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Who we are

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Padirac Innovation is a ** non-profit organization ** created in France in the middle of 2014. It aims to produce technological tools for human needs.

Here are its activities so far:

From 2014 to 2016, Padirac Innovation solved the following Innocentive problems:

  • Inventing a tool to deactivate abandoned chemical warfare,

  • Finding a drug for eczema that is currently without valid IP,

  • Helping Cleveland Clinic with new strategies to mitigate the adaptation of prostate cancer cells to new drugs.

In 2017, Padirac Innovation developed an early heart failure detector

This heart failure detector uses artificial intelligence to detect S4 sounds with a mini ultrasound Doppler.

From 2018, Padirac Innovation has worked specifically on cancer and ALS

  • In the first half of 2018, Padirac Innovation developed reliable information in French on health care related to cancer.
  • In the second half of 2018, Padirac Innovation designed several achievements related to ALS.

In 2019, Padirac Innovation is writing a book about ALS research

This book will be published at the end of 2019.

Study of Edaravone in ALS Korean patients.

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Purpose of this study

Edaravone was approved as a therapeutic drug against ALS in June 2015 in Japan and by the Korean Ministry of Food and Drug Safety in December 2015.

In this observational study, on ALS patients in the Korean population, patients treated with edaravone showed modest results on ALSFRS-R and lung function tests. Several previous studies on edaravone also reported quite low results in the treatment of ALS.

Results of this new study on Edaravone

The phase 3 clinical trial on edaravone showed an average decrease in ALSFRS-R of 5.01 points in 6 months in the treated group, and an average decrease of 7.50 points in 6 months in the control group.

The patients involved in this study showed an average decrease of 5.75 point.

The initial characteristics of ALS patients included in this study had an average ALSFRS-R of 34.25 and an average CVF of 75%, reflecting a more advanced stage of ALS patients in this new study compared to patients in the recent trial. phase 3 clinical trial.

It should be noted that a recent study of advanced ALS patients with a FVC of less than 60% showed no benefit of edaravone, reflecting the importance of early intervention in the treatment of ALS patients. The study in Korean patients also showed some efficacy in ALS patients with a mean CVF score of 75%.

In the present study, the results also indicate that the reduction is not limited to a specific area, but also relates to different areas of ALSFRS-R.

Castillo-Viguera et al. have suggested that removal of more than 20% of ALSFRS-R is clinically significant; The phase 3 clinical trial on edaravone had shown a 33% decrease in progression, but the present study showed only a slower progression of 23% after 6 months.

Adverse effects of Edaravone

Edaravone is known to cause frequent side effects, in up to 84% of patients. The most common side effects are bruising, constipation, contact dermatitis, dysphagia, eczema and inflammation of the upper respiratory tract (in order of decreasing frequency); 16% of patients experienced serious adverse events. In the present study, two patients presented with eczema and pruritus, which were well tolerated with oral antihistamine and steroidal therapy. Transient leukopenia should also be noted in a patient who has recovered after a few days of initial treatment. No deaths were encountered during the follow-up period.

The limitations of the present study are as follows. The study was observational, with no control group for comparison. The small number of patients recruited must be taken into account in the evaluation of the results.

Conclusions

This is a study of Korean patients on the open-label study of edaravone in patients with ALS. The treatment was well tolerated without significant adverse events. Consistent with previous studies in Japan, the United States and Europe, the present study shows that the treatment was well tolerated and showed only a slight improvement at a later stage of ALS.

The study is available here: https://doi.org/10.1007/s10072-019-04055-3

The majority of patients still do not respond to immune checkpoint treatments.

This type of treatment had been presented as revolutionary a few years ago. Unfortunately, medical research has accustomed us to these shattering announcements that never materialize. A subtype of macrophages, a form of immune cells with anti-inflammatory properties, protects rather than destroys cancer cells. Instead of directly attacking cancer cells, a new immunotherapy technique targets and removes this subtype of macrophages, after which the immune system begins to reduce cancer.

