DNA Treatment Could Delay Paralysis That Strikes Nearly All Patients With ALS

Summary: DNA designer therapeutics restored levels of a protein critical to motor neuron function, restoring the activity that is impaired as a result of ALS.

Source: UCSD

In virtually all persons with amyotrophic lateral sclerosis (ALS) and in up to half of all cases of Alzheimer’s disease (AD) and frontotemporal dementia, a protein called TDP-43 is lost from its normal location in the nucleus of the cell.

In turn, this triggers the loss of stathmin-2, a protein crucial to regeneration of neurons and the maintenance of their connections to muscle fibers, essential to contraction and movement.

Writing in the March 16, 2023 issue of Science, a team of scientists, led by senior study author Don Cleveland, PhD, Distinguished Professor of Medicine, Neurosciences and Cellular and Molecular Medicine at University of California San Diego School of Medicine, with colleagues and elsewhere, demonstrate that stathmin-2 loss can be rescued using designer DNA drugs that restore normal processing of protein-encoding RNA.

“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.

Cleveland is broadly credited with developing the concept of designer DNA drugs, which act to either turn on or turn off genes associated with many degenerative diseases of the aging human nervous system, including ALS, AD, Huntington’s disease and cancer.  

Several designer DNA drugs are currently in clinical trials for multiple diseases. One such drug has been approved to treat a childhood neurodegenerative disease called spinal muscular atrophy.

The new study builds upon ongoing research by Cleveland and others regarding the role and loss of TDP-43, a protein associated with ALS, AD and other neurodegenerative disorders. In ALS, TDP-43 loss impacts the motor neurons that innervate and trigger contraction of skeletal muscles, causing them to degenerate, eventually resulting in paralysis.

This shows DNA
Several designer DNA drugs are currently in clinical trials for multiple diseases. One such drug has been approved to treat a childhood neurodegenerative disease called spinal muscular atrophy. Image is in the public domain

“In almost all of instances of ALS, there is aggregation of TDP-43, a protein that functions in maturation of the RNA intermediates that encode many proteins. Reduced TDP-43 activity causes misassembly of the RNA-encoding stathmin-2, a protein required for maintenance of the connection of motor neurons to muscle,” said Cleveland. 

“Without stathmin-2, motor neurons disconnect from muscle, driving paralysis that is characteristic of ALS. 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.”

Specifically, the researchers edited genes in mice to contain human STMN2 gene sequences and then injected antisense oligonucleotides — small bits of DNA or RNA that can bind to specific RNA molecules, blocking their ability to make a protein or changing how their final RNAs are assembled — into cerebral spinal fluid.

The injections corrected STMN2 pre-mRNA misprocessing and restored stathmin-2 protein expression fully independent of TDP-43 function.

“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,” Cleveland said. 

Co-authors include: Michael W. Baughn, Jone López-Erauskin, Melinda S. Beccari, Roy Maimon, Sonia Vazquez-Sanchez, Jonathan W. Artates and Eitan Acks, all at Ludwig Institute for Cancer Research-UC San Diego and UC San Diego; Ze’ev Melamed, Ludwig Institute for Cancer Research-UC San Diego, UC San Diego, and The Hebrew University of Jerusalem; Karen Ling, Paayman Jafar-nejad, Frank Rigo and C. Frank Bennett, all at Ionis Pharmaceuticals; Aamir Zuberi, Maximilliano Presa, Elena Gonzalo-Gil and Cathleen Lutz, all at The Jackson Laboratory; Som Chaturvedi, Mariana Bravo-Hernández, Vanessa Taupin and Stephen Moore, all at UC San Diego; L. Sandra Ndayambaje and Ana R. Agra de Almeida Quadros, Harvard Medical School; Clotilde Lagier-Tourenne, Harvard University and Broad Institute of Harvard University and Massachusetts Institute of Technology.

About this genetics and ALS research news

Author: Scott LaFee
Source: UCSD
Contact: Scott LaFee – UCSD
Image: The image is in the public domain

Original Research: Closed access.
Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies” by Don Cleveland et al. Science


Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies


Nuclear clearance and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of neurodegenerative diseases called TDP-43 proteinopathies. This includes almost all instances of amyotrophic lateral sclerosis (ALS) and about half of frontotemporal dementia. In ALS, the motor neurons that innervate and trigger contraction of skeletal muscles degenerate, resulting in paralysis. One of the most highly abundant motor neuron mRNAs encodes stathmin-2, a protein necessary for axonal regeneration and maintenance of neuromuscular junctions (NMJs). Loss of functional TDP-43 is accompanied by misprocessing of the STMN2 RNA precursor, which is driven by use of cryptic splicing and polyadenylation sites, and producing a truncated RNA that encodes a nonfunctional stathmin-2 fragment.


Recognizing that stathmin-2 is essential for axonal recovery after injury and NMJ maintenance, a central interest in TDP-43 proteinopathies is to determine the mechanism through which TDP-43 enables correct processing of STMN2 mRNAs and to develop methods to restore stathmin-2 synthesis in neurons with TDP-43 dysfunction.


We found that TDP-43 binding to a 24-base, GU-rich motif within the first intron of the STMN2 pre-mRNA was required to suppress cryptic splicing and polyadenylation. Conversion of this GU-rich binding motif into a 19-base sequence bound by the MS2 bacteriophage coat protein (MCP) ablated TDP-43 binding and produced constitutive misprocessing of STMN2. Correct processing of this modified STMN2 pre-mRNA was restored by binding of MCP, suggesting that TDP-43 normally functions by sterically blocking access to the cryptic sites of RNA-processing factors. Further genome editing revealed that the cryptic 3′ splice acceptor, not the cryptic polyadenylation site, was essential for initiating STMN2 pre-mRNA misprocessing.

Rescue of stathmin-2 expression and axonal regeneration after injury in human motor neurons depleted of TDP-43 was achieved with steric binding antisense oligonucleotides (ASOs). Humanization (by insertion of the human STMN2 cryptic exon) sensitized mouse Stmn2 to TDP-43 expression level. Mice alternately humanized with the cryptic exon containing a disrupted TDP-43 binding site produced chronic Stmn2 pre-mRNA misprocessing independent of TDP-43 level. ASOs were identified that when injected into cerebral spinal fluid of mice with constitutive humanized Stmn2 RNA misprocessing, restored stathmin-2 mRNA and protein levels.


We determined that TDP-43 binding in the first intron of the STMN2 pre-mRNA sterically blocked access of RNA processing factors that would otherwise recognize and use a cryptic 3′ splice site. We identified RNA-targeted CRISPR effectors and ASOs that restored STMN2 levels despite reduced TDP-43. ASO injection into cerebral spinal fluid, an approach feasible for human therapy, rescued stathmin-2 protein levels in the central nervous system of mice with chronically misprocessed Stmn2 pre-mRNAs.

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