Spinal muscular atrophy (SMA) is a genetic disorder that is caused by the absence of the survival of motor neurons 1 (SMN1) protein-encoding gene on chromosome 5 (5q13). This is present in all cells and is important in the formation of the spliceosome, which is the body that is required for the splicing of the pre-mRNA transcript inside the cell.
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This is in some way especially important for the function of motor neurons. With low concentrations of the SMN1 protein in the motor neuron cells, the cells will die out and subsequently reduce the motor action units.
All patients with SMA lack normal activity of both alleles of the SMN1 gene on chromosome 5. In 95% of cases, this condition arises due to homozygous gene deletion or conversion from SMN1 to SMN2.
Comparatively, a small percentage of cases are due to subtle mutations. For example, SMA can arise as a result of the presence of the two alleles on the same chromosome with the same mutation on both, and no corresponding allele on the other chromosome. This condition can also be due to a de novo mutation on one allele.
The severity of the consequences of losing this gene is modified by the number of copies of the other form of the gene, namely, SMN2. The SMN2 gene has a nucleotide substitution which leads to the non-inclusion of exon 7 in the mRNA transcript. As a result, the formed protein becomes be nonfunctional and unstable. However, in 10-15% of the transcripts, exon 7 is present, which causes the formation of full-length mRNA and a normal protein.
The higher the number of SMN2 copies, therefore, the lower the chances of severe disease. However, this is not the only modifier and, in some cases, only 2 copies of the gene are present along with mild disease.
This phenomenon can be explained by the presence of another mutation that introduced a new exon splice enhancer causing higher production of the SMN protein. Thus, more work is required to elucidate the number and action of the genetic modifiers of the clinical severity of SMA. This is crucial to provide an accurate prognosis during genetic counseling and for the clinical usefulness of newborn SMA screening.
With the discovery of the SMN gene, many animal models were developed, which helped to understand the underlying changes in the motor action units. For instance, a combination of two SMN2 genes with no SMN1 genes caused severe phenotypes in mice; however, when eight SMN2 genes were introduced, the evidence of clinical disease disappeared.
In this way, it was found that by increasing the expression of the full-length SMN transcript, more of the functional protein could be produced. Furthermore, the phenotype could be altered towards the normal side of the spectrum.
This not only provided the proof of concept that such a therapeutic approach was rational and potentially effective, but also allowed for testing such treatment methods with investigation of resulting changes in the biochemical and molecular pathways in affected animals.
Many workers have focused on the development of drugs that increase SMN2 gene expression, such as HDAC inhibitors including sodium valproate, sodium butyrate, and trichstatin A, as well as aminoglycosides and quinazolone compounds.
These agents showed great promise in mouse models by activation of the SMN2 gene promoter, but failed to impact on humans. This lack of efficacy has led researchers to focus their next endeavors on determining the optimum time for treatment with these molecules, the right dose, and the appropriate duration of therapy.
Many other small molecules have also been developed and are being studied in relation to their specific effect on SMN2 gene splicing and hence increased full-length SMN mRNA transcription.
Many research groups are working on altering the process of SMN2 splicing to ensure that exon7 is included in the mRNA transcript.
Antisense oligonucleotides (ASOs) or synthetic RNA sequences, for example, can target a complementary sequence and change the splicing event in the SMN2 gene. This includes an ASO which silences intronic splicing in intron 7 (intron 7 intronic splicing silencer N1 (ISS-N1) so as to increase exon 7 inclusion. Another is aimed at intronic repressor Element 1, while yet another is bifunctional and targets the intron7-exon 8 junction. By this means, the ASOs activates hnRNP-A1, which, in turn, prevents exon 8 inclusion and increases the chances of exon 7 inclusion.
Yet another area of research is into the trans-splicing RNA (ts-RNA), which produces a hybrid mRNA from the pre-mRNA transcript by trans-splicing. This cuts out and replaces the mutated locus, thereby increasing the SMN protein level.
Work is now on to pass these molecules through human clinical trials to assess their usefulness and methods of optimum application.
The first SMA mouse rescue occurred by the introduction of an adenovirus-associated vector serotype 9 (scAAV9) and inspired tremendous work in this direction. The most important finding to date has been that the timing of the injection is crucial, since the effect reduces with advancing delay from the day of birth and appears to be nil after a period of time.
This may indicate a very short window of time for effective gene therapy in humans with type I SMA especially to prevent motor neuron loss. In ract, the success of this gene therapy could push up the importance of newborn screening in children following the birth of an affected sibling or to a carrier couple. However, more research needs to be done to determine the period of usefulness.
Stem cell therapy
Stem cells are also being explored for their potential ability to replace dying and dead motor neurons in SMA. Primary neural stem cells and pluripotent stem cells derived from embryonic stem cells have been used successfully in mice. In fact, induced pluripotent stem cells have now been produced from patient fibroblasts, which could speed up the development of compatible neuron replacements in this manner.
The challenges for the future remain to develop measures of outcome, determine the best markers of success, and conduct valid trials while still maintaining standards of supportive care for existing SMA patients. It will also be necessary to work out how to detect presymptomatic patients and to select the best therapies for further development.