Matthew J.A. Wood’s research while affiliated with University of Oxford and other places

Splice-switching oligonucleotides (SSOs) targeting BTK pre-mRNA offer a potential therapy for X-linked agammaglobulinemia (XLA). PMO-Pip6a conjugate treatment partially restores the B lineage phenotype, offering insights into splice correction in B cell maturation.

To understand the relative contributions of 5ʹ UTR elements to translation, we performed a comprehensive analysis of upstream open reading frames (uORFs) across a representative 5ʹ UTR. We selected the neurotrophin BDNF (Brain derived neurotrophic factor) as an exemplar as upregulation of this protein is a potential therapeutic approach for a plethora of neurodevelopmental, neurodegenerative, and neuropsychiatric disorder indications. Predicted uORFs were identified in 14 out of 17 BDNF RefSeq transcript isoforms, and experimentally confirmed to be exerting translation repression effects for five of these transcripts. These findings suggest that uORF elements play an important role in shaping the protein output from this locus. We explored several approaches to disrupt BDNF uORF function. Deletion of a 5ʹ UTR exon in BDNF v11 (containing eight predicted uORFs), in order to simulate an exon skipping outcome, resulted in pronounced upregulation in a reporter construct system. This effect was found to be partially uORF-dependent, but was also dependent on the disruption of an RNA secondary structure element. However, this transcript variant was found to not be expressed in human brain. Conversely, direct disruption of a single uORF start codon in the widely expressed BDNF v4 transcript variant using an adenine base editing approach resulted in a ~1.8-fold upregulation of endogenous BDNF protein expression in cell culture. This study describes novel BDNF regulatory mechanisms, and potential uORF-targeted modalities for therapeutic gene activation.

Cytotoxic attack particles released by CTLs and NK cells include diverse phospholipid membrane and glycoprotein encapsulated entities that contribute to target cell killing. Supramolecular attack particles (SMAPs) are one type of particle characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a proteinaceous shell including thrombospondin (TSP)-1. TSP-4 was also detected in bulk analysis of SMAPs released by CTLs; however, it has not been investigated whether TSP-4 contributes to distinct SMAP types or the same SMAP type as TSP-1 and, if in the same type of SMAP, whether TSP-4 and TSP-1 cooperate or compete. Here, we observed that TSP-4 expression increased upon CD8 ⁺ T cell activation while, surprisingly, TSP-1 was down-regulated. Correlative Light and Electron Microscopy and Stimulated Emission Depletion microscopy localized TSP-4 and TSP-1 in SMAP-containing multicore granules. Superresolution dSTORM revealed that TSP-4 and TSP-1 are usually enriched in the same SMAPs while particles with single-positive shells are rare. Retention Using Selective Hooks assays showed that TSP-4 localizes to the lytic granules faster than TSP-1 and promotes its accumulation therein. TSP-4 contributed to direct CTL-mediated killing, as previously shown for TSP-1. TSP-4 and TSP-1 were both required for latent SMAP-mediated cell killing, in which released SMAPs kill targets after removal of the CTLs. Of note, we found that chronic lymphocytic leukemia (CLL) cell culture supernatants suppressed expression of TSP-4 in CTL and latent SMAP-mediated killing. These results identify TSP-4 as a functionally important component of SMAPs and suggest that SMAPs may be targeted for immune suppression by CLL.

Recent years have seen enormous progress in the field of advanced therapeutics for the progressive muscle wasting disease Duchenne muscular dystrophy (DMD). In particular, four antisense oligonucleotide (ASO) therapies targeting various DMD-causing mutations have achieved FDA approval, marking major milestones in the treatment of this disease. These compounds are designed to induce alternative splicing events that restore the translation reading frame of the dystrophin gene, leading to the generation of internally-deleted, but mostly functional, pseudodystrophin proteins with the potential to compensate for the genetic loss of dystrophin. However, the efficacy of these compounds is very limited, with delivery remaining a key obstacle to effective therapy. There is therefore an urgent need for improved ASO technologies with better efficacy, and with applicability to a wider range of patient mutations. Here we discuss recent developments in ASO therapies for DMD, and future prospects with a focus on ASO chemical modification and bioconjugation strategies.

Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs (miRNAs) can be used to profile cell type and cell state, and are ideal inputs for RNA nanodevices. Here, we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.

Upstream open reading frames (uORFs) are cis-regulatory motifs that are predicted to occur in the 5′ UTRs of the majority of human protein-coding transcripts and are typically associated with translational repression of the downstream primary open reading frame (pORF). Interference with uORF activity provides a potential mechanism for targeted upregulation of the expression of specific transcripts. It was previously reported that steric block antisense oligonucleotides (ASOs) can bind to and mask uORF start codons to inhibit translation initiation, and thereby disrupt uORF-mediated gene regulation. Given the relative maturity of the oligonucleotide field, such a uORF blocking mechanism might have widespread therapeutic utility. Here, we re-synthesized three of the most potent ASOs targeting the RNASEH1 uORF described in a study by Liang et al. and investigated their potential for RNASEH1 protein upregulation, with care taken to replicate the conditions of the original study. No upregulation (of endogenous or reporter protein expression) was observed with any of the oligonucleotides tested at doses ranging from 25 to 300 nM. Conversely, we observed downregulation of expression in some instances. We conclude that previously described RNASEH1 uORF-targeting steric block ASOs are incapable of upregulating pORF protein expression in our hands.

