Drugs that may enter phase III (2019).

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ALS drugs that may enter a phase III trial in 2019


Arimoclomol is an experimental drug developed by CytRx Corporation. Arimoclomol is believed to function by stimulating a normal cellular protein repair pathway through the activation of molecular chaperones. Since damaged proteins, called aggregates, are thought to play a role in many diseases, CytRx believes that arimoclomol could treat a broad range of diseases.

Heat shock transcription factor 1 (HSF1), a central coordinator of the chaperone response, helps cells managing proteins that are misfolded or aggregated. HSF1, which is activated during times of stress, switches on multiple genes, including the gene encoding the disaggregase Hsp40.


NurOwn is an experimental cell-based therapy by BrainStorm Cell Therapeutics that contains autologous cultured mesenchymal bone marrow stromal cells secreting neurotrophic factors including BDNF, GDNF and HGF, as a possible treatment for patients with ALS.

Brainstorm’s NurOwn therapy consists of bone marrow stem cells taken from each individual person, differentiated into cells that make neuroprotective growth factors, and infused back into muscle or the spinal cord.

Brainstorm’s therapeutic differs from that of the better-known Neuralstem, Inc., of Rockville, Maryland, which is transfusing neural stem cells from fetal tissue into the spinal cords of people with ALS.

Though it has great potential for clinical applications, the differentiation of MSCs is precisely regulated and coordinated by mechanical and molecular signals from the extracellular environment and involves complex pathways at the transcriptional and post-transcriptional levels that remain largely unexplored.

MSC-NTF cells are Mesenchymal Stromal Cells (MSC) induced to express high levels of neurotrophic factors (NTFs) using a culture-medium based approach.


ODM-109 aims in part, to improve breathing in ALS by improving the performance of muscles in the diaphragm. The drug candidate, also known as oral levosimendan, increases the force of contraction of certain muscles by boosting the calcium sensitivity of troponin C.

The drug candidate is an oral formulation of levosimendan. An intravenous formulation of levosimendan, marketed under the name Simdax, is clinically approved in some countries for the treatment of acute heart failure. Levosimendan's positive inotropic and vasodilator effects are tied to its abilities to increase calcium sensitivity and open ATP-sensitive potassium positive ion (K+) channels (mitoKATPchannels)

Levosimendan favourably affects mitochondrial adenosine triphosphate synthesis, conferring cardioprotection and possible neuronal protection during ischemic insults. In a model of spinal cord injury, levosimendan has been reported to attenuate neurologic motor dysfunction. This finding is supported by the fact that the selective mitoKATPchannel opener, diazoxide, is an effective neuroprotectant, as has been demonstrated in an ischemia reperfusion study in rats.


IONIS-SOD1Rx is a generation 2.0 antisense drug specifically designed to inhibit production of mutant superoxide dismutase (SOD1). SOD1 mutations account for approximately 20% of familial ALS cases.

This drug is the result of a collaboration between Biogen and IONIS Pharmaceuticals (formerly ISIS). A Phase III clinical trial is recruiting participants as of April 2019. Study completion is expected in May 2020.

Researchers are also considering antisense treatment for another genetic form of ALS caused by expansions in the C9ORF72 gene. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. The antisense oligonucleotide works by targeting and attaching itself to the stretch of RNA with the mistake so that the protein cannot be formed,, so prevent accumulation of these outside the nucleus.

Part of Isis’ success stems from chemically modifying oligonucleotides to make them last longer in the body and bind more tightly to their target RNAs.

SOD1Rx, like Kynamro, includes 2′-O-methoxyethyl sugars on its backbone. This modification typifies Isis’ second generation of oligonucleotide chemistry, but the company has developed other options. For SOD1 antisense, they plan to make it more potent before starting further safety trials, probably with higher doses and longer treatment times.

Split hand syndrome and ALS

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What is the split hand syndrome?

