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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

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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

Intermittent usage of cancer drugs

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Researchers have already advocated intermittent treatments, either to provide a respite for example in the treatment of prostate cancer, or because models of cell ecology show the interest of pre-empting the development of the mutations of resistance.

Selumetinib, an experimental inhibitor of MEK 1/2 from AstraZeneca, has experienced numerous failures in its trials, including the most recent in non-small cell lung cancer (NSCLC) with KRAS mutation and thyroid cancer.

Despite this series of failures, selumetinib is still being tested in different types of cancer, both as monotherapy and in combination with checkpoint inhibitor drugs.

Nevertheless, the development of resistance has proven to be a problem with drugs that inhibit MEK and associated pathways. But the company is trying to turn these failures into opportunities by partnering with researchers at the Babraham Institute in the UK to figure out how cancer cells become resistant to treatment.

By studying the cellular signaling pathway that selumetinib was supposed to disrupt, the AZ-Babraham joint team discovered that the cancer cells were developing a bypass strategy to avoid the effects of the drug, but that this strategy was destroying the cancer cells when the drug was no longer applied.

The researchers made this discovery by exposing human colon cancer cells to selumetinib for several weeks. After a while, they became resistant to selumetinib by amplifying a gene called BRAF. This allowed them to maintain the production of growth factors.

But once the drug was removed, BRAF amplification became an obstacle, activating a pathway that caused rapid cancer cell cell aging. Apparently the amplification is not reversible and cells that do not have this mutation have a competitive advantage over cancer cells.

The researchers found that these cells were then susceptible to a second attack of selumetinib. They believe their findings would be applicable to melanoma and other cancers, they said.

Although in-vitro studies are rarely transposable to living beings, studies in mice have shown that intermittent administration can prolong the tumor's reduction effects of the drug.

It should be noted, however, that the mice had undergone xeno-grafting and therefore were not mice that would be models of the target cancer. Researchers believe that knowledge about resistance resistance should inform future dosing schedules.

They published their findings in the journal Nature Communications.

"Our results provide a clear rationale for intermittent drug therapy, to delay or defeat emerging resistance," they wrote in the study.

About 80% of lung cancers are non-small cell lung cancers (NSCLC / NSCLC) and about 15 to 20% of these cells carry epidermal growth factor receptor (EGFR) activating mutations.

The treatment of EGFR mutant CPNPC has improved considerably with the introduction of EGFR tyrosine kinase inhibitors (TKIs). Several TKIs targeting this receptor have been developed, including the first generation EGFR TKI, gefitinib (Iressa) and erlotinib (Tarceva).

Although EGFR-mutated NSCLC patients derive substantial benefits from EGFR TKI, their disease tends to evolve within a year because tumors eventually develop resistance to treatment.

Second-generation TKI EGFR includes afatinib (Gilotrif) and dacomitinib (Vizimpro), as well as third-generation neratinib and EGFR TKIs, including osimertinib (Tagrisso), and experimental therapeutics. like olmutinib and nazartinib.

The most common activating EGFR mutations in NSCLC include the deletion of exon 19 and L858R. Patients whose tumors harbor these EGFR-activating mutations are treated with first-generation EGFR TKIs. Although most patients initially responded to these treatments, more than 60% of them develop resistance by acquiring the "gatekeeper" point mutation, T790M.

Third-generation inhibitors, such as osimertinib, have been developed to target T790M mutations and to be active against the suppression of the original exon 19 and L858R. However, patients also develop resistance to this treatment, through alternative bridging mechanisms, as described in the table below.

enter image description here

The targeted treatment of lung cancer patients with EGFR mutations consisted solely of monotherapy with various EGFR tyrosine kinase inhibitors, although we have known for more than 10 years that cancers resistant to EGFR TKIs result from activation of the MET bypass pathway.

Resistance acquired by amplification of MET as a derivation pathway is observed in approximately 5 to 10% of patients whose disease progresses after treatment with first- or second-generation EGFR-TKI and in approximately 25% of those whose disease progresses after TKI treatment of third-generation EGFR. The resistance induced by the MET confers a more aggressive behavior to cancers with EGFR mutants.

Combination of osimertinib and savolitinib

A combination of osimertinib and savolitinib, a MET inhibitor, was tested in a first cohort of patients with EGFR mutant lung cancer with acquired resistance induced by MET amplification after ITK therapy. first or second generation EGFR. Patient tumors were also negative for the T790M mutation.

In the second cohort, the same combination was tested in patients with EGFR-mutant lung cancer with acquired resistance induced by MET metabolism enhancement following treatment with osimertinib or another third-generation experimental TKI. EGFR.

The goal sought through the study of patients who received previous first-generation and second-generation EGFR-TKIs and those who had previously received previous third generation EGFR-TKIs in two separate cohorts was to provide more accurate assessments of combined treatment in these two distinct groups of patients.

