Scientists manage to optimize an available therapy, based on in vivo trials, to treat spinal muscular atrophy

Headed by CONICET researchers and led by Alberto Kornblihtt, the study is the cover of the prestigious Cell journal.

The study led by CONICET researchers was selected to illustrate the cover of the scientific journal Cell. The design/illustration was made by Lucia Giono.
Luciano Marasco, Vanina Sánchez and Alberto Kornblihtt. Photo: CONICET Photography/ Verónica Tello.

Spinal muscular atrophy (SMA) is a rare neurodegenerative disease that affects motor neurons and is caused by mutations in both copies of the SMN1 gene. Although there are different types of SMA, which are determined by the severity of the disease and the time when symptoms appear, this disease is characterized by a progressive loss of muscle strength and can affect the ability to speak, walk, swallow and even breathe.

Until the end of 2016, the treatments available for SMA were only supportive and there was no pharmacological therapy that could stop or slow the progression of the disease. This situation changed in December of that year when the United States Food and Drug Administration (FDA) authorized the use of a drug called nusinersen, which was later also approved in Argentina. This drug, which was developed by the Uruguayan researcher Adrián Krainer at the Cold Spring Harbor Laboratory in New York, targets the SMN2 gene -which is very similar to SMN1. The administration of nusinersen to SMA patients increases the production of SMN protein and thus stops or slows the progressive death of motor neurons.

A recent study led by CONICET researchers suggests that the results of the treatment with nusinersen -based on in vitro and in vivo tests- could be optimized if they are combined with the administration of valproic acid, a drug that has the function of inhibiting histone deacetylases and which is currently used to treat cases of epilepsy.  The work led by Alberto Kornblihtt, CONICET researcher at the Institute of Physiology, Molecular Biology and Neurosciences (IFIBYNE, CONICET-UBA), was published today in Cell and chosen to illustrate the cover of the prestigious scientific journal.

Spinal muscular atrophy and alternative splicing of the SMN2 gene

The SMN2 gene, therapeutic target of nusinersen, differs from SMN1 only in 11 base pairs, out of a total of approximately 30 thousand. However, the difference in a single base pair in exon 7 makes that when SMN2 is transcribed from DNA to mRNA, this exon is mostly skipped -a phenomenon known as alternative splicing- and, as a consequence, the amount of SMN protein that is synthesized is insufficient. For this reason, the presence of SMN2 in the human genome cannot compensate for the mutations in the SMN1 gene that SMA patients have. Nevertheless, it is the existence of the SMN2 gene that allows fetuses with mutations in both alleles of the SMN1 gene to complete development and be born.

“Otherwise, mutations in SMN1 would directly cause embryonic lethality, as it happens with mice, animals that do not have the SMN2 gene,” explains Luciano Marasco, CONICET doctoral fellow at the IFIBYNE and first author of the study.

Nusinersen is an antisense oligonucleotide (ASO) that increases the inclusion of exon 7 in the transcript of the SMN2 gene and, consequently, increases the production of SMN protein to compensate for the absence of SMN1 gene. This means that the action of nusinersen consists of correcting the alternative splicing of SMN2 to obtain greater amounts of the SMN protein. When administered directly into the cerebrospinal fluid, ASO corrects the alternative splicing of SMN2, mainly at the central nervous system.

Shortly before the approval of this drug in the United States, relatives of patients with SMA grouped in ‘Familias AME’ (FAME Argentina) visited Kornblihtt’s laboratory, which has considerable experience in the analysis of the mechanisms that regulate splicing, to ask him to research into the subject from the point of view of basic science.

“We always had the dream that the subject be studied in our country. The great investigations were carried out in the United States, Europe or Canada and although these advances were very important for us, at the same time we felt far away. We considered that Kornblihtt was the right person because we knew his background in the study of splicing and we knew that he was in contact with Adrian Krainer, who had discovered the mechanism that was used for the first treatment of SMA. We focused on basic science research because we think that the more we know about a subject, the more likely it is that better therapies will be developed,” says Vanina Sánchez, president of FAME Argentina.

Although Kornblihtt’s first response to the request was reluctant, after verifying that the alternative splicing of SMN2 changes according to the speed of transcription (exon 7 is included less in the mature mRNA if the transcription is slow and more if the transcription is fast), paradigm of splicing that his laboratory has been studying for many years, accepted the proposal but warned that the study might not have satisfactory results.

A possible combined therapy

“It occurred to us that if the inclusion of exon 7 of the SMN2 gene increased along with the speed of transcription, by using drugs that open the chromatin of the gene, the enzyme responsible of the transcription -RNA polymerase- would go faster and contribute with nusinersen in the improvement of the inclusion of exon 7 and the increase of the production of the SMN protein. Part of the aim was for this to happen not only in the central nervous system, which is where nusinersen injections primarily work, but also in peripheral tissues where SMN2 is also expressed,” describes Kornblihtt, who is also an emeritus professor at the ‘Department of Physiology, Molecular and Cellular Biology of the Faculty of Exact and Natural Sciences of the University of Buenos Aires (FCEN, UBA).

“First we used artificial minigenes to see how nusinersen interacted with the inhibitors of histone deacetylase, which open chromatin. Afterwards, we continue working with the endogenous SMN2 gene in cultured cells, always aiming to increase the healthy isoform of the gene in the mature mRNA and to produce greater amounts of the SMN protein,” Marasco explains.

After conducting promising cell culture tests, Marasco spent two months in Adrian Krainer’s lab in New York where he analyzed the effects of the drug combination in mouse models of the disease that have the human SMN2 gene incorporated.

