Investigating the Impact of Delivery Routes for Exon Skipping Therapies in the CNS of DMD Mouse Models

Nucleic acid-based therapies have demonstrated great potential for the treatment of monogenetic diseases, including neurologic disorders. To date, regulatory approval has been received for a dozen antisense oligonucleotides (ASOs); however, these chemistries cannot readily cross the blood–brain barrier when administered systemically. Therefore, an investigation of their potential effects within the central nervous system (CNS) requires local delivery. Here, we studied the brain distribution and exon-skipping efficacy of two ASO chemistries, PMO and tcDNA, when delivered to the cerebrospinal fluid (CSF) of mice carrying a deletion in exon 52 of the dystrophin gene, a model of Duchenne muscular dystrophy (DMD). Following intracerebroventricular (ICV) delivery (unilateral, bilateral, bolus vs. slow rate, repeated via cannula or very slow via osmotic pumps), ASO levels were quantified across brain regions and exon 51 skipping was evaluated, revealing that tcDNA treatment invariably generates comparable or more skipping relative to that with PMO, even when the PMO was administered at higher doses. We also performed intra-cisterna magna (ICM) delivery as an alternative route for CSF delivery and found a biased distribution of the ASOs towards posterior brain regions, including the cerebellum, hindbrain, and the cervical part of the spinal cord. Finally, we combined both ICV and ICM injection methods to assess the potential of an additive effect of this methodology in inducing efficient exon skipping across different brain regions. Our results provide useful insights into the local delivery and associated efficacy of ASOs in the CNS in mouse models of DMD. These findings pave the way for further ASO-based therapy application to the CNS for neurological disease

Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes

Effects of low-dose gestational tcdd exposure on behavior and on hippocampal neuron morphology and gene expression in mice

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and toxic environmental pollutant. Gestational exposure to TCDD has been linked to cognitive and motor deficits, and increased incidence of autism spectrum disorder (ASD) traits in children. Most animal studies of these neurodevelopmental effects involve acute TCDD exposure, which does not model typical exposure in humans.publishedVersio

Structural insights into human brachyury DNA recognition and discovery of progressible binders for cancer therapy

Brachyury is a transcription factor that plays an essential role in tumour growth of the rare bone cancer chordoma and is implicated in other solid tumours. Brachyury is minimally expressed in healthy tissues, making it a potential therapeutic target. Unfortunately, as a ligandless transcription factor, brachyury has historically been considered undruggable. To investigate direct targeting of brachyury by small molecules, we determine the structure of human brachyury both alone and in complex with DNA. The structures provide insights into DNA binding and the context of the chordoma associated G177D variant. We use crystallographic fragment screening to identify hotspots on numerous pockets on the brachyury surface. Finally, we perform follow-up chemistry on fragment hits and describe the progression of a thiazole chemical series into binders with low µM potency. Thus we show that brachyury is ligandable and provide an example of how crystallographic fragment screening may be used to target protein classes that are difficult to address using other approaches

Networking to Optimize Dmd exon 53 Skipping in the Brain of mdx52 Mouse Model

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the open reading frame and thus prevent production of functional dystrophin proteins. Recent advances in DMD treatment, notably exon skipping and AAV gene therapy, have achieved some success aimed at alleviating the symptoms related to progressive muscle damage. However, they do not address the brain comorbidities associated with DMD, which remains a critical aspect of the disease. The mdx52 mouse model recapitulates one of the most frequent genetic pathogenic variants associated with brain involvement in DMD. Deletion of exon 52 impedes expression of two brain dystrophins, Dp427 and Dp140, expressed from distinct promoters. Interestingly, this mutation is eligible for exon skipping strategies aimed at excluding exon 51 or 53 from dystrophin mRNA. We previously showed that exon 51 skipping can restore partial expression of internally deleted yet functional Dp427 in the brain following intracerebroventricular (ICV) injection of antisense oligonucleotides (ASO). This was associated with a partial improvement of anxiety traits, unconditioned fear response, and Pavlovian fear learning and memory in the mdx52 mouse model. In the present study, we investigated in the same mouse model the skipping of exon 53 in order to restore expression of both Dp427 and Dp140. However, in contrast to exon 51, we found that exon 53 skipping was particularly difficult in mdx52 mice and a combination of multiple ASOs had to be used simultaneously to reach substantial levels of exon 53 skipping, regardless of their chemistry (tcDNA, PMO, or 2'MOE). Following ICV injection of a combination of ASO sequences, we measured up to 25% of exon 53 skipping in the hippocampus of treated mdx52 mice, but this did not elicit significant protein restoration. These findings indicate that skipping mouse dystrophin exon 53 is challenging. As such, it has not yet been possible to answer the pertinent question whether rescuing both Dp427 and Dp140 in the brain is imperative to more optimal treatment of neurological aspects of dystrophinopathy

Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment

The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3′ untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex “carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT),” which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3′-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay

Open drug discovery in Alzheimer\u27s disease.

Alzheimer\u27s disease (AD) drug discovery has focused on a set of highly studied therapeutic hypotheses, with limited success. The heterogeneous nature of AD processes suggests that a more diverse, systems-integrated strategy may identify new therapeutic hypotheses. Although many target hypotheses have arisen from systems-level modeling of human disease, in practice and for many reasons, it has proven challenging to translate them into drug discovery pipelines. First, many hypotheses implicate protein targets and/or biological mechanisms that are under-studied, meaning there is a paucity of evidence to inform experimental strategies as well as high-quality reagents to perform them. Second, systems-level targets are predicted to act in concert, requiring adaptations in how we characterize new drug targets. Here we posit that the development and open distribution of high-quality experimental reagents and informatic outputs-termed target enabling packages (TEPs)-will catalyze rapid evaluation of emerging systems-integrated targets in AD by enabling parallel, independent, and unencumbered research

FOXN1 forms higher-order nuclear condensates displaced by mutations causing immunodeficiency

The transcription factor FOXN1 is a master regulator of thymic epithelial cell (TEC) development and function. Here, we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multimolecular nuclear condensates essential for the factor’s transcriptional activity. FOXN1’s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are altered in a patient with a mutant FOXN1 that is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog selectively impairs mouse TEC differentiation, revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer\u27s disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer\u27s disease (AD) traits and microglia. These proteins are more abundant in Alzheimer\u27s patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PI