Many scientists subscribe to the “dying back” hypothesis, whereby degeneration begins at the neuromuscular junction when motor neurons retreat from the synapse. A few others, prefer the “dying forward” or upper motor neuron hypothesis. They believe ALS begins in the brain, before spreading to lower motor neurons. In medicine, the split hand syndrome is a neurological syndrome in which thumb hand muscles undergo mass loss, while the muscles on the side of the little finger are spared. This makes it difficult to grab small objects between thumb and forefinger. If there are no lesions affecting the branches of the ulnar nerve that are directed to the unused muscles, it is almost certain that the lesion is located in the anterior horn of the spinal cord at C8-T1.This area is often associated with ALS and is the place where higher motor neurons join lower motor neurons. This syndrome has been proposed as a relatively specific sign of amyotrophic lateral sclerosis, but it can also occur in other anterior horn disorders, such as spinal muscular atrophy, Charcot-Marie-Tooth disease, poliomyelitis and progressive muscle atrophy. The phenomenon is observed in more than half of ALS patients, and the underlying mechanism is not fully understood. To a certain extent, these characteristics can also be observed during normal aging. The term split-hand syndrome was coined for the first time in 1994 by a Cleveland Clinic researcher named Asa J. Wilbourn.

How does this syndrome relate to ALS?

Composite motor action potential (CMAP) is an electromyographic study (electrical study of muscle function). Several studies have shown a significant reduction in the amplitude of motor action potentials during low frequency repetitive nerve stimulation (RNS) of muscles involved in ALS.

The motor plate is a type of synapse that allows the transmission of a nerve message from a nerve fiber to a muscle fiber in the form of a chemical message by neurotransmitters that will bind to the specific receptors on the surface. muscle fibers. It is not known if the dysfunction of the motor plate is involved in the formation of the divided hand.

A study showing that neuromuscular junction degradation is linked to this syndrome

Dong Zhang, Yuying Zhao, Yan Chuanzhu, Lili Cao and Wei Li have studied the dysfunctions of the neuromuscular junction in different muscles of the hand in patients with ALS, to determine if these dysfunctions are related to the phenomenon of the divided hand. This clinical study at Shandong University's Qilu Hospital enrolled 51 ALS patients, 24 patients with myasthenia gravis had a decrease in RNS, and 20 patients with Lambert Eaton Myasthenia Gravis Syndrome (LEMS).

Who were the patients?

The mean age at onset of the 51 patients with ALS was 58 years old. This group included 23 women and 28 men. The evolution of their disease has varied from 5 to 24 months. Of these, 36 patients had upper limbs, 10 lower limbs and 5 patients had a bulbar form. Patients with myasthenia gravis included 9 men and 15 women, and the mean age was 44 years. The LEMS patients included 16 men and 4 women, and the average age was 59 years old.

What did this study find?

Among the fifty-one ALS patients, thirty-one patients had a split of the hand, 24 patients with the upper limb form and 6 patients with the lower limb form. There was no statistical difference in the frequency of hand splitting between the upper limb group and the lower limb group. There was no hand fracture in patients with bulbar-type ALS. This study showed that more than 60% of the hand muscles of ALS patients had a negative △ D similar to that of patients with myasthenia gravis, but significantly different from that of patients with LEMS, suggesting that Postsynaptic abnormalities could play a major role.


A dysfunction of neuromuscular transmission has been found in the hand muscles of patients with ALS, it is confirmed that the abductor pollicis brevis (short abductor muscle of the thumb) is involved in this syndrome. The dysfunction of the neuromuscular transmission of this muscle could be involved in the formation of the split hand phenomenon. While not a breakthrough, this study highlight a disease starting at the neuromuscular junction, not at the interface between upper neurons and lower neurons in the spine.

