DNA treatment can slow the paralysis that affects almost all patients with ALS

Resume: Drugs from DNA designers restored levels of a protein essential to motor neuron function and restored activity compromised due to ALS.

Source: UCSD

In virtually all individuals with amyotrophic lateral sclerosis (ALS) and in more than 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 cell’s nucleus.

This, in turn, causes the loss of stathmin-2, a protein critical to the regeneration of neurons and maintaining their connections to muscle fibers, essential for contraction and movement.

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

“Using mouse models that we designed to misprocess their stathmin-2-encoding RNAs, as 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 body.” nervous system,” Cleveland said.

Cleveland is widely credited with developing the concept of designer DNA drugs, which turn on or 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 is approved to treat a neurodegenerative disease in children called spinal muscular atrophy.

The new study builds on 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, the loss of TDP-43 affects the motor neurons that innervate and contract skeletal muscles, causing them to degenerate and ultimately result in paralysis.

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Several designer DNA drugs are currently in clinical trials for multiple diseases. One such drug is approved to treat a neurodegenerative disease in children called spinal muscular atrophy. The image is in the public domain

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

“Without stathmin-2, motor neurons disconnect from muscles, leading to the paralysis that is characteristic of ALS. What we have now found is that we can mimic TDP-43 function with a designer DNA drug, restoring the proper stathmin-2 RNA and protein level in the mammalian nervous system.”

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

The injections corrected the misprocessing of STMN2 pre-mRNA and completely restored stathmin-2 protein expression 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 taking 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 from the 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 of Ionis Pharmaceuticals; Aamir Zuberi, Maximilliano Presa, Elena Gonzalo-Gil, and Cathleen Lutz, all from 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-Turenne, 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 cases of amyotrophic lateral sclerosis (ALS) and about half of frontotemporal dementia. In ALS, the motor neurons that innervate skeletal muscles and cause contraction degenerate, resulting in paralysis. One of the most abundant motor neuron mRNAs encodes stathmin-2, a protein required for axonal regeneration and maintenance of neuromuscular junctions (NMJs). Loss of functional TDP-43 is associated with mishandling of the STMN2 RNA precursor, which is driven by the use of cryptic splicing and polyadenylation sites and produces a truncated RNA encoding a non-functional 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 by which TDP-43 enables proper processing of STMN2 mRNAs and to develop methods to restore stathmin-2 synthesis in neurons with TDP-43 dysfunction.


We found that TDP-43 binds to a 24-base GU-rich motif in 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) destroyed TDP-43 binding and caused constitutive misprocessing of STMN2. Correct processing of this change STMN2 pre-mRNA was restored by MCP binding, suggesting that TDP-43 functions normally 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 post-injury axonal regeneration in human motor neurons lacking TDP-43 was achieved with sterically binding antisense oligonucleotides (ASOs). Humanization (by introducing 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 caused chronic Stmn2 pre-mRNA misprocessing independent of TDP-43 level. ASOs were identified that constitutively humanized when injected into mouse cerebrospinal fluid Stmn2 RNA misprocessing, restored stathmin-2 mRNA and protein levels.


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

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