the brain. SMN-C3 was given to the mouse for 10 days and the level of SMN was measured on Day 1 & 10. 24 hours after the first administration, SMN protein level in quadriceps and brain increased. After 10 days of treatment, the SMN level is 2-2.5 times of that on Day 0, and the level remains higher than Day 0 even 7 days after the treatment stopped.

 

This study shows the SMN-C1, C2, C3 compounds can inhibit alternative splicing of SMN2 mRNA to generate the mutant ∆7 responsible for SMA, while not significantly changing the mRNA expression of SMN2. In addition, when SMN-C3 was given to SMA mouse, it was shown that it mainly acted in the brain. The effect includes an increased level of SMN protein in quadriceps and the brain. 

 

This study introduced some interesting compounds that can potentially be used in treating SMA, by inhibiting the mutation caused by the alternative splicing of SMN2. In addition, in vivo experiments in mice also showed promising result, with the level of full length SMN protein increasing in quadriceps and the brain.

 

One concern is that SMN-C3 can lead to the over- or under-expression of some genes, which might have unexpected consequences. These genes need to be identified and the effect of the change of transcription studied. Also, possible side effects should be studied both in mouth and in clinical trials. 

Jones et al. went on to investigate SMN-C3 further; in their study on how SMN-C3 impacted gene transcription, it was observed that aside from no significant change in mRNA abundance for SMN2, 6 genes were upregulated while 6 were downregulated. This poses a potential risk for treatment with SMN-C3, as the change in regulation for functional genes could have unexpected side effects.

Further studies in an in vivo mouse model showed that the oral administration of SMN-C3 resulted in an increase in SMN protein over a period of 10 days, showing it has the intended biological effect. It also suggest that doses should continue to see a long-term effect as once the doses stopped being administered SMN levels dropped. This suggests the protein is unstable.

SMN-C3 has potential to be a treatment option for SMA, though it should be noted that long-term use could have side effects in human trials.

   Fibroblasts taken from SMA patients were treated with the aforementioned drugs and a PCR was carried out to see the levels of full length (FL) mRNA and mutated spliced SMN2 genes (∆7), found in the cells. It was found that increased SMN-C1 dosage contributed to a lower expression of mutated SMN2 proteins. Similarly, SMN-C2 and C3 showed a similar trend in the decrease of ∆7 transcription and an increase in FL transcription.

 When tested in vivo, SMN C-3 was shown to increase the SMN protein expression in both the peripheral and central nervous system over a period of 10 days from the initial consumption. This could have adverse effects on the peripheral nervous system which was not the target region. The drug is therefore not a specific and targeted treatment which could cause neuronal damage in other unaffected areas of the body.

From figure 3, we can see as time post-dose increases, less FL SMN2 is made and more alternative spliced SMN2 is made. Since the alternative spliced SMN2 is missing exon 7, it eventually becomes degraded which is why the level of SMN protein decreases. However, after 10 days of treatment the concentration of full length SMA protein increased significantly from baseline, and it took 168 hours for full length protein levels to decrease to normal. This implies that continuous use of the drug could lead to an increase in SMN protein production. 

This research could prove to be useful in developing treatment for patients with SMA.  

When Jones et al. administered SMN-C3 in vivo to a mouse model, it was seen that the C3 was absorbed in much higher concentrations in the brain compared to the plasma. This could be a concern, as this increases the likelihood of the motor neurons within the brain being affected and of an affected individual developing SMA. A higher concentration of SMN-C3 was seen in the quadriceps as compared to the brain, and this could again imply that the motor neurons within these muscles could be significantly affected in the disease.

These findings highlight that all three forms of SMN can be implicated within SMA and could be considered potential therapeutical targets in treatment of SMA. It would also be valuable to further research the 14 genes affected by SMN-3, to evaluate any other potential risks.

However, with SMN-C3, the amount of SMN2 gene is not significantly affected compared with 11713 other genes. However, with repeated administration of SMN-C3, SMN protein is increasingly expressed due to increasing start concentrations of SMN proteins. In day 10 of administration, SMN protein levels are still 25% higher than the baseline after 168 hours, which shows that SMN-C3 helps the production of normal SMN protein (which is not rapidly degraded). These data collectively show the positive correlation of SMN-C3 dosage and normal SMN protein levels, which may suggest that SMN-C3 is a feasible drug for the disease. 

However, with SMN-C3, the amount of SMN2 gene is not significantly affected compared with 11713 other genes. However, with repeated administration of SMN-C3, SMN protein is increasingly expressed due to increasing start concentrations of SMN proteins. In day 10 of administration, SMN protein levels are still 25% higher than the baseline after 168 hours, which shows that SMN-C3 helps the production of normal SMN protein (which is not rapidly degraded). These data collectively show the positive correlation of SMN-C3 dosage and normal SMN protein levels, which may suggest that SMN-C3 is a feasible drug for the disease. 