Tumor-associated macrophages

Tumor-associated macrophages are the most abundant immune cells present in solid tumors, and the fact that they contribute significantly to tumor progression is well documented.

In addition to their trophic functions favoring angiogenesis, invasion, and metastasis, tumor-associated macrophages may inhibit proliferation and activation of T-cells by various mechanisms.

Bad clinical results, but not everywhere

The important functions of macrophages in relation to tumor progression have led to a substantial interest in the development of new therapeutic strategies for targeting tumor-associated macrophages.

However, despite a strong correlation between tumor-associated macrophage accumulation and poor clinical trial results, in some cases the accumulation of macrophage subsets associated with specific tumors may be associated with a good prognosis.

An example is the frequency of HLA-DR + tumor-associated macrophages, which has been associated with beneficial effects in several studies (de Vos van Steenwijk et al., 2013, Ino et al., 2013), perhaps reflecting the role of macrophages in orchestrating protective immune responses (Mantovani and Allavena, 2015).

In fact, recent studies using a coupled monocell analysis by mass cytometry and RNA sequencing have revealed an unprecedented level of diversity within the tumor-associated macrophage compartment in patients with lung adenocarcinoma and cell carcinoma kidney (Chevrier et al., 2017; Lavin et al., 2017). In the case of renal cell carcinoma, 17 different tumors-associated macrophage phenotypes have been documented (Chevrier et al., 2017).

Diversity of tumor-associated macrophage subsets

Researchers still lack basic knowledge about the functions of different subsets of tumor-associated macrophages and their respective contributions to tumor progression.

However, it is tempting to think that selective targeting of tumor-associated macrophage subsets with protumoral functions, while preserving the immune functions of other tumor-associated macrophage subsets, could offer significant clinical benefits .

CD163 protein as a marker of bad proostic

The expression of CD163 by tumor-associated macrophages has been shown to be a particularly potent indicator of poor prognosis in several human cancers (Komohara et al., 2014), including melanoma (Jensen et al., 2009; Bronkhorst et al. al., 2011; Lee). et al., 2018).

CD163 is a macrophage and monocyte-specific transmembrane protein that acts as a receptor for haptoglobin-hemoglobin complexes formed during intravascular hemolysis (Kristiansen et al., 2001). Hemoglobin is contained in red blood cells or red blood cells. In case of destruction of the latter (physiologically or not), the hemoglobin-haptoglobin complex is removed from the plasma mainly at the level of the spleen.

CD163 expression is induced by tumor-promoting cytokines such as IL-6 and IL-10, whereas inflammatory stimuli, including LPS, TNFα, and IFNγ, result in rapid expression regulation. and removal of the CD163 membrane via proteolytic excretion (Etzerodt et al., 2010; Etzerodt and Moestrup, 2013).

This, combined with the generation of heme anti-inflammatory metabolites from hemoglobin scavenging, has led to the association of CD163 + macrophages with anti-inflammatory functions (Etzerodt and Moestrup, 2013).

Caution is needed

Indeed, the link between CD163 accumulation and tumor-associated macrophages and tumor progression is based exclusively on correlations with clinical evolution, and experimental evidence of specific promotion functions. the tumor are still missing.

Moreover, the recent development of immune check point inhibitors (immune control point), such as anti-PD-1, has had a huge impact on the treatment of cancer, particularly in malignant melanoma (Robert and al., 2015; Ugurel et al., 2017). The blocking of PD-1 / PD-L1 signaling has resulted in unprecedented tumor regression rates (Tumeh et al., 2014), but only for a small number of patients.

New therapeutic strategies

Therefore, new therapeutic strategies to enhance antitumor immunity, to overcome immune checkpoint resistance or to improve serious adverse side effects, are absolutely necessary.

Researchers conducted extensive characterization of tumor-associated macrophage subsets in a clinically relevant mouse model of melanoma resistant to anti-PD-1 treatment.