The studies with mice also aimed to increase the expression of the healthy isoform of SMN2  mature mRNA, as well as the expression of the protein in all organs. The results of these tests with animal models exceeded the expectations of the research team.

“The mice with SMA that are given ASO (nusinersen) with an inhibitor of deacetylases, such as valproic acid, have longer survival, gain more weight and have better motor properties and greater strength in the forelimbs than mice given ASO alone.  If they are turned over, mice treated only with nusinersen take a long time to right themselves, but those treated with the combination of the two drugs do so immediately,” Marasco explains.

“Although there were other groups that tested this combination, no one had explored the molecular combination or had done so until now with animal models, but only in cell cultures,” says Kornblihtt.

The researchers also tried combining ASO with another histone deacetylase inhibitor, such as trichostatin A and had similar results, but unlike trichostatin, valproic acid is already approved for clinical use.

Besides, the scientists found that the mice treated with valproic acid alone died within seven days, as did the control mice treated with physiological solution. This means that, in in vivo tests, only when combined with ASO does the action of histone deacetylase inhibitor have relevant effects.

An important discovery

In addition to verifying in mice with SMA that the opening of the chromatin of the SMN2 gene improves the therapeutic effect of nusinersen, by favoring a greater inclusion of exon 7 in the mature nRNA, the scientists also discovered that this drug has the effect of compacting the chromatin and thus slow down the rate of transcription. This action of the ASO goes exactly in the opposite direction of what is looked for because it contributes to inhibiting the inclusion of exon 7 in the transcription of SMN2 gene.

“This mechanism is completely new because no one who works with ASOs in different diseases imagined or demonstrated that these therapies with ASOs can modify the chromatin. In other genes, this modification could be, as in this case, the opposite of the desired effect, but it could also be synergistic,” Kornblihtt affirms.José Stigliano, CONICET doctoral fellow in Kornblihtt’s team and also co-author of the study, continues to work on this finding.

“Valproic acid compensates for the side effect that ASO has. It is worth mentioning that despite this side effect that we seek to correct, the therapy is super good”, Marasco affirms.

Additionally, the researchers confirmed that this combined therapy has no deleterious effects on the genome. “The studies have shown that from 15,000 genes that we analyzed, it only affects about 60, a very insignificant number,” Kornblihtt says.

The value of basic science and the expectation of clinical continuity

Both the researchers and the families who are members of FAME hope that eventually, and with the preliminary studies that need to be done, the combination of nusinersen and valproic acid can be tested in clinical trials.

“We would like someone to continue working on the results of this study and that at some point it can go to the clinical trial stage so as to see if it is possible to improve the results of therapy with nusinersen,” Vanina Sánchez says.

“After the publication of this study we hope that groups interested in testing the therapy will appear. It is not easy because it is a very rare disease, but I think we have contributed an important seed, not only to the knowledge of this disease, but also of how ASOs work,” says Marasco.

In addition to the expectation of optimizing the treatment by combining it with another drug, there is also the expectation of making it more economical. “The injection of nusinersen is very expensive, whereas valproic acid is very cheap. So, if adding valproic acid could also reduce the necessary doses of ASO, the total cost of the therapy would be cheaper,” Kornblihtt affirms.

For the scientist, one of the lessons of this study is that research in basic science is essential to discover and understand mechanisms for the design of new therapies. “Our country cannot afford to focus only on applied research or repeat things that are already known from other parts,” he says.

For Vanina Sanchez, everything that came after Kornblihtt accepted the challenge of studying the alternative splicing of the SMN2 gene was good news. “Although six years passed before this publication, Alberto informed us about the intermediate discoveries, which were communicated in scientific meetings. We are very proud and eternally grateful to Alberto, Luciano and the entire team. The fact that Argentina holds this place in the research on SMA was an unthinkable dream for us. It was also very exciting to go to the laboratory and meet the other side, the researchers who are not usually in contact with patients. And I know that it was also important for them to meet the families who hope that the results of all these studies will mean better possibilities for their children,” she concludes.

Research grants

This research work was supported by a grant given jointly by FAME Argentina and the association that brings together relatives of SMA patients in the United States, CureSMA, and another grant from the Lounsbery Foundation, also from the United States. Besides, the study had the support the University of Buenos Aires (UBACYT 20020170100046BA), the Agencia Nacional de Promoción Científica y Tecnológica of Argentina (PICT-2019 862) y el CONICET (PUE 22920170100062CO).

The recognition of a Nobel

Phil Sharp, Nobel Prize winner in Physiology or Medicine for his discovery of splicing in 1993 and professor at the Massachusetts Institute of Technology (MIT), spoke about the work in glowing terms:

“This study is fascinating because it opens up new fundamental questions about gene activity and relates them directly to current medical treatments,” the scientist said.


Cell cover

The study led by Alberto Kornblihtt and whose first author is Luciano Marasco was selected to illustrate the cover of Cell journal.

The cover, designed by Lucia Giono and inspired by the mobiles of the sculptor Alexander Calder (1898-1976), symbolizes the mechanism of the combined therapy for SMA proposed in the article. Nusinersen, the drug designed by Adrián Krainer, acts by promoting the inclusion of exon 7 (E7) in SMN2 SMN2 mRNA, restoring SMN protein levels. In this issue, Marasco et al. show that a nusinersen like oligonucleotide also causes histone methylation at the SMN2 gene (H3K9me2, green plate), mitigating its effect on E7 inclusion. However, drugs that stimulate histone acetylation (H3K9Ac, red plate) counterbalance this effect, favoring E7 (white plate) inclusion between E6 (blue plate) and E8 (yellow plate), which supports a potential combined therapy for SMA.

By Miguel Faigón

Translation: Cintia González