CD4 T helper cells (TH cells) play a vital role in coordinating and amplifying the immune response to specific pathogens. They constitutively produce different kinds of extracellular vesicles (EVs), which mediate cell-to cell communication and play diverse roles in immune regulation and inflammatory processes. Here we provide a resource documenting the composition of activated TH cell EVs and demonstrating their ability to instigate pro-inflammatory response in antigen-presenting cells (APCs). EVs were characterized by lipidomics, proteomics, and NanoFCM. The activated TH cells derived EVs (act-EVs) were found to be enriched in TH cell-specific proteins, transmembrane and cytosolic EV marker proteins, and HLA proteins relative to resting CD4 T cells EVs (rest-EVs). The pro-inflammatory effect of act-EVs vs rest-EVs on donor matched APCs were characterized by chemokine and cytokine profiling and flow cytometry analysis. There was no discernible contrast in endotoxin levels between act-EVs and rest-EVs. Functional distinctions were seen to arise from variations in the content and composition of these EVs. Moreover, we validated our findings with an in-vivo investigation in mice, demonstrating the recruitment of monocytes, dendritic cells (DCs), neutrophils, and NK cells in the spleen, accompanied by the release of pro-inflammatory cytokines in the serum after administering act-EVs. In summary, this study sheds light on the role of TH cell released EVs in modulating the immune response during pro-inflammatory responses and this resource provides a foundation for development of novel therapeutics on EV based scaffolds.

Polyanionic antisense oligonucleotides hold great promise as RNA targeting drugs but issues with bioavailability hinder their development. Uncharged phosphorus-based backbones are promising alternatives but robust methods to produce them are limited. We report the synthesis and properties of oligonucleotides containing charge-neutral LNA alkyl phosphothiotriester backbones combined with 2′-O-methyl phosphorothioate nucleotides for therapeutic applications. The nature of the triester alkyl group dictates the success of solid-phase synthesis; tertiary alkyl groups are lost during the P(III) oxidation step, whereas primary alkyl groups are partially cleaved during deprotection. In contrast, oligonucleotides containing secondary phosphothiotriester linkages are stable, and large numbers of triesters can be incorporated. The modified oligonucleotides have excellent duplex stability with complementary RNA and exhibit strong nuclease resistance. To expand synthetic flexibility, oligonucleotides containing multiple internal alkynyl phosphothiotriesters can be conjugated to lipids, carbohydrates, or small molecules through CuAAC click chemistry. Oligonucleotides containing LNA-THP phosphothiotriesters exhibit high levels of pre-mRNA splice switching in eukaryotic cells.

Background Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease. Accumulating evidence strongly suggests that intrinsic muscle defects exist and contribute to disease progression, including imbalances in whole-body metabolic homeostasis. We have previously reported that tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor inducible 14 (Fn14) are significantly upregulated in skeletal muscle of the SOD1G93A ALS mouse model. While antagonising TWEAK did not impact survival, we did observe positive effects in skeletal muscle. Given that Fn14 has been proposed as the main effector of the TWEAK/Fn14 activity and that Fn14 can act independently from TWEAK in muscle, we suggest that manipulating Fn14 instead of TWEAK in the SOD1G93A ALS mice could lead to differential and potentially improved benefits. Methods We thus investigated the contribution of Fn14 to disease phenotypes in the SOD1G93A ALS mice. To do so, Fn14 knockout mice (Fn14−/−) were crossed onto the SOD1G93A background to generate SOD1G93A;Fn14−/− mice. Investigations were performed on both unexercised and exercised (rotarod and/or grid test) animals (wild type (WT), Fn14−/−, SOD1G93A and SOD1G93A;Fn14−/−). Results Here, we firstly confirm that the TWEAK/Fn14 pathway is dysregulated in skeletal muscle of SOD1G93A mice. We then show that Fn14-depleted SOD1G93A mice display increased lifespan, myofiber size, neuromuscular junction endplate area as well as altered expression of known molecular effectors of the TWEAK/Fn14 pathway, without an impact on motor function. Importantly, we also observe a complex interaction between exercise (rotarod and grid test), genotype, disease state and sex that influences the overall effects of Fn14 deletion on survival, expression of known molecular effectors of the TWEAK/Fn14 pathway, expression of myosin heavy chain isoforms and myofiber size. Conclusions Our study provides further insights on the different roles of the TWEAK/Fn14 pathway in pathological skeletal muscle and how they can be influenced by age, disease, sex and exercise. This is particularly relevant in the ALS field, where combinatorial therapies that include exercise regimens are currently being explored. As such, a better understanding and consideration of the interactions between treatments, muscle metabolism, sex and exercise will be of importance in future studies.