The researchers show that the specific targeting of a minor subset of macrophages associated with tumors expressing CD163 is sufficient to induce tumor regression in this model.

It is important to note that the specific macrophage depletion associated with CD163 + tumors results in increased recruitment of effector T cells and CCR2-dependent inflammatory monocytes, both of which contribute to tumor regression.

These studies are the first to demonstrate the selective targeting of macrophages associated with tumors and CD163 and their specific contribution to tumor progression.

An inhibitor of RPK1 has been tested for safety in healthy people

Why take an interest in RPK1?

Serine / threonine protein kinase 1 (RIPK1) interacting with receptors is an intracellular protein involved in the regulation of inflammation and cell death. RIPK1 is activated in response to several inflammatory stimuli, including tumor necrosis factor alpha (TNF-α) signaling by the TNF 1 receptor. When activated, RIPK1 elicits multiple cellular responses, including cytokine release, microglial activation, and necroptosis, a regulated form of cell death.

The early role of RIPK1 in this signaling cascade led to the hypothesis that inhibition of RIPK1 signaling could be beneficial in diseases characterized by excess cell death and inflammation such as amyotrophic lateral sclerosis (ALS).

Indeed, inhibition of RIPK1 activity has been shown to protect against necroptotic cell death in vitro over a range of cell death models (see below).

In animal models of diseases ranging from ulcerative colitis to multiple sclerosis, inhibition of this pathway protects against pathology and cell death. These non-clinical findings, coupled with observations of increased activity of RIPK1 in human diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and multiple sclerosis, suggest that inhibition of RIPK1 could be beneficial in many different chronic diseases.

What problems are there with RPK1 inhibitors?

Inhibitors of RIPK1 are currently being evaluated as treatments for systemic inflammatory diseases, including inflammatory bowel disease and psoriasis, but there is no evidence that previously studied inhibitors in humans enter the system. central nervous system (CNS). To evaluate the potential for inhibition of RIPK1 as a therapeutic for chronic neurodegenerative diseases, it is necessary to study the pharmacokinetics (PK), pharmacodynamics (PD) and safety profile of a molecule capable of entering in the CNS at effective concentrations.

DNL104 is a selective inhibitor of CNS penetrable RIPK1 activity developed by Denali Therapeutics as a potential treatment for neurodegenerative disease. Denali, a CNS biotechnology company, is made up of veterans from Genentech, and joined the RIK1 program in 2016 with the acquisition of Incro Pharmaceuticals. Sanofi paid $ 125 million (€ 110 million) by the end of 2018 for participation in two developing RIPK1 inhibitors in Denali. The agreement covers small molecules designed to treat several neurodegenerative and systemic inflammatory diseases.

What is the current knowledge on the subject?

Inhibition of phosphorylation of RIP K 1 shows protection against pathology and inflammation in vitro and in animals, induced by various challenges, including in animal models with CNS disease (AD and ALS).

What question did this study address?

The safety, tolerability, pharmacokinetic, and pharmacodynamic effects of the CNS-penetrating RIP1 kinase inhibitor D NL104 were tested in randomized, placebo-controlled, increasing dose placebo-controlled trials.

What does this study add to our knowledge?

The results show that DNL104 inhibits phosphorylation of RIPK1 in healthy healthy volunteers with no effect on central nervous system safety, but liver toxicity issues have been raised in the multiple-dose-escalation study, in which 37.5 % of subjects (6 subjects) developed high liver function tests. related to the drug, of which 50% (3 subjects) were classified in the category inducing a drug-induced liver injury (DILI).

Why focus on necroptosis?

In 2014, we knew for a long time that the origin of ALS was not in motor neurons, but in other cells. But 8 years after the discovery of TDP-43 and 3 years after the discovery of C9orf72, most knowledge about the mechanisms of motor neuron degeneration in ALS still came from studies on SOD1-type mouse models. A clear conclusion from these studies is that non-neuronal cells play a critical role in the neurodegeneration related to SOD1 mutations. Indeed, the presence of healthy glial cells significantly delayed the onset of motor neuron degeneration, increasing the life without disease by 50%.