A measure of the difference in total transcription expression of 11,714 genes was then conducted to identify if the expression of any genes was affected following SMN-C3 treatment. SMN-C3 was shown to have no significant effect on the expression of the SMN2 gene, however it was shown to have a significant effect on the expression of 12 (0.001%) genes, with half of these being significantly upregulated and the other half being significantly downregulated.

Figure 2 shows that 7 genes are upregulated, and 7 genes are downregulated by SMN-C3, while SMN2 is not significantly affected. Those genes that have an altered expression level could potentially affect the splicing of SMN2 giving the result of Figure 1. 

From in vivo model, it is suggested that SMN-C3 could actually reach the brain and have an effect over SMN protein level in living organisms. Also, the fact that SMN protein level gradually decreases after the day 10 dose indicates that the protein is not very stable and decomposes overtime. This study suggests potential treatment for SMA. However, the fact that 7 genes were upregulated and 7 genes were downregulated posts a potential threat to the body. 

 

SMN compounds have shown to upregulate the full length gene and downregulate the alternatively form, so they could be a potential therapeutic target. However, SMN-C3 showed to have significant effect on 14 off-target genes imposing a high risk as the brain has a high absorption. Further studies needed to investigate the associated risk by the off-targeting effect on genes.

SMN-C3 has no significant change in total mRNA abundance. This suggests one of the gene did have altered expression could be mediating the change in SMN2 splicing detected.

A mouse model with SMA was also investigated by given oral doses of SMN-C1, SMN-C2 and SMN-C3 (10mg/kg) for 10 days. Within a few hours after the dose, the SMN protein level at both quadriceps and brain increases significantly. The level of SMN protein was then degrades gradually. The difference between day 1 and day 10 is that the overall level of SMN protein increases and the speed of SMN degradation slowed down. We can conclude that the three SMN compounds can affect the alternative splicing and could be a possible treatment for the SMA. However, side effects may occur as the SMN-C3 alters the splicing of 12 different genes shown in the second figure.

The mechanism by which SMN-C3 is increased is not entirely known, but there was upregulation and downregulation of certain genes. It could be possible that those genes could affect the splicing mechanism. Finally, it was shown that at higher concentrations of SMN-C3 there were higher percentages of healthy SMN protein.

However, the results should be taken with a pinch of salt. Firstly because the experiments were done in mice models. Therefore, it is not entirely possible to predict the consequences of SMN-C3 on humans. Secondly, SMN-C3 intake changed the expression of several genes. Although they could be involved in the splicing mechanism, they could also produce unwanted side effects.

In vivo models were then investigated, where SMA mice were given oral doses of the SMN-C3 (see figure 3). Plasma and brain tissues were collected and the compound concentrations were quantified over a 24 hour period. The results showed a spike in concentration in the first 6 hours, followed by a decrease. When quantifying SMN protein in quadriceps and brain after once daily dose on days 1 and 10, it was clear that the level of SMN protein was higher in day 10 than day 1. 

The data discussed above shows a great potential for those compounds to offer a new potential treatment for spinal muscular atrophy (SMA). 

 

Jones et al. then used an mouse model to examine the impact of SMN-C1~3 in vivo. Once daily oral doses of SMN-C3 (10 mg/kg) was given to mice and the concentration of SMN-C3 and functional SMN was measured on day 1 and 10. The level of SMN quickly increased in 12 hours then wear off, which matches the trend of concentration of SMN-C3 in mouse. However, SMN-C3 did exhibit an accumulating influence on the level of SMN. The peak concentration of SMN after intake of SMN-C3 on day 10 was two times higher than the peak concentration on day 1.

 

Overall, SMN-C1~3 seems like a potential treatment for SMA, but treatment must be long-term to maintain sufficient level of SMN. However, SMN-C1~3 did impacted the expression level of around 15 other genes in fibroblasts which must be looking into for following consequences. And the effect of SMN-C1~3 on splicing mechanisms of other genes was not examined in the experiment.

Then, Jones et al. looked more closely and the regulation of gene expression when SMA patients are treated with SMA-C3. By comparing to a control of SMA patients treated with DMSO, the treatment with SMA-C3 does not have a significant on most genes (within the fibroblasts) transcription levels. However, 7 genes were upregulated and 7 were downregulated with SMA-C3 treatment. Looking specifically at the SMA2 gene, it’s expression was not affected by SNM-C3, suggesting that the normal expression of the gene is not affected, but comparing it to the previous figure, SMN-C3 only changes the way that SMN2 is spliced and not overall the gene expression.  

A mouse was used as an in vivo model to give Jones et al. an idea of specifically SNM-C3's effect on a larger scale, compared to just looking at fibroblasts. The treatment goes to the mice brain in the first place, as the plasma concentration of SNM-C3 in the brain rises only a few hours after the dose. This is beneficial as the drug can signal to the rest of the body to treat the disease.