Orphan drug designation to APB-102

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FDA has granted orphan drug designation to APB-102, a gene therapy for SOD1 ALS

Familial ALS, which represents about 10% of all ALS cases, is inherited as a dominant trait. About 20% of these cases arise from mutations in the gene encoding cytosolic Cu/Zn superoxide dismutase 1 (SOD1 ). An estimated 12 to 23% of patients with familial ALS and 1 to 3% of patients with sporadic ALS carry a mutation in this gene; 185 mutations in SOD1 have been identified. So if ALS is a rare disease, each mutation of SOD1 is an extremely improbable case, roughly one out of 100,000,000!

An elusive disease

Multiple mechanisms have been proposed to explain why mutant SOD1 proteins are neurotoxic, including the observation that mutant SOD1 acquires toxicity via conformational instability, misfolding, and some degree of aggregation. In turn, this activates multiple adverse events that include the unfolded protein response, endoplasmic reticulum (ER) stress, mitochondrial damage, heightened cellular excitability, impaired axonal transport, and some elements of apoptotic and necrotic cell death. Some data suggest that misfolded mutant SOD1 protein can spread from cell to cell in a prion-like fashion. Additionally, it is proposed that mutant SOD1 can cause toxic misfolding of wild-type SOD1.

Toward a therapy for ALS

The therapeutic silencing of SOD1 has been pursued by many groups, using various modalities: antisense oligonucleotides (ASOs), RNA interference (RNAi), viral vector-delivered RNAi, and CRISPR-Cas9. From a clinical perspective, one of the major disadvantages of ASOs and small interfering RNAs is the repeated dosing of the patients, whereas rAAV-mediated gene therapy (including gene transfer and RNAi-based gene silencing) relies on a one-time dosing paradigm.

side effects

Technological improvements allow the ASO doses to be less frequent than in the past, for example, by nusinersen (Spinraza), a recently approved ASO developed as a treatment for spinal muscular atrophy (SMA) by Biogen and Ionis Pharmaceuticals. With this drug, a typical patient would receive three intrathecal doses yearly upon completion of the loading doses In contrast, as an example, AVXS-101, a gene therapy treatment developed by AveXis as a treatment for SMA type 1, has a therapeutic effect for up to 24 months after a single intravenous injection of a rAAV9 vector. However AVXS-101 has side effects like asymptomatic liver enzyme elevations. These types of adverse events have been observed with other gene therapy trials.

What did Apic Bio?

A potential therapy for SOD1 is to suppress the expression of the mutant gene, whatever its mutation. Indeed SOD1 has a role and suppressing its expression, even the mutant one, will create side effects. Apic Bio investigated silencing of SOD1, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1 .

In recent years, Apic Bio and others have investigated this strategy in depth using various modalities. Apic Bio have previously demonstrated the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis ) using an AAV serotype for delivery that has been shown to be safe in clinical trials. They optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30° during intrathecal infusion. Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1 -linked ALS. They selected a recombinant adeno-associated viral vector serotype rh.10 (rAAVrh.10) because of its excellent central nervous system (CNS) transduction and safety profile in nonhuman primates. The presence of GFP in their vectors caused mild liver toxicity, as previously described, and a cellular immune response in two of eight animals. The fact that the immune response is not detected in all the injected animals can be explained by the early sacrifice point (22 days).

Orphan drug designation

The U.S. Food and Drug Administration (FDA) has granted orphan drug designation to APB-102, a gene therapy soon to be in clinical development for the treatment of genetic SOD1 amyotrophic lateral sclerosis (ALS). The U.S. FDA Orphan Drug program provides orphan designation to novel drugs that are intended for the treatment of rare diseases (those affecting fewer than 200,000 people in the United States). The designation provides sponsors with development and commercial incentives including seven years of market exclusivity in the US, consultation by FDA on clinical study design, potential for expedited drug development, and certain fee exemptions and reductions.

What is next?

Having an orphan drug designation in ALS is not a so big deal, several dozens drugs got it for ALS and it was withdrawn a few years later by FDA, when it was obvious they were not efficient at all. The important thing is now to wait for clinical trials. For that Apic Bio needs money, so probably they will solicit investors. And in this perspective, having an orphan drug designation will help them a lot.