Since the work of the Jean-Pierre Julien Group in 2005, it has been suggested several times that interneurons, myelinating Schwann cells of the peripheral nervous system and endothelial cells of the vascular system could be at the origin of ALS. But other studies have suggested instead that astrocytes could cause spontaneous degeneration of motor neurons. For example, in 2003, researchers led by Don Cleveland of the University of California at San Diego involved astrocytes in motor neuron death, showing that administering SOD1 to these non-neuronal cells still resulted in motor neuron disease.

Agnostic research on the cause of ALS

Usually when a scientist decides to set up an experiment, he wants to test a hypothesis. The hypothesis itself is based on a model of the disease. A new trend in biology is to do research without having a preconceived idea (the model of the disease). It is believed that this is a difficult way to achieve results that could not have been achieved by conventional procedures.

In order to determine whether astrocytes from sALS patients can kill motoneurons independently without being exposed to SOD1, the Przedborski group decides to study the mix of different types of cells after they have been exposed to ALS, without prejudging of what causes ALS. For that they decide to design "their" in-vitro model of ALS. This well-cited article (100 times), however, contradicts many other studies.

Diane Re and Virginia Le Verche isolate astrocytes derived from post mortem motor cortex and spinal cord tissue from six SALS patients and 15 controls. They realize that after one month of culture, astrocytes have dominated other cultures. The researchers then mixed these astrocytes with motor neurons derived from human embryonic stem cells. While neurons thrived when co-occurring with non-sALS control astrocytes, their number began to fall after only four days of culturing with sALS astrocytes. All of this clearly shows that astrocytes from SALS patients specifically kill motor neurons, unlike control astrocytes.

However, other types of neurons than the motoneurons were resistant to the deleterious signals delivered by sALS astrocytes, and the fibroblasts of sALS patients also did not destroy the motoneurons, indicating that the toxic relationship was astrocyte-specific. and SALS motor neurons. To determine the role of SOD1 the researchers inhibited the expression of this protein in astrocytes using four small hairpin RNAs. The treatment failed to protect the motor neurons. The decrease in TDP-43 expression in astrocytes did not save them either.

Controversial research

These results contradict a study conducted by a team of Brian Kaspar, who found that astrocytes derived from neural progenitor cells taken from sALS patients needed SOD1 to destroy motor neurons, even though sALS patients showed no evidence of mutation of this gene (Haidet-Phillips et al., 2011). But in 2014, in the same issue as the publication of the Przedborski group, the Haidet-Phillips group publishes an article1 that is very similar to that of the Przedborski group, except that it incriminates NF-κB and therefore a mechanism for apoptosis rather than necroptosis, but in any case SOD1 is no longer supposed to be the primary cause of ALS.

For this team the inactivation of SOD1 in human astrocytes of patients with SALS does not preserve the motor neurons. How ALS astrocytes become toxic remains completely obscure. No known ALS-related mutations were identified in their samples and yet the toxic phenotype persisted even after several passages of adult astrocytes in culture. The authors suggest that necroptosis is the dominant mode of cell death in their in vitro model of sALS.

In 2019 it is difficult to say who is right between all these contradictory studies. Apoptosis and necroptosis are major mechanisms of cell death that usually result in opposite immune responses. Apoptotic death usually leads to immunologically silent responses, while death by necroptosis releases molecules that promote inflammation, a process called necrosis.

Drugs that may enter phase III (2019).

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ALS drugs that may enter a phase III trial in 2019

Arimoclomol

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

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

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.

Tofersen

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.