For the cohort of patients who have previously received first- or second-generation EGFR-TKI, treatment with the osimertinib-savolitinib combination covers both the current dominant resistance mutation (based on MET amplification) and a likely future mutation resistance (based on T790m). "In this branch, we hypothesized that the response rate would be high," Sequist said in an interview.

enter image description here

On the other hand, in the cohort of patients who have already received third-generation EGFR-TKIs (osimertinib or one of the other third-generation EGFR experimental TKIs), their cancer probably acquired resistance via T790M after treatment with TKI EGFR generation, and following treatment with a third-generation EGFR TFR covering T790M, a second mutation driven by MET amplification appeared, distinguishing their disease from that of the first cohort.

In the cohort of 46 patients who had previously received a first- or second-generation EGFR TKA, savolitinib-treated osimertinib treatment yielded an objective response rate (ORR) of 52%, with 24 partial responses (PR). ). The median duration of response (DOR) was 7.1 months.

In the cohort of 48 patients who received a third-generation EGFR TKI, savolitinib-treated osimertinib treatment resulted in a ROP of 28%, with 12 RPs. The median DOR was 9.7 months.

"This discovery illustrates the value of careful patient selection in targeted therapy studies," said Sequist, adding, "These clinically significant responses also demonstrate that, as different heterogeneous mutation resistance appear, they can their turn to be controlled by adapting the therapy. "

Few people reach the age of 80 without a kind of neuropathology developing in their brains, and multi-pathology is the norm. Neuropathologists commonly observe cytoplasmic inclusions of phosphorylated TDP-43 in post-mortem brain samples with or without Aβ plaques and neurofibrillary tangles that define Alzheimer's disease.

enter image description here

Inclusions containing this RNA binding protein were first implicated in ALS and FTD more than 10 years ago. Shortly after, neuropathologists identified the TDP-43 pathology in people who had symptoms of Alzheimer's disease, not FTD (Amador-Ortiz et al., 2007). It often accompanied the hippocampal sclerosis, a disease characterized by a narrowed hippocampus ravaged by gliosis. More than a decade ago, Dickson had described curious cases of hippocampal sclerosis in people with dementia without the pathology of Alzheimer's disease (Dickson et al., 1994).

Researchers now suspect that many of these cases, as well as more recent cases, were caused by TDP-43 proteinopathy.

Over the last ten years, several studies have described the clinical and pathological evolution of this TDP-43 proteinopathy, recently called LATE. A study of TDP-43 in post-mortem brain samples of nearly 1,000 deceased participants in the Rush Memory and Aging Project and RUD-MAP (ROS-MAP) project (James et al. , 2016). Half of the participants had TDP-43 pathology and 37% had both TDP-43 and Alzheimer's disease. People with both conditions were more likely to have clinical dementia than those with only one condition. This study proposed a pathological staging pattern, in which the pathology TDP-43 reached the amygdala at stage 1, then the hippocampus and cortex entorhinal at stage 2, and finally the neocortex stage at 3. Once the pathology extends beyond the amygdala, it correlates well with cognitive impairment.

LATE is a proposal for a new common neurodegenerative disease, which has been named so by an international group of pathologists, clinicians and epidemiologists in a consensus report published April 30 in the Brain journal.

This international group predicts that this disease is a clinical diagnosis of Alzheimer's disease over five diagnosis. In their article, written after a workshop in Atlanta last fall, Peter Nelson's researchers at the University of Kentucky at Lexington summarized the decades of research that preceded the term. They describe the characteristics of LATE, propose ways to classify and diagnose it, and call for the development of specific biomarkers and therapies.

A typical case of predominantly age-related TDP-43 encephalopathy in limbic (LATE), would be that of an 86-year-old woman who would develop classic amnesic symptoms of Alzheimer's disease in her last years of life, but at autopsy, his brain would only carry a modest burden of Aβ plates and tau entanglement. Instead, the inclusions of TDP-43 would have invaded its limbic regions.

The LATE pathology often coincides with the hippocampal sclerosis, but the latter is not necessary for a diagnosis.

How often is LATE? ROSMAP researchers, led by Rush's Julie Schneider, analyzed their cohort data again to take into account the proposed neuropathological staging. They estimate the impact of LATE at about half that of Alzheimer's disease in the elderly and equal to the combined impact of all vascular neuropathologies. This would make LATE about 100 times more widespread than the FTD.

enter image description here

The co-occurrence of Alzheimer's disease and LATE neuropathologies in older people complicates the interpretation of therapeutic trials specific to Alzheimer's disease and may even mask positive results, Nelson noted.

The question is: what is the usefulness of introducing a new definite disease that can be diagnosed so far only through post-mortem neuropathological findings?

How will researchers differentiate the proposed new disease from Alzheimer's disease? There are no fluid biomarkers or PET tracers to detect the pathology of TDP-43, although researchers are interested. The intracellular localization of TDP-43 and its low pathological burden complicate the development of biomarkers. For the moment, the best the diagnoser can do is to go through a process of elimination.

Once biomarkers exist, they will facilitate targeted drug discovery efforts on LATE, for example on its TDP-43 aggregates. Given that comorbid neuropathologies in LATE at risk could potentially interact (Robinson et al., 2018), Trojanowski has proposed an immunotherapeutic cocktail of antibodies to Aβ, tau, TDP-43 and α-synuclein.

