Evaluation de trois approches de thérapie génique pour le traitement des dysferlinopathies (miniprotéine, compensation et trans-épissage)

Les dysferlinopathies sont des maladies musculaires dues à une déficience en protéine dysferline, codée par le gène DYSF. Dans ce travail de thèse, trois approches thérapeutiques ont été évaluées pour ces pathologies, sur des modèles cellulaires et murins. Un variant transcriptionnel court de la dysferline a été vectorisé dans un AAV8r et injecté dans le modèle murin Bla/J, déficient en dysferline. L analyse des muscles des animaux traités montre une augmentation de la résistance des fibres musculaires au stress mécanique, mais n apporte pas de correction histologique. Cette étude souligne également la toxicité de cette miniprotéine. L anoctamine 5, impliquée dans des pathologies et des activités similaires à la dysferline, a été testée en tant que protéine compensatrice. L anoctamine 5 surexprimée dans le modèle Bla/J ne permet pas la restauration d un phénotype normal. La compensation de DYSF par ANO5 n est donc pas une voie thérapeutique à exploiter pour les dysferlinopathies. Enfin, une thérapie génique par chirurgie de l ARN dysferline a été évaluée en utilisant le trans-épissage médié par le splicéosome (SMaRT). La preuve de principe de la reprogrammation d un minigène dysferline a été faite (ARN et protéine trans-épissée obtenus in vitro). L efficacité du SMaRT dans un contexte endogène s est en revanche révélée faible, et n a pas permis la restauration d une protéine dysferline fonctionnelle dans des myoblastes humains. De plus, l observation de l expression de protéines directement à partir du RTM (RNA-trans-splicing molecule) a fait apparaître des limites à l utilisation du SMaRT pour la thérapie génique, et en particulier pour les dysferlinopathies.Dysferlinopathies are muscular diseases due to mutations in DYSF gene, inducing dysferlin protein deficiency. In this thesis, three therapeutic approaches have been investigated for these pathologies, on cell or mice models. A short transcriptional dysferlin variant has been injected into Bla/J dysferlin deficient mouse model, using AAV8r vector. Muscle fibers of treated animals displayed an increased resistance to mechanical stress without therapeutic benefit. These experiments also pointed out the toxicity of this strategy. A protein compensation approach has been tested using anoctamin 5, known to be involved in pathologies and activities similar to dysferlin s ones. AAVr mediated Anoctamin 5 overexpression in Bla/J model does not rescue their muscle phenotype. Overexpression of ANO5 does not seem to be a valuable therapeutic strategy for dysferlin deficiency. Dysferlin RNA surgery was evaluated as a possible genetic therapy using Spliceosome-Mediated RNA Trans-splicing (SMaRT). On a Minigene target, SMaRT is able to induce RNA reprogramming by trans-splicing, and produce the corresponding protein. But efficiency is by far decreased in endogenous context and not good enough to restore functional dysferlin in human myoblasts. Moreover, we described proteins resulting from RNA-trans-splicing molecule (RTM) self-expression, limiting the value of SMaRT as therapeutic strategy, especially for dysferlinopathies.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Evaluation of three approaches of gene therapy for the treatment of dysferlinopathies : miniprotein, compensation and trans-splicing