Using gene therapy to treat multiple dystrophies

Congenital Muscular Dystrophy Type 1A (MDC1A) is caused by mutations in the Lama2 gene that cause muscle wasting and destruction of the protective myelin coating around the peripheral nerves. CRISPR has already shown positive preclinical results in Duchenne muscular dystrophy, mainly by targeting mutations in the gene that produces dystrophin, a protein essential for muscle support. Ronald Cohn and his colleagues at Toronto's Hospital for Sick Children (SickKids) used earlier CRISPR-cas9 to eliminate dysfunctional dystrophin exons (inclusion-exon strategy), leaving a shortened but still functional gene. The therapy has been effective for more than a year in mice. In another study researchers applied CRISPR to target exon 51 and successfully restored dystrophin levels to 92% of normal in models of Duchenne muscular dystrophy in dogs.

The problem of the heterogeneity of muscular dystrophies

There are more than 350 nonsense pathogen, missense, splice site and deletion mutations in LAMA2 reported to date.One of the problems in developing a therapy for the treatment of MDC1A is that the heterogeneity of the mutations often leads to varying severity and progression of the disease. Therefore, it is urgent to develop a universal, mutations-independent strategy that offers a therapeutic approach to all patients with MDC1A.

How to extend the application range of CRISPR-Cas9?

Given the number of genomic alterations causing MDC1A, the Crispr-Cas9-mediated correction would require the design and in-depth analysis of several single-guide RNAs (sgRNAs) specific to each mutation, which means that as many therapies of this type should be designed and evaluated thoroughly for each mutation. In addition, safety concerns regarding the potential mutagenic nature of the CRISPR-Cas9 system and the presence of non-targeted effects after gene editing remain, which together may be difficult from the point of view of safety and security. regulation.

A recent study has described the use of the CRISPR transcription activation system to induce expression of target genes in skeletal, renal and hepatic tissues, resulting in a phenotypic increase such as increased muscle mass and improvement. substantial pathophysiology of the disease. However, it relied almost exclusively on a transgenic mouse model expressing Cas9 or on local intramuscular treatments and so it is difficult to extrapolate the efficacy of this strategy to models relevant to the disease.

On the other hand, attenuation of disease pathogenicity by targeted modulation of disease-modifying gene expression would be a potentially safer and more beneficial alternative for all individuals with MDC1A.

An indirect strategy to address all congenital muscular dystrophies

Neuromuscular diseases have provided excellent examples for demonstrating the role of disease modifiers. One of the most potent disease modifiers reported for MDC1A is laminin 1, which is structurally similar to laminin 2. However, laminin 1 is not expressed in skeletal muscle or Schwann cells.

Previous studies have demonstrated that overexpression of transgenic Lama1 healed from myopathy and peripheral neuropathy in mouse models.

Although these studies established a compensation function for laminin 1 in MDC1A, the use of this modifier as postnatal genomic therapy is hampered by the size of the Lama1 cDNA, which exceeds the packaging capacity of AAV vectors. CRISPR-Cas9 technologies have offered opportunities for regulating gene expression and creating epigenetic alterations without introducing double-strand breaks in DNA, known as the CRISPR transcription activation system.

The strategy uses nuclease-deficient Cas9 (dCas9), which is unable to cut DNA due to mutations in the nuclease domains and retains the ability to specifically bind to DNA when guided by sgRNA.

CRISPR activation uses modified versions of dCas9, a mutation of Cas9 without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs). Like a standard CRISPR-Cas9 system, dCas9 activation systems rely on similar components such as Cas9 variants for modulation or modification of genes, gRNAs to guide Cas9 to intended targets, and vectors for introduction into cells. However, while a standard CRISPR-Cas9 system relies on creating breaks in DNA through the endonuclease activity of Cas9 and then manipulating DNA Repair mechanisms for gene editing, dCas9 activation systems are modified and employ transcriptional activators to increase expression of genes of interest.

Using previously described Streptococcus pyogenes (Sp dCas9) fused to multiple copies of the VP16 transcription enhancer, Canadian and other researchers have demonstrated the use of the CRISPR-dCas9 system to positively regulate the expression of the modifying genes. vitro.