Is it possible that a medication treating congestive heart failure can improve the breathing of people with ALS? Or that a drug used to treat cancer could reduce motor neuron inflammation and possibly slow the progression of the disease?

The reuse of drugs is not a new idea. Many drugs have found a new function - for example tamoxifen, originally developed to treat breast cancer, is now used in the treatment of bipolar disorder.

So, how can a medicine that treats a disease, act for another disease?

Obviously, once a drug enters the body, we have little control over its delivery. Although it can be designed to treat, for example, kidney cells, it also travels and interacts in other places. It is these "non-targeted" effects that cause the side effects of drugs. Sometimes, however, this disruption can have positive effects and it is these beneficial results that drug reuse attempts to exploit.

However, we can not take an approved medicine and give it to people with another disease simply because we think it could work for them. Pre-clinical tests and clinical trials are still needed. A safe dose must be established for the new therapeutic target, a certain degree of efficacy must be established, and we need to understand the benefits and risks before the drug can be made available as a new treatment.

MIROCALS - IL-2: From cancer treatment to motor neuron protection

This trial tests interleukin-2 (IL-2), a drug already used to treat some forms of cancer. IL-2 is naturally produced by the body. Its main role is to promote the production of regulatory T cells (or Tregs) - a part of the immune system that is thought to play a role in protecting nerve cells from damage. IL-2 can increase blood levels of Treg and could protect motor neurons in ALS, slowing the progression of the disease.

Studies have already identified the lowest dose of IL-2 that still triggers an increase in Treg without serious side effects.

The goal of this phase 2 trial is to evaluate the safety and efficacy of IL-2 and to confirm that altering the immune response by increasing the Treg rate will slow down the progression of ALS. The study will recruit 216 participants and the results are expected in autumn 2021.

TUDCA - a treatment for liver disease that could protect motor neurons from programmed cell death

Tauroursodeoxycholic acid (TUDCA) is a bile acid. Bears contain large amounts of TUDCA in their bile.

TUDCA prevents apoptosis of cells through its inhibitory role in the transport of BAX to mitochondria.

TUDCA is a water-soluble bile salt used in the treatment of cholestasis, a liver disease in which bile acid accumulates in an unhealthy liver, damaging cells by destroying membranes and signaling cell death. TUDCA also appears to reduce the stress of the endoplasmic reticulum (ER), an organelle of the cell that facilitates the folding of proteins. By reducing the stress of the endoplasmic reticulum, TUDCA can protect against neurological damage.

The aim of this phase 3 trial is to evaluate the safety and efficacy of TUDCA as a complementary therapy to riluzole, as measured by ALSFRS-R scores, in 440 people with ALS. complete in the summer of 2022. The ALSFRS-R is used to assess and monitor functional changes in a person with ALS over time. It consists of 12 questions that deal with aspects of the person's daily life, each of which is rated by the person from 4 to 0, with 4 being "normal".

You can find out more about the TUDCA clinical trial on the TUDCA website and on clinicaltrials.gov.

Perampanel - antiepileptic drug that could prevent the toxic accumulation of TDP-43

It was the first antiepileptic drug in the class of selective noncompetitive AMPA receptor antagonists. This medication can lead to serious psychiatric and behavioral changes; it can cause homicidal or suicidal thoughts. In a mouse model of ALS, Perampanel has been shown to prevent motor neuron death by stopping the toxic accumulation of TDP-43 protein. Long-term Perampanel therapy also resulted in a visible improvement in motor function in treated mice.

The aim of this phase 2 trial is to evaluate the effect of Perampanel on disease progression (measured by ALSFRS-R) in 60 people with sporadic ALS. The results are expected for the winter of 2022. To learn more about this trial, go to clinicaltrials.gov.