Les dysferlinopathies sont des maladies musculaires dues à une déficience en protéine dysferline, codée par le gène DYSF. Dans ce travail de thèse, trois approches thérapeutiques ont été évaluées pour ces pathologies, sur des modèles cellulaires et murins. Un variant transcriptionnel court de la dysferline a été vectorisé dans un AAV8r et injecté dans le modèle murin Bla/J, déficient en dysferline. L’analyse des muscles des animaux traités montre une augmentation de la résistance des fibres musculaires au stress mécanique, mais n’apporte pas de correction histologique. Cette étude souligne également la toxicité de cette miniprotéine. L’anoctamine 5, impliquée dans des pathologies et des activités similaires à la dysferline, a été testée en tant que protéine compensatrice. L’anoctamine 5 surexprimée dans le modèle Bla/J ne permet pas la restauration d’un phénotype normal. La compensation de DYSF par ANO5 n’est donc pas une voie thérapeutique à exploiter pour les dysferlinopathies. Enfin, une thérapie génique par chirurgie de l’ARN dysferline a été évaluée en utilisant le trans-épissage médié par le splicéosome (SMaRT). La preuve de principe de la reprogrammation d’un minigène dysferline a été faite (ARN et protéine trans-épissée obtenus in vitro). L’efficacité du SMaRT dans un contexte endogène s’est en revanche révélée faible, et n’a pas permis la restauration d’une protéine dysferline fonctionnelle dans des myoblastes humains. De plus, l’observation de l’expression de protéines directement à partir du RTM (RNA-trans-splicing molecule) a fait apparaître des limites à l’utilisation du SMaRT pour la thérapie génique, et en particulier pour les dysferlinopathies.Dysferlinopathies are muscular diseases due to mutations in DYSF gene, inducing dysferlin protein deficiency. In this thesis, three therapeutic approaches have been investigated for these pathologies, on cell or mice models. A short transcriptional dysferlin variant has been injected into Bla/J dysferlin deficient mouse model, using AAV8r vector. Muscle fibers of treated animals displayed an increased resistance to mechanical stress without therapeutic benefit. These experiments also pointed out the toxicity of this strategy. A protein compensation approach has been tested using anoctamin 5, known to be involved in pathologies and activities similar to dysferlin’s ones. AAVr mediated Anoctamin 5 overexpression in Bla/J model does not rescue their muscle phenotype. Overexpression of ANO5 does not seem to be a valuable therapeutic strategy for dysferlin deficiency. Dysferlin RNA surgery was evaluated as a possible genetic therapy using Spliceosome-Mediated RNA Trans-splicing (SMaRT). On a Minigene target, SMaRT is able to induce RNA reprogramming by trans-splicing, and produce the corresponding protein. But efficiency is by far decreased in endogenous context and not good enough to restore functional dysferlin in human myoblasts. Moreover, we described proteins resulting from RNA-trans-splicing molecule (RTM) self-expression, limiting the value of SMaRT as therapeutic strategy, especially for dysferlinopathies

Le corps en travail : des "portraits à la Despatin/Gobeli"

International audienc

Cis -splicing and Translation of the Pre- Trans -splicing Molecule Combine With Efficiency in Spliceosome-mediated RNA Trans -splicing

International audienceMuscular dystrophies are a group of genetically distinct diseases for which no treatment exists. While gene transfer approach is being tested for several of these diseases, such strategies can be hampered when the size of the corresponding complementary DNA (cDNA) exceeds the packaging capacity of adeno-associated virus vectors. This issue concerns, in particular, dysferlinopathies and titinopathies that are due to mutations in the dysferlin (DYSF) and titin (TTN) genes. We investigated the efficacy of RNA trans-splicing as a mode of RNA therapy for these two types of diseases. Results obtained with RNA trans-splicing molecules designed to target the 3' end of mouse titin and human dysferlin pre-mRNA transcripts indicated that trans-splicing of pre-mRNA generated from minigene constructs or from the endogenous genes was achieved. Collectively, these results provide the first demonstration of DYSF and TTN trans-splicing reprogramming in vitro and in vivo. However, in addition to these positive results, we uncovered a possible issue of the technique in the form of undesirable translation of RNA pre-trans-splicing molecules, directly from open reading frames present on the molecule or associated with internal alternative cis-splicing. These events may hamper the efficiency of the trans-splicing process and/or lead to toxicity

Removal of the calpain 3 protease reverses the myopathology in a mouse model for titinopathies

International audienceThe dominant tibial muscular dystrophy (TMD) and recessive limb-girdle muscular dystrophy 2J are allelicdisorders caused by mutations in the C-terminus of titin, a giant sarcomeric protein. Both clinical presentationswere initially identified in a large Finnish family and linked to a founder mutation (FINmaj). To furtherunderstand the physiopathology of these two diseases, we generated a mouse model carrying the FINmajmutation. In heterozygous mice, dystrophic myopathology appears late at 9 months of age in few distalmuscles. In homozygous (HO) mice, the first signs appear in the Soleus at 1 month of age and extend tomost muscles at 6 months of age. Interestingly, the heart is also severely affected in HO mice. The mutationleads to the loss of the very C-terminal end of titin and to a secondary deficiency of calpain 3, a partner oftitin. By crossing the FINmaj model with a calpain 3-deficient model, the TMD phenotype was corrected,demonstrating a participation of calpain 3 in the pathogenesis of this disease

The α1,6-Fucosyltransferase Gene (<i>fut8</i>) from the <i>Sf</i>9 Lepidopteran Insect Cell Line: Insights into <i>fut8</i> Evolution