When the load is too big for viral vectors

A major challenge for in vivo applications is the large size of Sp dCas9 and its derivatives, which exceed the packaging capacity of the AAV genome. Targeting Lama1 presents a particular challenge: the gene is too large to be contained in the viral vectors traditionally used to administer gene therapy. To account for this limitation, the researchers adapted the transcriptional regulatory system and used a considerably smaller Cas9 protein, derived from mStaphylococcus aureus (Sa 9), to positively regulate Lama1.

What results were obtained?

By increasing the expression of Lama1, the treatment not only prevented paralysis in pre-symptomatic mice, but also reversed the progression of the disease in previously symptomatic animals. The treatment resulted in a reduction of fibrosis and an increase in the size of the muscle fiber, thus preventing the appearance of symptoms. More importantly, in mice already suffering from paralysis, the treatment also allowed the animals to get up and move. The researchers also observed a significant increase in nerve conduction velocity, showing a restoration of the myelin sheath and an improvement in neuromuscular function.

Application to Duchenne muscular dystrophy

The Cohn team believes that its strategy of using the CRISPR provided by AAV to upregulate Lama1 could also be applied to Duchenne muscular dystrophy. To achieve greater efficiency, the system could be used in combination with another technology that corrects the mutation. Its application as a combinatorial therapeutic approach, involving simultaneous up-regulation of protective disease-modifying genes and downregulation of harmful genes would represent a new paradigm for reducing disease phenotypes.

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.

Conclusion

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.

Familial ALS represents a small percentage of all ALS cases and C9ORF72 is the most common mutated gene in familial ALS.

An interesting study identifies PAF1C, as a transcription complex needed to read the C9ORF72 gene expansion RNA in flies and yeast. PAF1C components were upregulated in brain samples from people with C9ORF72-FTD, and bound to the C9ORF72 promoter. The role of PAF1C in expression of the expanded repeats suggests its human homolog may be a target for mitigating C9ORF72 toxicity. It used is about fruit flies, yeast and human tissues. Indeed fruit flies and yeast even more stranger to human CNS than genetically engineered mice. But it is this study that stroked my interest, because they found a link with human FTD where C9 is implicated.

Lindsey Goodman suggests that a potential route to turning off C9orf72-repeat toxicity may be to avert its transcription in the first place.

Using a genetic screen in fruit flies, the scientists sought genes that temper the toxicity of the GGGGCC hexanucleotide repeat that is found in C9orf72 and that lead to the transcription of the C9ORF72-encoded poly(GR) dipeptide. enter image description here

They were interested in several components of the PAF1C RNA polymerase III complex, which is especially good at transcribing GC-rich DNA. They identified several PAF1C components as modifiers of C9orf72-associated disease.

Transgenic downregulation of PAF1C components disrupted the transcription of G4C2 RNA in both Drosophila and Saccharomyces cerevisiae and resulted in reduced toxicity in Drosophila. in Drosophila, the PAF1C components Paf1 and Leo1 appear to be selective for the transcription of long, toxic repeat expansions, but not shorter, nontoxic expansions. In yeast, PAF1C components regulate the expression of both sense and antisense repeats. PAF1C is upregulated following (G4C2) 30+ expression in flies and mice. In humans, PAF1 is also upregulated in C9+ -derived cells, and its heterodimer partner, LEO1, binds C9+ repeat chromatin. In human C9+ FTD, PAF1 and LEO1 are upregulated and their expression positively correlates with the expression of repeat-containing C9orf72 transcripts. These data indicate that PAF1C activity is an important factor for transcription of the long, toxic repeat in C9+ FTD.

You can find more information here.

A plea for a gene therapy for ALS

- Posted in English by

Introduction

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:

QKTSDLIVLG LPWKTTEQDL KEYFSTFGEV LMVQVKKDLK TGHSKGFGFV
RFTEYETQVK VMSQRHMIDG RWCDCKLPNS K

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 related 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

Conclusion

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)

https://padiracinnovation.org/

References

[ 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.

