Genetic Knock-Down of Hdac3 Does Not Modify Disease-Related Phenotypes in a Mouse Model of Huntington's Disease

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expansion of a CAG/polyglutamine repeat for which there are no disease modifying treatments. In recent years, transcriptional dysregulation has emerged as a pathogenic process that appears early in disease progression and has been recapitulated across multiple HD models. Altered histone acetylation has been proposed to underlie this transcriptional dysregulation and histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), have been shown to improve polyglutamine-dependent phenotypes in numerous HD models. However potent pan-HDAC inhibitors such as SAHA display toxic side-effects. To better understand the mechanism underlying this potential therapeutic benefit and to dissociate the beneficial and toxic effects of SAHA, we set out to identify the specific HDAC(s) involved in this process. For this purpose, we are exploring the effect of the genetic reduction of specific HDACs on HD-related phenotypes in the R6/2 mouse model of HD. The study presented here focuses on HDAC3, which, as a class I HDAC, is one of the preferred targets of SAHA and is directly involved in histone deacetylation. To evaluate a potential benefit of Hdac3 genetic reduction in R6/2, we generated a mouse carrying a critical deletion in the Hdac3 gene. We confirmed that the complete knock-out of Hdac3 is embryonic lethal. To test the effects of HDAC3 inhibition, we used Hdac3+/− heterozygotes to reduce nuclear HDAC3 levels in R6/2 mice. We found that Hdac3 knock-down does not ameliorate physiological or behavioural phenotypes and has no effect on molecular changes including dysregulated transcripts. We conclude that HDAC3 should not be considered as the major mediator of the beneficial effect induced by SAHA and other HDAC inhibitors in HD

Oligonucleotide Therapeutics: From Discovery and Development to Patentability

Following the first proof of concept of using small nucleic acids to modulate gene expression, a long period of maturation led, at the end of the last century, to the first marketing authorization of an oligonucleotide-based therapy. Since then, 12 more compounds have hit the market and many more are in late clinical development. Many companies were founded to exploit their therapeutic potential and Big Pharma was quickly convinced that oligonucleotides could represent credible alternatives to protein-targeting products. Many technologies have been developed to improve oligonucleotide pharmacokinetics and pharmacodynamics. Initially targeting rare diseases and niche markets, oligonucleotides are now able to benefit large patient populations. However, there is still room for oligonucleotide improvement and further breakthroughs are likely to emerge in the coming years. In this review we provide an overview of therapeutic oligonucleotides. We present in particular the different types of oligonucleotides and their modes of action, the tissues they target and the routes by which they are administered to patients, and the therapeutic areas in which they are used. In addition, we present the different ways of patenting oligonucleotides. We finally discuss future challenges and opportunities for this drug-discovery platform

Mécanismes d’action et brevetabilité des oligonucléotides thérapeutiques

International audienceLes oligonucléotides sont des petits acides nucléiques de synthèse capables de moduler l’expression de gènes cibles et leurs transcrits. Largement utilisés par les chercheurs comme outils de recherche pour moduler l’expression des gènes dont ils cherchent à décrypter les fonctions, les oligonucléotides peuvent également servir d’agents thérapeutiques pour réguler des cibles d’intérêt. Après l’arrivée sur le marché du premier oligonucléotide thérapeutique en 1998, le domaine a connu peu de succès cliniques jusqu’en 2016, date à laquelle le Spinraza® devient le premier médicament autorisé pour le traitement de l’amyotrophie spinale. Il deviendra dans les années suivantes le premier « blockbuster »1 de cette classe de molécules. Depuis lors, une dizaine d’oligonucléotides ont reçu des autorisations de mise sur le marché (AMM), et de nombreux autres font actuellement l’objet d’un développement clinique. Dans cet article, nous décrivons différents oligonucléotides thérapeutiques, ainsi que leurs modes d’action et leur brevetabilité

Oligonucleotide Therapeutics: From Discovery and Development to Patentability

Following the first proof of concept of using small nucleic acids to modulate gene expression, a long period of maturation led, at the end of the last century, to the first marketing authorization of an oligonucleotide-based therapy. Since then, 12 more compounds have hit the market and many more are in late clinical development. Many companies were founded to exploit their therapeutic potential and Big Pharma was quickly convinced that oligonucleotides could represent credible alternatives to protein-targeting products. Many technologies have been developed to improve oligonucleotide pharmacokinetics and pharmacodynamics. Initially targeting rare diseases and niche markets, oligonucleotides are now able to benefit large patient populations. However, there is still room for oligonucleotide improvement and further breakthroughs are likely to emerge in the coming years. In this review we provide an overview of therapeutic oligonucleotides. We present in particular the different types of oligonucleotides and their modes of action, the tissues they target and the routes by which they are administered to patients, and the therapeutic areas in which they are used. In addition, we present the different ways of patenting oligonucleotides. We finally discuss future challenges and opportunities for this drug-discovery platform

Differential aggregation and functional impairment induced by polyalanine expansions in FOXL2, a transcription factor involved in cranio-facial and ovarian development

