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Development of new chemical tools to combat ALS

Periodic Reporting for period 2 - Combat_ALS (Development of new chemical tools to combat ALS)

Periodo di rendicontazione: 2021-03-01 al 2022-02-28

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease that produces progressive neurodegeneration. Great efforts have been dedicated to find a cure for this disease with no success up to date.
The precise molecular mechanisms responsible of ALS remain unknown. However, mutations in several genes have been identified in the genome of affected individuals. Among the genetic defects related to ALS found so far, one of them appears in a higher proportion of cases: a mutation in the C9ORF72 gene.
This gene, located in the chromosome IX, contains a sequence of six nucleotides (5’-GGGGCC-3’/3’-CCCCGG-5’) which appears repeated a number of times. In individuals not suffering of ALS, the number of repeats is frequently two and sometimes can go up to 20. However, it was recently discovered that in a high proportion of ALS patients, the number of repeats of that sequence is much higher, reaching hundreds or even thousands.
Although not completely understood, the most prominent mechanism suggested to explain the deleterious effect of the expansion is the toxicity mediated by transcription of RNA containing the expansion of the repeated motif. These RNAs associate inside the cell forming accumulations of RNA called RNA foci. Some proteins with affinity for RNA are trapped in the RNA foci and, consequently, their function is precluded. Moreover, the RNAs containing the repeat expansion are translated giving rise to aberrant proteins formed by the successive repeat of only two kind of aminoacids. These proteins, called dipeptide repeat proteins (DPR), have also a toxic effect for the cell.
The main purpose of Combat_ALS action is to develop molecules able to counteract the deleterious effects produced by the presence of RNAs containing the repeat expansion of C9ORF72. To that end, two kind of molecules are being developed: (I) The so-called antisense oligonucleotides (ASO) and (II) small organic compounds with affinity for the RNAs.
The antisense therapy is based on the formation of a hybrid double helix where one strand is RNA and the other is a complementary oligonucleotide designed to hybridize with the RNA. The hybrid duplex is recognized inside the cells by the RNAseH machinery which degrades the RNA molecule involved in the hybrid duplex. Alternatively, the hybrid duplex does not activate the RNAseH but blocks RNA processing mechanism such as translation or splicing. In order to be stable in the cellular context and to increase their affinity for the RNA, the ASO need to contain chemical modifications. In this project, we are preparing ASO containing chemical modifications in some sugars of the nucleotides. Our ASO are designed to target the repeated part of the RNAs transcribed from C9ORF72 gene. We test the efficiency of these molecules by analyzing the effect of their use on cells having the repeat expansion defect in the C9ORF72 gene.
In addition, this project aims to discover specific small molecules with affinity for RNA structures formed by the repeated sequence expanded in C9ORF72 gene. These ligands may be of therapeutic interest since they can disrupt pathological interactions between RNA structures and RNA binding proteins. Also, the selective interaction of these compounds with the RNA may affect abnormal processes such as RNA aggregation or DPR production. In order to find small molecules with affinity for these RNAs, we are using state-of-the art techniques that allows screening of a great number of compounds.
We are also working in the preparation and use of human cerebral organoids as models to test the efficiency of both antisense and lead compounds from the screenings toward rescuing healthy phenotypes. Cerebral organoids are miniature organ-like structures which represent a novel system with huge advantages in recapitulating the architecture of the brain and with great potential to study human brain development and pathology. We plan to characterize the phenotype of human cerebral organoids carrying the repeat expansion in C9ORF72 gene and use them to evaluate the efficiency of the molecules resulting from the described previous studies.
A set of 20 antisense oligonucleotides (ASO) having different chemical modifications (most of them containing Fluorine in the sugar moiety) and targeting the sense and the antisense repeat expanded C9ORF72 transcripts has been produced and their activity has been evaluated in cells. In parallel, structural studies have been performed to characterize the structure adopted by the ASO as well as to confirm the formation and stability of the ASO:RNA hybrid duplex in vitro. Four of the tested ASO show significant activity reducing RNA accumulations in cells containing the genetic defect in the C9ORF72 gene. Additionally, these ASO act as blockers of translation.
Long repeat-containing RNAs (96-mer) formed by the repetitive unit appearing mutated in C9ORF72 gene have been prepared and characterized by different biophysical techniques. Microscopy studies have been carried out to analyse the structure of these RNAs and their aggregates. We have observed that RNA aggregation is favored under certain conditions. Considering the structural information that we have gathered, we have used the RNAs as targets in a screening of small fluorinated compounds performed by 19-NMR spectroscopy. Two libraries (a total of 636 compound) have been screened using both RNAs. Further screenings will be performed under conditions where RNA is in a different aggregation state. Hit compounds are being validated by H-NMR and CD spectroscopy.
In collaboration with Prof. Berninger's group (King's College, London, UK) we have developed cerebral organoids from patient cells and thus containing the repeat expansion in the C9ORF72 gene. A phenotypic characterization of the organoids is being currently carried out. Once completed, the most promising ASO mentioned before will be tested for their efficiency to rescue healthy phenotype features.
Results of this project have been presented in a virtual international meeting as poster communication and will be further disseminated in a three more meeting along the current year. Two articles containing these results are now under preparation and will be published in due time.
Some of the results obtained so far establish the efficiency of the particular sugar modifications present in our set of ASO. These modifications have never been tested before in correcting phenotype defects of ALS affected cells. We have observed that some of these molecules show significant efficiency in decreasing the number of RNA foci and blocking translation. Similarly, finding small compounds with selective affinity for the repeat-expanded RNAs involved in ALS may help the development of efficient drugs to stop ALS. We expect that further tests and evolution of our molecules will lead to promising agents to be considered in the treatment of ALS, a disease with no cure affecting one in 50,000 individuals per year worldwide.
On the other hand, the structural characterization of aggregates of repeated RNA resulting from C9ORF72 transcription may greatly contribute to understand a recurrent process that takes place not only in ALS but in many other neurodegenerative diseases.
Finally, we expect to establish cerebral organoids carrying a repeat expansion in C9ORF72 gene as a an ideal and novel model to test therapies against ALS.
Graphical description of the Combat_ALS action