[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

[4] Ashley E. Frakes, Lyndsey Braun, Laura Ferraiuolo, Denis C. Guttridge, and Brian K. Kaspar Additive amelioration of ALS by co‐targeting independent pathogenic mechanisms Ann Clin Transl Neurol. 2017 Feb; 4(2): 76–86. Published online 2017 Jan 11. doi: 10.1002/acn3.375

[5] Spencer B, Emadi S, Desplats P, Eleuteri S, Michael S, Kosberg K, et al. ESCRT-mediated uptake and degradation of brain-targeted alpha-synuclein single chain antibody attenuates neuronal degeneration in vivo. Mol Ther. 2014;22(10):1753-67.

[6] Tommaso Iannitti, Joseph M. Scarrott, Shibi Likhite, Ian R.P. Coldicott, Katherine E. Lewis, Paul R. Heath, Adrian Higginbottom, Monika A. Myszczynska, Marta Milo, Guillaume M. Hautbergue, Kathrin Meyer, Brian K. Kaspar, Laura Ferraiuolo, Pamela J. Shaw, and Mimoun Azzouz Translating SOD1 Gene Silencing toward the Clinic: A Highly Efficacious, Off-Target-free, and Biomarker-Supported Strategy for fALS Mol Ther Nucleic Acids. 2018 Sep 7; 12: 75–88. Published online 2018 May 3. doi: 10.1016/j.omtn.2018.04.015

[7] Maria Grazia Biferi, Mathilde Cohen-Tannoudji, Ambra Cappelletto, Benoit Giroux, Marianne Roda, Stéphanie Astord, Thibaut Marais, Corinne Bos, Thomas Voit, Arnaud Ferry, and Martine Barkats A New AAV10-U7-Mediated Gene Therapy Prolongs Survival and Restores Function in an ALS Mouse Model Mol Ther. 2017 Sep 6; 25(9): 2038–2052. Published online 2017 Jun 26. doi: 10.1016/j.ymthe.2017.05.017

[8] Butler DC, and Messer A. Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments. PLoS One. 2011;6(12):e29199.

[9] Ghadge GD, Pavlovic JD, Koduvayur SP, Kay BK, and Roos RP. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1. Neurobiol Dis. 2013;56:74-8.

[10] Patel P, Kriz J, Gravel M, Soucy G, Bareil C, Gravel C, et al. Adeno-associated virus-mediated delivery of a recombinant single-chain antibody against misfolded superoxide dismutase for treatment of amyotrophic lateral sclerosis. Mol Ther. 2014;22(3):498-510.

11 Tamaki Y, Shodai A, Morimura T, Hikiami R, Minamiyama S, Ayaki T, et al. Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals. Sci Rep. 2018;8(1):6030.

[12] Ghadge GD, Kay BK, Drigotas C, and Roos RP. Single chain variable fragment antibodies directed against SOD1 ameliorate disease in mutant SOD1 transgenic mice. Neurobiol Dis. 2018;121:131-7.

[13] Dong QX, Zhu J, Liu SY, Yu XL, and Liu RT. An oligomer-specific antibody improved motor function and attenuated neuropathology in the SOD1-G93A transgenic mouse model of ALS. Int Immunopharmacol. 2018;65:413-21.

[14] Andrew P. Tosolini and James N. Sleigh Motor Neuron Gene Therapy: Lessons from Spinal Muscular Atrophy for Amyotrophic Lateral Sclerosis Front Mol Neurosci. 2017; 10: 405. Published online 2017 Dec 7. doi: 10.3389/fnmol.2017.00405

[15] Chiang, Chien-Hao, Grauffel, Cédric, Wu, Lien-Szu, Kuo, Pan-Hsien, Doudeva, Lyudmila G., Lim, Carmay, Shen, Che-Kun James, Yuan, Hanna S. Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation Nature Scientific Reports https://doi.org/10.1038/srep21581



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