DNA treatment could delay paralysis that affects almost all patients with ALS

Summary: DNA designer therapeutics restored levels of a protein critical to motor neuron function and restored activity impaired as a result of ALS.

Source: UCSD

In virtually all people 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 loses its normal position in the nucleus.

This, in turn, triggers the loss of stathmin-2, a protein critical to regenerating neurons and maintaining their connections to muscle fibers, which are 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 a stathmin-2 Losses that occur can be salvaged using designer DNA drugs that restore normal processing of protein-coding RNA.

“Using mouse models that we constructed to misprocess their stathmin-2-encoding RNAs, as in these human diseases, we show that administering one of these designer DNA drugs to the fluid lining the brain and spinal cord surrounding that restores normal stathmin-2 levels throughout the nervous system,” Cleveland said.

Cleveland is widely credited with developing the concept of designer DNA drugs that either 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 has been approved for the treatment of a childhood neurodegenerative disease called spinal muscular atrophy.

The new study builds on ongoing research by Cleveland and others on the role and loss of TDP-43, a protein implicated in ALS, AD and other neurodegenerative diseases. In ALS, TDP-43 loss affects the motor neurons that innervate skeletal muscles and cause them to contract, causing them to degenerate and eventually lead to paralysis.

Several designer DNA drugs are currently in clinical trials for multiple diseases. One such drug has been approved for the treatment of a childhood neurodegenerative disease 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 encode many proteins. Decreased TDP-43 activity causes misassembly of the RNA-encoding stathmin-2, a protein required to maintain motor neuron connectivity to muscle,” Cleveland said.

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

Specifically, the researchers engineered genes in 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 the species and alter the way their final RNAs are assembled – into the cerebrospinal fluid.

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

“Our findings form the basis of a clinical trial of delaying 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 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 Jafarnejad, 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 Harvard University Broad Institute and Massachusetts Institute of Technology.

About this news from genetics and ALS research

Author: Scott LaFee
Source: UCSD
Contact: Scott LaFee-UCSD
Picture: 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 a hallmark of neurodegenerative diseases termed 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 muscle and trigger contraction degenerate, leading to paralysis. One of the most abundant motoneuron mRNAs encodes stathmin-2, a protein necessary for axonal regeneration and maintenance of neuromuscular junctions (NMJs). The loss of functional TDP-43 is accompanied by incorrect processing of the STMN2 RNA precursor powered by the use of cryptic splicing and polyadenylation sites, producing a truncated RNA encoding a nonfunctional stathmin-2 fragment.


Given that stathmin-2 is essential for axonal repair after injury and maintenance of the NMJ, a key interest in TDP-43 proteinopathies is to determine the mechanism by which TDP-43 enables the 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 binds to a 24-base GU-rich motif within the first intron of STMN2 Pre-mRNA was required to suppress cryptic splicing and polyadenylation. Conversion of this GU-rich binding motif to a 19-base sequence bound to the MS2 bacteriophage coat protein (MCP) disrupted TDP-43 binding and resulted in a constitutive misprocessing of STMN2. Correct processing of these changed STMN2 Pre-mRNA was regenerated 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 3′ cryptic splice acceptor, not the cryptic polyadenylation site, was essential for initiation STMN2 Pre-mRNA misprocessing.

Using steric-binding antisense oligonucleotides (ASOs), stathmin-2 expression and axonal regeneration were rescued after injury in TDP-43-deprived human motor neurons. Humanization (by inserting the human STMN2 cryptic exon) sensitized mouse Stmn2 to the TDP-43 expression level. Mice alternately humanized with the cryptic exon containing a disrupted TDP-43 binding site produces chronic Stmn2 Pre-mRNA misprocessing independent of TDP-43 level. ASOs were identified that became constitutively humanized when injected into the cerebrospinal fluid of mice Stmn2 RNA misprocessing, restored stathmin-2 mRNA and protein levels.


We have found that TDP-43 binding occurs 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-directed CRISPR effectors and ASOs that were restored STMN2 Values ​​despite reduced TDP-43. ASO cerebrospinal fluid injection, an approach amenable to human therapy, rescued stathmin-2 protein levels in the central nervous system of mice with chronic misprocessing Stmn2 pre-mRNAs.

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