Ranolazine - the drug against angina pectoris that can be neuroprotective

Used to treat angina pectoris (chest pain), ranolazine works by inhibiting the accumulation of sodium and calcium ions in cells, although the way it treats angina is not fully understood. Calcium ions play an important role in hyperexcitability when neurons "trigger" more than they would normally, causing fasciculations (muscle contractions), one of the first symptoms of ALS. Ranolazine may have a neuroprotective effect by reducing neuronal hyperexcitability, thereby slowing the progression of the disease and reducing the frequency of cramps.

The Phase 2 trial will evaluate the safety and efficacy of ranolazine in 20 people with ALS and is expected to be completed in the summer of 2019. For more information, see clinicaltrials.gov.

Pimozide - an antipsychotic that could improve muscle function

Pimozide is used in the treatment of schizophrenia and in the reduction of uncontrolled muscle tics associated with Tourette's syndrome. It works by decreasing the activity of dopamine, a neurotransmitter that sends messages between brain cells. In people with ALS, motor neuron damage results in disruption of communication between neurons and muscles at the neuromuscular junction (NMJ). Pimozide has been shown to improve communication with NMJ in mice and fish for the purpose of improving muscle function.

This phase 2 study will examine whether pimozide can help slow the progression of ALS in 100 people with the disease. The trial should be completed by the end of 2019 and you can find out more on clinicaltrials.gov.

Rapamycin - the anti-rejection drug that can prevent neurodegeneration

Used to prevent rejection of transplanted organs, rapamycin works by weakening the body's immune system to accept transplanted organs more easily. The neuron's inability to eliminate the accumulation of proteins in the cytoplasm, and an imbalanced function of the immune system that damages motor neurons by neurotoxicity rather than protecting them, are two potential influences in the development of ALS. These two mechanisms represent important therapeutic targets. In neurodegeneration models, rapamycin has been shown to suppress inflammatory neurotoxic responses caused by T cells (T cells are part of the immune system and generally protect nerve cells from damage) and aid in protein breakdown. accumulated in the cytoplasm.

The goal of this phase 2 trial, which will involve 63 people with ALS, is to obtain predictive information for a larger study. Its completion is scheduled for autumn 2019. For more information, see clinicaltrials.gov.

A plea for a gene therapy for ALS

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This draft document is an open call to the pharmaceutical industry to create a drug targeting TDP-43 proteinopathies such as Amyotrophic Lateral Sclerosis (ALS).

It describes how such a drug could be realistically produced with common laboratories technologies like antibodies or transfection. The recently approved AVXS-101 for Spinal muscular atrophy (SMA) probably shows the pathway for designing this new drug.

enter image description here

How this TDP-43 drug would work?

  • One or several therapeutics goals and molecular targets are defined in order to alter the production of mutated TPD-43.
  • Epitopes are defined for those targets.
  • Antibodies are designed from those epitopes.
  • Plasmids are then produced, that encode all different combinations of heavy and light chains purified from the selected hybridoma cell.
  • These plasmids are inserted in AAV viral vectors.
  • Once inserted behind the BBB, those viral vectors infect cells that were producing mutated TPD-43.

Now the infected cell produces TDP-43 which is modified according to the therapeutic goal defined in the first step.

What is the state of art in genetic therapy for TDP-43?

This proposal is motivated by several successes in mice models of ALS that were published in the last five years 1 and [9-11]. Similar reports have been made in a drosophila model of ALS 2. Related works have been done for SOD1 mice models [6][7][10] [12, 13] and even macaques [3]. In total, some 100 articles have been published since 2007 on these topics.

What next steps are recommended?

The next step should be human trials of ALS gene therapies, or at least experimentations in pigs model of ALS. While there are currently no clinical ALS gene therapies, nusinersen, was recently approved for SMA. AVXS-101 another gene therapy, demonstrated a dramatic increase in survival and even improvements in SMA. SMA and ALS share a number of pathological, cellular, and genetic features suggesting that clinical insights into one disorder may have value for the other [14]. Hopefully this essay could provide some impetus for experimentations to reduce levels of mutated TDP-43 in pigs model of ALS and point to a pathway toward human trials.

About ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective degeneration of both upper and lower motor neurons. Midlife patients present to the clinician with a muscle-related symptomatology. Disease then progresses to muscle atrophy, followed by complete paralysis, and death generally occurs by respiratory failure after 3 to 5 years from symptoms onset. Ninety percent of cases have sporadic origin (sALS) whereas 10 % have familial inherited mutations (fALS).

Single chain antibodies and ALS

Single-chain variable fragment (scFv), have been introduced two decades ago, through the generation of a variety of recombinant antibodies binding to various epitopes of pathological proteins implicated in the field of neurodegenerative diseases. The clinical demonstration of their efficacy in ameliorating pathological symptoms is well established.

Some single chain antibodies are have been studied for ALS [9-11] but only the scFv targeting misfolded SOD1 proved to be effective in vivo in ameliorating pathological changes and slowing down disease progression in a mouse model with ALS-linked SOD1 mutation [10, 12, 13].

The generation of a scFv antibody against TDP-43, and its therapeutic effect when delivered in ALS/FTD patients with TDP-43 pathology was reported recently [ 1] .

About TDP-43

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein, highly and ubiquitously expressed, with main localization in the nucleus of cells. TDP-43 consists of an N-terminal domain (NTD) and two tandem RNA recognition motifs, RRM1 and RRM2, followed by a C-terminal glycine-rich region (G). Thanks to its two RNA-recognition domains (RRM1 and RRM2) the protein is a multifunctional factor involved in different aspects of RNA metabolism such as transcription, splicing, stabilization and transport.

TDP-43 and ALS

Although mutations in TDP-43 are very rare, occurring in 3% of fALS and 1.5% of sALS, more than 90% of ALS cases (fALS and sALS) show a pathological behavior of this protein called TDP-43 proteinopathy. This event was first described in 2006 as a consistent mislocalization and aggregation of the protein in the cytoplasm where TDP-43 can form hyperphosphorylated, fragmented and ubiquitinated inclusions that impair the physiological function of the protein.

TDP-43 and other pathologies

TDP-43 proteinopathy is not exclusive to ALS. It is indeed present in 50% of frontotemporal lobar dementia (FTLD) patients. FTLD or FTD (frontotemporal dementia) is a midlife onset disease, clinically heterogeneous, characterized by changes in behavior, personality and/or language.

Because of TDP-43 proteinopathy, ALS and FTD are now considered as a disease continuum with 50% of ALS patients presenting cognitive impairment and 15% of FTD patients having motor impairments. Interestingly, TDP-43 proteinopathy has also been observed in other neurodegenerative disorders.

TDP-43 domains and proteinopathies.

Different studies have highlighted the sensitivity of the RRM1, RRM2 or C terminal domain in inducing TDP-43 proteinopathy. Oxidation or misfolding of this domain results in cytosolic mislocalization with irreversible protein aggregation. Apart from the RNA metabolism, the RRM1 domain is also responsible for the interaction with the p65 subunit of NF-κB, so targeting RRM1 would also diminish inflammation. SMA studies highlighted the importance of simultaneously treating multiple disease pathways. Like in SMA, it is thus clear that prognosis can be improved in ALS models by attempting a multifaceted gene therapy approach [4].

For example the genetic suppression of the NF-κB pathway in microglia and shRNA-mediated knockdown of SOD1 via systemic AAV9 administration resulted in an additive amelioration in all assessed phenotypes. The median mutant mouse lifespan was expanded from 137 to 188 days with a maximum survival of 204 days, which is one of the best extensions reported to date [4].

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls transcription of DNA, cytokine production and cell survival. NF-κB is found in almost all animal cell types and is involved in cellular responses to stress. Both TDP-43 and NF-κB proteins are over-expressed in sporadic ALS patients and down-regulating TDP-43 can reduce NF-κB activation.

Single chain (scFv) antibodies to inhibit TDP-43

Scientists have described the generation of single chain (scFv) antibodies specifically against the RRM1 domain of TDP-43 with a dual aim:

  • (i) to block TDP-43/p65 interaction reducing NF-κB activation
  • (ii) to interfere with protein aggregation.

The same method could be used against the RRM2 domain or the C-terminal glycine-rich region where ALS-causing mutations are located.

A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.

ScFvs have many uses, e.g., flow cytometry, immunohistochemistry, and as antigen-binding domains of artificial T cell receptors. Unlike monoclonal antibodies, which are often produced in mammalian cell cultures, scFvs are more often produced in bacteria cell cultures such as E. coli.

Due to their small size, good tissue penetration and low immunogenicity, scFv antibodies have been produced for different neurodegenerative disorders [9-11].

What specific design problems do we have to solve?

In addition of generic problems that are encountered while designing gene therapies, we have to solve some specific problems:

  • There are several isoforms of TDP-43
  • We need to design antibodies that target epitopes belonging to several domains, separately or together.
  • We need to design antibodies for each mutation of TDP-43 that are relevant in ALS.
  • We may extend this work to other proteins that are implicated in ALS, such as FUS.
  • We may extend this approach to SOD1, where there is already a significant body of related work.
  • While our main target is ALS, there are many other proteinopathies which would require other antibodies.

The RMM1 RNA recognition motif starts at position 101 and ends at position 191. So from Uniprot isoform 1 (there is another isoform), this gives this sequence for the wild type:


About fifty missense mutations in TARDBP have been identified in familial and sporadic ALS, most of which are located in the C-terminal G-rich region with only two exceptions to-date, A90V in the NTD and D169G in the RRM1.

enter image description here

There are several online predictor for B cells, like ABCpred Prediction Server, that can suggest linear epitopes. But as most interactions between antigens and antibodies rely on binding to conformational epitopes, it may be preferable to use a conformational epitope prediction server like the CEP server (http://bioinfo.ernet.in/cep.htm). From those epitopes it is possible to computationally deduce paratopes and antibodies.

ALS gene therapy and humans

Consideration for AAV gene therapy vector in ALS. AAV is safe Despite limited packaging capacity (≈4.5 kb for single-stranded and ≈2.4 kb for self-complementary AAV), AAV has become the most promising vector for gene delivery in neurological disease; it establishes stable nuclear episomes, thus reducing the risk of integrating into the host genome and causing insertional mutagenesis, it can transduce both dividing and non-mitotic cells, and it maintains exogenous gene expression for extended periods (Murlidharan et al., 2014).

AAV is successfully used in a close disease

A gene therapy for SMA, called AVXS-101, which delivers the SMN1 gene using scAAV9, has shown significant clinical potential. AVXS-101 is administered intravenously or intrathecally. Upon administration, the self-complimentary AAV9 viral vector delivers the SMN1 transgene to cell nuclei where the transgene begins to encode SMN protein, thus addressing the root cause of the disease.

With approximately twice the capacity of AAV, lentivirus has also been employed as a proof-of-concept vector in pre-clinical models of SMA (Azzouz et al., 2004a) and ALS, however, given that lentivirus can randomly insert into the host genome, there are major safety issues associated with its clinical application (Imbert et al., 2017). The advantages of AAV led to scAAV9 being chosen for SMN1 delivery in the AveXis gene therapy, AVXS-101.

Multiple AAV serotypes have been used in SMA mice (Foust et al., 2010; Passini et al., 2010; Tsai et al., 2012), but serotype 9 was selected for AVXS-101 because of its comparatively strong tropism toward LMNs throughout the spinal cord in a range of species (Foust et al., 2009; Bevan et al., 2011; Federici et al., 2012).

