LATE — Une protéinopathie TDP-43 qui frappe après 80 ans

- Posted by admin in Français

Peu de personnes atteignent l’âge de 80 ans sans qu’une sorte de neuropathologie ne se soit développée dans leur cerveau, et la multi-pathologie est la norme. Les neuropathologistes observent couramment des inclusions cytoplasmiques de TDP-43 phosphorylé dans des échantillons de cerveau post-mortem avec ou sans les plaques Aβ et les enchevêtrements neurofibrillaires qui définissent la maladie d'Alzheimer.

enter image description here

Les inclusions contenant cette protéine de liaison à l'ARN ont été impliquées pour la première fois dans la SLA et la FTD il y a plus de dix ans. Peu de temps après, des neuropathologistes ont repéré la pathologie TDP-43 chez des personnes qui avaient des symptômes de maladie d'Alzheimer, et non de FTD (Amador-Ortiz et al., 2007). Elle accompagnait souvent la sclérose de l'hippocampe, un hippocampe rétréci ravagé par la gliose. Plus d'une décennie auparavant, Dickson avait décrit des cas curieux de sclérose de l'hippocampe chez des personnes atteintes de démence sans pathologie de la maladie d'Alzheimer (Dickson et al., 1994).

Les chercheurs soupçonnent maintenant qu'une grande partie de ces cas, ainsi que des cas plus récent, étaient causés par une protéinopathie TDP-43.

Au cours des dix dernières années, plusieurs études ont décrit l’évolution clinique et pathologique de cette protéinopathie TDP-43, récemment baptisée LATE. On peut citer une étude sur TDP-43, dans des échantillons de cerveau post-mortem de près de 1 000 participants décédés, dans le cadre du projet Rush Memory and Aging Project et RUD-MAP (ROS-MAP) (James et al., 2016). La moitié des participants étaient atteints de la pathologie TDP-43 et 37% des participants étaient atteints à la fois de la pathologie TDP-43 et de la maladie d'Alzheimer. Les personnes ayant les deux pathologies étaient plus susceptibles d'être atteinte de démence clinique que celles qui étaient atteinte d'une seule pathologie. Cette étude proposait un schéma de stadification pathologique, dans lequel la pathologie TDP-43 atteignait l'amygdale au stade 1, puis l'hippocampe et le cortex entorhinal au stade 2, et enfin le néocortex au stade 3. Une fois que la pathologie s'étend au-delà de l'amygdale, elle se corrèle bien avec une déficience cognitive.

LATE est une proposition d'une nouvelle maladie neurodégénérative courante, qui a été baptisée ainsi par un groupe international de pathologistes, cliniciens et épidémiologistes dans un rapport de consensus publié le 30 avril dans Brain.

Ce groupe international prédit que cette maladie concerne un diagnostic clinique de maladie d'Alzheimer sur cinq. Dans leur article, rédigé à la suite d’un atelier tenu à Atlanta à l’automne dernier, les chercheurs de Peter Nelson, de l’Université du Kentucky à Lexington, ont résumé les décennies de recherche qui ont précédé l’appellation du terme. Ils décrivent les caractéristiques de LATE, proposent des moyens de la classer et de la diagnostiquer, et appellent au développement de biomarqueurs et de thérapies spécifiques.

Un cas typique d’encéphalopathie TDP-43 liée à l’âge prédominant dans le limbique (LATE), serait celui d'une femme de 86 ans qui développerait les symptômes amnésiques classiques de la maladie d’Alzheimer au cours de ses dernières années de vie, mais à l’autopsie, son cerveau ne porterait plus qu’un fardeau modeste de plaques Aβ et de tau enchevêtrement. Au lieu de cela, les inclusions de TDP-43 auraient envahi ses régions limbiques.

La pathologie LATE coïncide souvent avec la sclérose de l'hippocampe, mais cette dernière n'est pas nécessaire pour un diagnostic.

Quelle est la fréquence de LATE? Les chercheurs de ROSMAP, dirigés par Julie Schneider de Rush, ont analysé à nouveau les données de leur cohorte afin de prendre en compte la mise en scène neuropathologique proposée. Ils estiment l’impact du LATE à environ la moitié de celui de la maladie d'Alzheimer chez les personnes âgées et égal à l’impact combiné de toutes les neuropathologies vasculaires. Cela rendrait LATE environ 100 fois plus répandu que le FTD.

enter image description here

La co-occurrence des neuropathologies maladie d'Alzheimer et LATE chez les personnes âgées complique l'interprétation des essais thérapeutiques spécifiques à la maladie d'Alzheimer et peut même masquer des résultats positifs, a noté Nelson.

On peut se demander quelle est l'utilité de l'introduction d'une nouvelle maladie définie et pouvant être diagnostiquée jusqu'à présent uniquement grâce aux résultats neuropathologiques obtenus post mortem?

Comment les chercheurs vont-ils différencier la nouvelle maladie proposée de la maladie d'Alzheimer? Il n’existe pas de biomarqueurs de fluide ni de traceurs PET pour détecter la pathologie de TDP-43, bien que des chercheurs s’y intéressent. La localisation intracellulaire du TDP-43 et son faible fardeau pathologique compliquent le développement de biomarqueurs. Pour le moment, le mieux que le diagnostiqueur puisse faire est de suivre un processus d'élimination.

Une fois que les biomarqueurs existeront, ils faciliteront les efforts de découverte de médicaments ciblés sur LATE, par exemple sur ses agrégats de TDP-43. Étant donné que les neuropathologies comorbides chez les personnes à risque de LATE pourraient potentiellement interagir (Robinson et al., 2018), Trojanowski a proposé un cocktail immunothérapeutique d'anticorps dirigés contre Aβ, tau, TDP-43 et α-synucléine

A plea for a gene therapy for ALS

- Posted by admin in English

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

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

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

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

[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


Please, help us continue to provide valuable information: