Manipulating SMN2 gene expression as a potential treatment for spinal muscular atrophy




Esther Montrose

Introduction

Spinal muscular atrophy (SMA) is a disease that affects the motor nerve cells in the spinal cord that control voluntary muscle movement. It is the most common cause of death in children under the age of two from a genetic disease. SMA is caused by the homozygous loss of the SMN1 gene. This gene contains nine exons that code for the survival motor neuron (SMN) protein. This protein is essential to the maintenance of motor neurons, and without functional SMN, the nerve cells can die, leading to fatal muscle weakness. There are children who lack the SMN1 gene, yet they are still surviving. Their DNA was analyzed and it was found that they carry an amplification of the number of copies of the SMN2 gene. The more copies of SMN2 that an individual had, their severity of SMA symptoms diminished.

The SMN2 gene has an identical exonic sequence to SMN1, except for one C to T nucleotide difference within exon 8. This SNP does not change the amino acid sequence encoded by the gene; however, it alters where the mRNA is spliced. The majority of the transcript generated lacks exon 8 (Figure 1).

Only 10-15% of the SMN protein encoded by SMN2 is functional. Around 90% of what is produced by SMN2 is a truncated protein that rapidly is degraded. If one copy of SMN2 can produce around 10% of functional SMN, the children with multiple copies of SMN2 are surviving due to the total amount of the functional protein produced from all of their SMN2 genes together being high enough to maintain their motor neurons. If the splicing process can be altered such that the transcript generated from the SMN2 allele contains the exon 8 sequence, this can have a positive impact on the survival of these individuals. The impact of chemical compounds on the alternative splicing of exon 8 is evaluated in a minigene containing the SMN2 genomic DNA spanning exons 7 through 9.

Materials and Methods
HEK cells were transfected with an SMN2 minigene in a PCI vector containing genomic DNA spanning from exon 7 through exon 9, and treated with 11 different nutraceuticals.

The total RNA was purified from the cell lysates.

RT-PCR was performed on all samples using primers designed to check for the inclusion of exon 8.

RT-PCR products were visualized using gel electrophoresis on a 1% agarose gel.

The desired bands were purified, sequenced, and aligned with reported transcripts.

Results
Figure 2 shows the visualization of the RT-PCR products obtained by using primers to check for the inclusion of exon 8. Lane 1 is the product from the untreated cells, and lanes 2-12 are the products from the cells exposed to various different nutraceuticals.
Treatment 5 facilitates the production of a larger PCR product, whose size suggests the inclusion of exon 8.
Both PCR products generated from the RNA isolated from cells treated with nutraceutical 5 were sequenced. The sequence purified from the lower band aligned perfectly with the sequence of exon 7 and exon 9, but not exon 8 (Figure 3). The sequence of the upper band aligned perfectly with exon 7, exon 8, and exon 9 (Figure 4).

Discussion
The results from the RT-PCR in Figure 2 clearly demonstrate that treatment 5 facilitates the production of an SMN2 transcript that includes the exon 8 encoded sequence (Figure 1). Both the exon 8 lacking and containing sequences were verified using Sanger sequencing. The sequence of the 326 base pair PCR product in both of the samples matched the sequence of exon 7 and exon 9, but not exon 8 (Figure 3). This, therefore, is the dominant SMN2 transcript that splices out exon 8. The sequence of the 380 base pair PCR product in both samples matched the sequences of exon 7, exon 8, and exon 9, indicating exon 8 inclusion (Figure 4). There are small amounts of the full SMN2 transcript produced in vivo, so it makes sense that there is this upper band even in the untreated sample. The upper band is much stronger in treatment 5, though, indicating that this treatment is able to manipulate the splicing machinery of SMN2 and induce it to include exon 8 more frequently.

Further studies can observe the effects of treatment 5 on cells derived from patients who have SMA and lack the SMN1 gene to see if it impacts those cells in the same way. This study also represents a proof of principle, that the inclusion of exon 8 can be manipulated by chemical compounds, and it encourages a further screening of a large bank of compounds to see if additional ones may facilitate the inclusion of exon 8. This analysis may also reveal the presence of compounds that facilitate the exclusion of exon 8. Identifying these compounds can inform individuals with SMA on which ones to avoid.

References
What is SMA | Cure SMA.

SMA Foundation | About SMA.

Wi 2. Wirth B, Brichta L, Schrank B et al. Mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number.

Lorson CL, Hahnen E, Androphy EJ, Wirth B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy


Figures


Figure 1-Splicing diagram of SMN1 and SMN2


Figure 2-Gel electrophoresis results from RT-PCR


Figure 3-Sequence alignment of the lower band. The top line is the sequence of SMN2 transcript and the bottom line is the sequence of the 326 base pair PCR product


Figure 4-Sequence alignment of the upper band. The top line is the sequence of SMN2 transcript and the bottom line is the sequence of the 380 base pair PCR product


Abstract
Spinal muscular atrophy (SMA) is a devastating disease that affects the motor nerve cells in the spinal cord that control voluntary muscle movement, compromising one’s ability to walk, eat, and breathe. Approximately 1/6,000 children are born each year with this disease. SMA is caused by the homozygous loss of the SMN1 gene, which codes for the survival motor neuron (SMN) protein. Lacking this functional protein leads to debilitating and potentially fatal muscle weakness. The gene SMN2 has a nearly identical exonic sequence to SMN1, other than a C to T base pair change within exon 8. This SNP doesn’t change the encoded amino acid sequence, but it causes for the majority of the transcripts to lack exon 8 sequence. This transcript that lacks this 54 base pair exon produces a truncated protein that is rapidly degraded. This project aimed to test the ability of various nutraceuticals to modulate the alternative splicing of exon 8. One compound was shown to increase the level of exon 8 inclusion, which could lead to an increase in the production of functional SMN protein, and therefore serve as a potential treatment for spinal muscular atrophy.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin for providing me the opportunity to participate in this project. I would also like to thank Anthony Evans and Devin Rocks for their support and patience. All of their constant availability, direction, and guidance is greatly appreciated, and without it, this project would not have been possible.


This document was last modified 05/14/2019.
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