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.

Acellular systems rapidly develop the potential of synthetic biology, opening the way for a new wave of applications in living organisms.

When engineering an organism, most laboratories synthesize genes, insert them into cells, and see if the desired effect occurs. There are many limitations to this approach. The process can be time consuming and often the genes do not work as expected. As a result, many in the field now view cell-free systems - an in vitro tool for studying biology - as an easily accessible approach for prototype genes before they are inserted into a living cell. Cell-free systems have some crucial advantages over living organisms and can be made from whole cell extracts or individually purified components, such as PURE.

Cell-free systems can be used to produce toxins in high yields, unlike living cells, and components can generally be added or removed without consequence, while the deletion in vivo of an protein could kill the cell.

Protein Production 2.0: Unblock unnatural chemicals

But then many still do not see the potential of cell-free systems: It's an incredibly powerful approach to dissecting complex biological problems. Synthetic biology laboratories now exploit acellular systems to produce proteins with unnatural chemical properties, to create prototypes of metabolic pathways and even to detect biomolecules of clinical importance in just a few minutes.

Cell-free systems have long been used to produce natural proteins because the preparation of an extract takes only a few days and toxic proteins can be produced while the chemical environment is tightly controlled. But some laboratories are looking beyond the production of natural proteins.

In living organisms, unnatural amino acids are usually incorporated into proteins with a method called Stop Codon Suppression. In this method, a rarely used stop codon, typically UAG, is reaffected to produce another protein. In this way, an orthogonal tRNA that recognizes UAG can be expressed, but instead of signaling the translation to stop, it incorporates an unnatural amino acid. More than 100 unnatural amino acids have been incorporated into proteins using this approach 3.

Compared to living cells, acellular systems have a higher tolerance to toxicity caused by unnatural components, no cellular membrane barrier limits the transport of unnatural amino acids, more flexible control of the reaction is possible by freely adjusting the composition of the system and there is a higher incorporation efficiency non-natural amino acids.

Beyond Protein Production: Acellular Systems for Prototyping Lanes

While synthetic biologists adopt acellular systems to produce proteins that are decorated with unnatural amino acids, others are eagerly applying new capabilities to simultaneously probe tens of interacting proteins.

Acellular systems are more than a prototyping tool of autonomous genetic parts.

These are tools to test entire metabolic pathways. By creating numerous cell extracts, each with only a portion of the expressed pathway, it is possible to transform cell-free systems into a modular system that can be used to assemble any desired pathway in vitro. This smart approach, combined with automation and machine learning, could dramatically accelerate the way scientists test combinations of metabolic pathways. Nevertheless, there are fundamental limitations to our ability to apply the results of cell-free systems to living organisms.

Sensors on demand

The abundance of data allowed by this approach even allows us to go beyond the metabolic pathways; this could also be useful for the creation of acellular systems for the clinical biosonde.

The cells constantly detect their environment, react to the signals and act with caution. It follows naturally that the brilliant reactivity and programmability of biology could then be used to detect molecules of clinical importance for humans.

Cell-free systems have been developed for biosensing applications, with the goal of accurately diagnosing disease in minutes rather than hours.

Biosensors have focused on the application of acellular systems to design genetically encoded biosensors that measure biomarkers in clinically relevant samples. Although many models of biosensors work in the laboratory, few have been tested on real clinical specimens.

The reason that cell-free systems are preferable to living systems [as biosensors] in some contexts is that tests are economical, fast, quantitative, scalable and automated, and reproducible. They also offer advantages for biocapture in that they can be lyophilized on surfaces such as paper, they are not genetically modified organisms and are therefore more acceptable for use in clinical and field environments.

In the coming years, cell-free systems will be increasingly used in the clinic, especially in situations where a rapid pre-test is desirable.

A more distant future without cells

Yuan Lu, for example, is considering the use of cell-free systems in areas other than biology. "For this to happen, cell-free systems can not simply focus on biological transcription and translation," he says. "To achieve revolutionary development, cell-free systems must be strongly integrated with materials science, neuroscience, electronic engineering, 3D printing, artificial intelligence and other next-generation technologies. . "

Ashty Karim believes that cell-free systems will be increasingly used for "direct-to-use" applications. "We will begin to look at cell-free sensors as diagnostics in agriculture, defense and medicine, and we will see on-demand biofabrication of therapies, vaccines and commodities," she said. he said, pointing out that these advances are made possible by improvements in cell-free preparations and mixtures, such as "cell-free systems that can glycosylate proteins and cell-free systems containing orthogonal transcription factors".

The most ambitious application of cell-free systems, exemplified by the Build-a-Cell Consortium, aims to build a minimal synthetic cell from scratch. According to Paul Freemont, a member of the consortium, this effort will be facilitated by cell-free systems. He explains, "If we build a series of modules that mimic various aspects of living systems [in cell-free systems] like motility, detection and regulation, then the real challenge will be how to interface these different modules to produce a more complex synthetic cell. ".

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 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

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

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[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.

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[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