MDA 2023: Designer DNA drug for ALS shows promise in mouse studies

New treatment targets stathmin-2 protein, tied to nerve cell dysfunction

Marisa Wexler, MS avatar

by Marisa Wexler, MS |

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An illustration for the MDA Clinical & Scientific Conference shows a bold MDA acronym against a background of cells and synapses.

In most cases of amyotrophic lateral sclerosis (ALS), dysfunction of a protein known as TDP-43 leads to abnormally low levels of another protein, stathmin-2, which is sufficient to drive nerve cell dysfunction, new data suggest.

In an aim to restore stathmin-2 levels, researchers created a designer DNA treatment. Now, that experimental therapy has shown promise in early mouse studies.

Results from the research were shared by Clotide Lagier-Tourenne, MD, PhD, a professor at Massachusetts General Hospital and Harvard Medical School, at the Muscular Dystrophy Association’s MDA Clinical & Scientific Conference, held March 19-22 in Dallas and virtually. The talk was titled “Stathmin-2: an emerging therapeutic target in TDP-43 proteinopathies.”

The data also were detailed in the study, “Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies,” which was published in Science.

“Our findings lay the foundation for a clinical trial to delay paralysis in ALS by maintaining stathmin-2 protein levels in patients using our designer DNA drug,” Don Cleveland, PhD, a professor at the University of California San Diego (UCSD) and co-author of the study, said in a university press release.

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Creating a designer DNA therapy to restore stathmin-2

Nearly all cases of ALS are characterized by abnormalities with the protein TDP-43. Normally, TDP-43 is located in the nucleus, the cellular compartment that houses DNA, but in ALS, the protein moves outside of the nucleus, disrupting its normal function.

TDP-43 has long been known to be involved in processing messenger RNA (mRNA) — an intermediary molecule made when genes are read to make protein. In particular, TDP-43 plays essential roles in mRNA splicing.

Within a cell’s DNA, genes contain exons — the parts of the genetic code that have instructions for making proteins — that are interspersed with introns. While introns don’t provide instructions for making protein, they have other regulatory functions.

When a gene is read, the entire sequence normally is copied over into mRNA, and then the mRNA undergoes splicing to remove all the introns and string the exons together. The final mature mRNA sequence is then sent to the cell’s protein-making machines, which can read the code to make protein.

“Now, as a field, we know that TDP-43 binds thousands of targets, and actually leads to hundreds if not thousands of RNA alterations and RNA splicing,” Lagier-Tourenne said.

In cell experiments, the researchers showed that reducing TDP-43 levels leads to a change in splicing for the gene STMN2, which provides instructions for making the protein stathmin-2. Specifically, the lack of TDP-43 led to the inclusion of an extra sequence called a cryptic exon, which is normally removed from the mature code.

The researchers showed that TDP-43 normally binds to the cryptic exon sequence, preventing it from being included in the mature mRNA. Without TDP-43, the cryptic exon gets included in the mature sequence, but other exons get left out, resulting in a shortened mRNA that cannot be used to produce the stathmin-2 protein. The end result is that dysfunctional TDP-43 leads to reduced levels of stathmin-2.

In their study, the team found high levels of the shortened STMN2 mRNA containing the cryptic exon in spinal cord samples from people with sporadic ALS. However, the abnormal mRNA was not present in people without ALS. Consistently, levels of stathmin-2 protein were decreased in the sporadic ALS patients, but not in spinal cords unaffected by ALS.

The abnormal mRNA also was present in people with ALS caused by mutations in the C9ORF7 gene, which is known to cause TDP-43 abnormalities. However, patients with mutations in the gene SOD1 — a rare genetic form of ALS that typically doesn’t cause abnormalities with TDP-43 — did not have the abnormal mRNA.

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The stathmin-2 protein is known to play roles in providing structural support to nerve cells and axons — the long fibers that nerves use to connect with each other. The researchers showed that motor neuron cells lacking stathmin-2 were unable to regrow after an axon injury. Cells lacking TDP-43 and, consequently, with low stathmin-2 levels, also were not able to regrow.

However, when the researchers engineered cells lacking TDP-43 so that they could express higher levels of stathmin-2 protein, the cells’ ability to regrow was restored.

“What was very surprising was that, in these motor neurons where we have TDP-43 knock-down, we know there are hundreds if not thousands of [RNA] expression and splicing alterations, and restoring stathmin-2 was sufficient to completely rescue the regeneration ability of these motor neurons,” Lagier-Tourenne said.

In mice, reducing levels of stathmin-2 in the nervous system led to progressive motor weakness and dysfunction, with poorer connections between nerve and muscle cells. These findings collectively implied that increasing stathmin-2 levels could be therapeutic in ALS.

Our findings lay the foundation for a clinical trial to delay paralysis in ALS by maintaining stathmin-2 protein levels in patients using our designer DNA drug.

The researchers created a designer DNA drug, called an antisense oligonucleotide, which basically aims to mimic the function of TDP-43, thereby correcting splicing of STMN2 mRNA and restoring production of stathmin-2 protein.

A similar type of therapy is approved in the U.S. and other countries to treat spinal muscular atrophy, a genetic disorder that, like ALS, affects motor neurons.

“What we have now found is that we can mimic TDP-43 function with a designer DNA drug, thereby restoring correct stathmin-2 RNA and protein level in the mammalian nervous system,” Cleveland said.

To test the experimental therapy, the researchers used mice engineered to express a version of the human STMN2 gene that lacked sites for TDP-43 to bind — meaning that these mice always produced the abnormal, truncated mRNA, and had low stathmin-2 levels as a result. Treatment with the experimental DNA therapy restored stathmin-2 levels.

“With mouse models we engineered to misprocess their stathmin-2 encoding RNAs, like in these human diseases, we show that administration of one of these designer DNA drugs into the fluid that surrounds the brain and spinal cord restores normal stathmin-2 levels throughout the nervous system,” Cleveland said.