DNA treatment could delay the paralysis that strikes nearly all ALS patients

Summary: DNA design therapies have restored levels of a protein critical to motor neuron function, restoring activity that is 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 is lost from its normal location in the cell nucleus.

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

Written in the March 16, 2023 issue of Sciencea team of scientists, led by lead study author Don Cleveland, PhD, professor emeritus of medicine, neuroscience, and cellular and molecular medicine at the University of California San Diego School of Medicine, with colleagues and elsewhere demonstrate that the loss of stathmin-2 can be rescued using designer DNA drugs that restore normal protein-coding RNA processing.

“With mouse models that we have designed to misprocess their RNAs encoding stathmin-2, as in these human diseases, we show that administering one of these DNA-based drugs in the fluid that surrounds the brain and spinal cord restores normal levels of stathmin-2 throughout life. nervous system,” Cleveland said.

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

Several tailor-made 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 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 motor neurons that innervate and trigger contraction of skeletal muscles, causing them to degenerate, eventually resulting in paralysis.

This shows DNA
Several tailor-made 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 public domain

“In almost all cases of ALS, there is aggregation of TDP-43, a protein that functions in the processing of RNA intermediates that code for many proteins. Reduced activity of TDP-43 causes improper assembly of stathmin-2 encodes RNA, a protein necessary to maintain motor neuron connection to muscle,” Cleveland said.

“Without stathmin-2, motor neurons disconnect from muscle, causing the characteristic paralysis of ALS. What we have now discovered is that we can mimic TDP-43 function with a designer DNA drug, thereby restoring the correct level of stathmin-2 RNA and protein to the nervous system of mammals.

Specifically, the researchers edited genes in mice to contain human STMN2 gene sequences, 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 altering the way their final RNAs are assembled – in the cerebrospinal fluid.

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

“Our findings lay the groundwork for a clinical trial to delay paralysis in ALS by maintaining stathmin-2 protein levels in patients using our DNA-based 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 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 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 La Fee – UCSD
Picture: Image is in public domain

Original research: Access closed.
“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 are hallmarks 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, motor neurons that innervate and trigger skeletal muscle contraction degenerate, resulting in paralysis. One of the most abundant motor neuron mRNAs codes for stathmin-2, a protein necessary for axonal regeneration and maintenance of neuromuscular junctions (NMJs). Loss of functional TDP-43 is accompanied by poor processing of the STMN2 RNA precursor, which is driven by the use of cryptic splice and polyadenylation sites, and produces truncated RNA that encodes a non-functional stathmin-2 fragment.


Recognizing that stathmin-2 is essential for axonal recovery after injury and maintenance of NMJ, a central interest in TDP-43 proteinopathies is to determine the mechanism by which TDP-43 enables proper processing of STMN2 mRNA 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 to a 19-base sequence bound by bacteriophage MS2 coat protein (MCP) suppressed TDP-43 binding and produced constitutive misprocessing of STMN2. Proper handling of this change STMN2 pre-mRNA was restored by MCP binding, suggesting that TDP-43 functions normally by sterically blocking access to cryptic sites of RNA processing factors. Further genome editing revealed that the 3′ cryptic splice acceptor, not the cryptic polyadenylation site, was essential for initiating STMN2 pre-mRNA processing error.

Rescue of stathmin-2 expression and axonal regeneration after injury in TDP-43-depleted human motor neurons has been achieved with sterically bound antisense oligonucleotides (ASO). Humanization (by inserting the human STMN2 cryptic exon) sensitized mouse Stmn2 at the TDP-43 expression level. Humanized mice alternating with the cryptic exon containing a disrupted TDP-43 binding site produced a Stmn2 pre-mRNA processing error independent of TDP-43 level. ASOs have been identified which, when injected into the cerebrospinal fluid of mice with constitutive humanization Stmn2 Improper RNA processing, restoration of stathmin-2 mRNA and protein levels.


We have determined that the binding of TDP-43 in the first intron of the STMN2 the pre-mRNA sterically blocked the 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 despite the reduction in TDP-43. Cerebrospinal fluid injection of ASO, a feasible approach for human therapy, rescued stathmin-2 protein levels in the central nervous system of chronically abused mice Stmn2 pre-mRNA.

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