Timing, site and dosage of the treatment

The successful treatment of any disorder is more likely to occur when a therapy is administered during early pathogenesis rather than at later time points and, in particular, at disease end stage. Whilst intuitive, this highlights the importance of earlier diagnosis, especially for ALS where it is estimated that most ALS are already very advanced when diagnosed.

AAV9-based approaches for some neurodegenerative diseases such as ALS are less efficient at an older age, which is a challenge given that ALS typically occurs at a mild-age (Foust et al., 2010).

It has been considered safest to use vectors derived from viruses that normally infect humans, but that comes with the price that the immune system may recognize them as pathogens and try to eliminate them. These immune responses have the effect of removing transduced cells and limiting gene therapy efficacy. It is therefore critical when translating AAV9-mediated gene therapy for clinical applications, to first determine whether the patient has pre-existing immunity to AAV and to then mitigate the development of potentially damaging immune responses to therapy, particularly when the gene therapy is to be delivered intravenously.

Toxicities associated with AAV accumulation are likely to arise. The immune reaction may only starting late in the treatment, when the increase in viral load reaches a certain threshold.

AAV9 displays neuronal tropism and can mediate stable, long-term expression with a single administration, which is important given immunogenicity issues associated with viruses (Lorain et al., 2008). This contrasts with the multiple, invasive intrathecal injections of nusinersen, which can have adverse side effects (Haché et al., 2016).

Hence, there is a fine balance between administering sufficient gene therapy to ensure correct targeting in effective quantities without causing systemic toxic accumulation and adverse side effects. It is difficult to monitor benefit if the natural history of the disease is variable and the phenotypic traits are not quantitative and are protracted over time. There is a strong need for reliable ALS biomarkers to discern sufficient target engagement and correct dosing [6].

It should also be remembered that once an AAV has been delivered, relatively little can be done to regulate transgene expression


This draft document is a plea and an open proposal to the pharmaceutical industry to create a drug targeting TDP-43 in Amyotrophic Lateral Sclerosis (ALS). It describes how such a drug could be realistically produced now with common laboratories technologies like antibodies or transfection. Hopefully new experimentations to reduce levels of mutated TDP-43, with the technologies summarized in this paper, will be done soon on pigs model of ALS. Next steps could be: - design antibodies that target other domains in TDP-43. - design antibodies for each mutation of TDP-43 that are relevant in ALS. - extend this work to other proteins that are implicated in ALS, such as FUS. - extend this approach to SOD1, where there is already a significant body of related work.

Jean-Pierre Le Rouzic

retired engineer from FT R&D

jeanpierre.lerouzic at wanadoo.ch (replace the .ch with .fr)



[ 1] Pozzi S, Thammisetty SS, Codron P, Rahimian R, Plourde KV, Soucy G, Bareil C, Phaneuf D, Kriz J, Gravel C, Julien JP. Viral-mediated delivery of antibody targeting TAR DNA-binding protein 43 mitigates associated neuropathology. J Clin Invest. 2019 Jan 22. pii: 123931. doi: 10.1172/JCI123931. [Epub ahead of print]

2 Gao, N., Huang, Y.-P., Chu, T.-T., Li, Q.-Q., Zhou, B., Chen, Y.-X., … Li, Y.-M. (2019). TDP-43 specific reduction induced by Di-hydrophobic tags conjugated peptides. Bioorganic Chemistry, 84, 254–259. doi:10.1016/j.bioorg.2018.11.042

[3] Kevin D Foust, Desirée L Salazar, Shibi Likhite, Laura Ferraiuolo, Dara Ditsworth, Hristelina Ilieva, Kathrin Meyer, Leah Schmelzer, Lyndsey Braun, Don W Cleveland, and Brian K Kaspar Therapeutic AAV9-mediated Suppression of Mutant SOD1 Slows Disease Progression and Extends Survival in Models of Inherited ALS Mol Ther. 2013 Dec; 21(12): 2148–2159. Published online 2013 Oct 15. Prepublished online 2013 Sep 6. doi: 10.1038/mt.2013.211

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