<div><p>The core alpha1,6-fucosyltransferase (FUT8) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the innermost asparagine-linked <i>N</i>-acetylglucosamine residue of glycoproteins. In mammals, this glycosylation has an important function in many fundamental biological processes and although no essential role has been demonstrated yet in all animals, FUT8 amino acid (aa) sequence and FUT8 activity are very well conserved throughout the animal kingdom. We have cloned the cDNA and the complete gene encoding the FUT8 in the <i>Sf9</i> (<i>Spodoptera frugiperda</i>) lepidopteran cell line. As in most animal genomes, <i>fut8</i> is a single-copy gene organized in different exons. The open reading frame contains 12 exons, a characteristic that seems to be shared by all lepidopteran <i>fut8</i> genes. We chose to study the gene structure as a way to characterize the evolutionary relationships of the <i>fut8</i> genes in metazoans. Analysis of the intron-exon organization in 56 <i>fut8</i> orthologs allowed us to propose a model for <i>fut8</i> evolution in metazoans. The presence of a highly variable number of exons in metazoan <i>fut8</i> genes suggests a complex evolutionary history with many intron gain and loss events, particularly in arthropods, but not in chordata. Moreover, despite the high conservation of lepidoptera FUT8 sequences also in vertebrates and hymenoptera, the exon-intron organization of hymenoptera <i>fut8</i> genes is order-specific with no shared exons. This feature suggests that the observed intron losses and gains may be linked to evolutionary innovations, such as the appearance of new orders.</p></div

Analysis of intron positions shared with <i>S. frugiperda fut8</i> (i<i>number</i>l) in arthropoda and chordata orthologs <i>fut8</i> genes.

<p>IS: intronic sequence, Nb: number. The intron phase was defined as follows: phase 0 introns are inserted between two codons; phase 1 introns are inserted after the first nucleotide of the codon, and phase 2 introns are inserted between the second and the third nucleotide of the codon.</p><p>(*) The gene is truncated and the 5′ exon is missing.</p><p>Analysis of intron positions shared with <i>S. frugiperda fut8</i> (i<i>number</i>l) in arthropoda and chordata orthologs <i>fut8</i> genes.</p

Schematic illustration of the correlation between animal <i>fut8</i> gene phylogeny, intron gain/loss and intron position (gene organization).

<p>This analysis was carried out by considering only the intron insertion sites in the <i>fut8</i> gene sequences encoding the conserved region of FUT8 proteins, between i<i>3</i>l and the stop codon. On the right, column 1 shows the total number of intron insertion sites (IS) identified in the different <i>fut8</i> genes, and column 2 shows the number of order- or family-specific intron insertion sites. Intron gains and losses are highlighted (grey and white boxes, respectively) as well as putative intron sliding in “near-intron-pairs” (light grey boxes). Putative ancestral introns are in a dark grey box.</p

Conserved aa and motifs found in all the α1,6-fucosyltransferases sequences.

<p>Schematic representations of the FUT8 protein showing the cytoplasmic (C), transmembrane (T) and stem region (S) characteristic of α1,6-fucosyltransferases. The catalytic domain is in white and motifs I, II and III in grey. In addition, a region found only in α1,6-fucosyltransferase with conserved cysteine residues is indicated by dashed lines and was named “Cys-rich” domain. Conserved aa and those implicated in the enzymatic activity are highlighted with orange stars. The conserved peptide sequences used to generate the motif I, motif II and motif III sequence logos were extracted from multiple alignments of 96 α1,6-fucosyltransferase sequences identified in the databases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422.s004" target="_blank">Table S2</a>) and visualized at the Weblogos site at Berkeley, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Felgner1" target="_blank">[63]</a>. In the logos, aa are colored according to their chemical properties: polar aa (G, C, S, T, Y) are green, basic (K, R, H) are blue, acidic (D, E) are red, hydrophobic (A, V, L, I, P, W, F, M) are black and neutral polar aa (N, Q) are pink. The overall height of the stacks indicates the sequence conservation at a given position, while the height of the symbol within the stack indicates the relative frequency of each aa at that position. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Nei1" target="_blank">[69]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Crooks1" target="_blank">[70]</a>.</p

Intron positions shared by <i>S. frugiperda fut8</i> (i<i>number</i>l) and with <i>fut8</i> genes identified in chordata (i<i>number</i>c).

<p>IS: Intronic Sequence.</p><p>(X): Presence of lepidopteran intron insertion site.</p><p>(<b>+</b>): Presence of chordata intron insertion site.</p><p>(*) The gene is truncated and the 5′ exon is missing.</p><p>Intron positions shared by <i>S. frugiperda fut8</i> (i<i>number</i>l) and with <i>fut8</i> genes identified in chordata (i<i>number</i>c).</p