International audiencePolyalanine (polyAla) tract expansions have been associated with an increasing number of human diseases. Here, we have undertaken a functional study of the effects of polyAla expansions in the context of the transcription factor FOXL2, involved in cranio-facial and ovarian development. Using two cellular models, we show that FOXL2 polyAla expansions lead to protein mislocalization and aggregation in a length-dependent manner. The fraction of cells containing cytoplasmic staining displays a sigmoidal relationship with respect to the length of the polyAla tract, suggesting the existence of a threshold length above which protein mislocalization occurs. The existence of such a threshold might be rationalized if we consider that the longer the polyAla tract is, the higher its tendency to misfolding or to inducing spurious interactions with cytoplasmic components. To study the intranuclear dynamics of polyAla-expanded FOXL2, we performed fluorescence recovery after photobleaching experiments. The most unexpected result concerned the pathogenic protein containing 19 Ala residues in the run, which was virtually immobile, although this variant does not present a classical aggregation pattern. Luciferase assays and real time RT-PCR of many potential target genes showed that polyAla expansions induce different losses of activity according to the target promoters tested. We provide molecular explanations for these findings. Although our main focus is the mechanisms of pathogenesis of polyAla-expanded proteins, we discuss the potential relevance of polyAla length variation in micro- and macroevolution because polyAla-containing proteins tend to be transcription factors

<i>Hdac3</i> genetic reduction does not reverse transcriptional dysregulation in R6/2.

<p>(A) Expression of <i>Bdnf</i> transcripts from different promoters (<i>Bdnf I</i>, <i>IV</i> and <i>V</i>) and the coding region (<i>Bdnf B</i>) in the cortex are represented as a percent of WT expression levels. With the exception of a slight decrease in <i>Bdnf</i> V, <i>Hdac3</i> reduction did not affect <i>Bdnf</i> expression. (B) Expression levels of genes specifically altered in the cerebellum of R6/2 mice are represented as a percent of WT expression. No significant difference was induced by <i>Hdac3</i> genetic reduction. (C) Expression levels of genes specifically altered in the striatum of R6/2 mice are represented as a percent of WT expression. A significant decrease in the expression of <i>Cnr1</i> in non-transgenic animals was observed as well as a slightly significant increase in <i>Cnr1</i> expression in R6/2 striata. Expression of the R6/2 transgene in Dbl brains is represented as a percent of that in R6/2 brains for cortex (D), cerebellum (E) and striatum (F). <i>Hdac3</i> reduction did not induce a significant change in transgene expression. Error bars correspond to S.E.M. (n = 8) *p<0.05. The same color code (blue = WT; red = Hdac3; green = R6/2 and purple = Dbl) was used for all the graphs. <i>Bdnf I, IV V</i>, brain derived neurotrophic factor promoter I, IV, V; <i>Bdnf B</i>, brain derived neurotrophic factor coding exon B; <i>Igfbp5</i>, insulin-like growth factor binding protein 5; <i>Kcnk2</i>, potassium channel subfamily K, member 2; <i>Nr4a2</i>, nuclear receptor subfamily 4, group A, member 2; <i>Pcp4</i>, Purkinje cell protein 4; <i>Uchl1</i>, ubiquitin C-terminal hydrolase L1; <i>Cnr1</i>, cannabinoid receptor 1; <i>Darpp32</i>, dopamine and cAMP regulated neuronal phosphoprotein; <i>Drd2</i>, dopamine D2 receptor; <i>Penk1</i>, proenkephalin.</p

Generation of an <i>Hdac3</i> convention knock-out allele.

<p>(A) Strategy to generate an <i>Hdac3</i> conventional knock-out allele. The genomic structure and the targeting vector are shown. The <i>Hdac</i>3 gene contains 15 exons (blue rectangles). LoxP sites (red triangles) were introduced upstream exon 11 and within exon 15. The vector contains a 5′ homology arm covering the exonic and intronic region from intron 3–4 to intron 10–11 and a 3′ homology arm covering a part of exon 15 and the 3′UTR (green rectangle). The conditional knock-out region (yellow rectangle) covers exon 11 to 14 and 5′ end of exon 15. This conditional allele was introduced by homologous recombination in ES cells. The neomycine cassette (pink rectangle) flanked by 2 LoxP sites was removed by electroporation of Cre recombinase in ES cells and the cells containing the allele corresponding to a complete deletion of exon 11 to 14 were selected. Primers used for genotyping are represented as black arrows. F1 = forward 1; F2 = forward 2; R = Reverse. (B) Representative genotyping PCR on mouse genomic DNA. Duplex PCR with F1, F2 and R primers detects both the WT (250 bp band with primers F2/R) and the knock-out (500 bp with primers F1/R) allele.</p

<i>Hdac3</i> genetic reduction does not modify R6/2 phenotypes.

<p>(A) Weight loss in males (left panel) and females (right panel) are shown between 4 and 15 weeks of age. <i>Hdac3</i> genetic reduction did not induce a significant increase of the body weight in R6/2 (B) RotaRod performance is represented as the average latency to fall in each group at 4, 8, 10, 12 and 14 weeks. <i>Hdac3</i> genetic reduction did not ameliorate the impairment in RotaRod performance in R6/2 (C) Average grip strength in each group is represented at 4, 11, 12, 13 and 14 weeks. <i>Hdac3</i> genetic reduction did not induce a significant improvement in the grip strength in R6/2 mice (D) Average activity for each genotype is shown at 5 (upper panel) and 13 (lower panel) weeks of age. <i>Hdac3</i> genetic reduction did not reverse the hypoactivity observed in R6/2 mice (E) Average brain weight for each group was measured at 15 weeks of age. <i>Hdac3</i> genetic reduction did not modify the brain weight loss in R6/2 but a slight increase in brain weight was observed in WT animals **p<0.01. Error bars correspond to SEM (n>12). The same color code (blue = WT; red = Hdac3; green = R6/2 and purple = Dbl) was used for all measured parameters.</p