Wave Life Sciences (United States)

Liposomes are considered among the most versatile and advanced nanoparticle delivery systems used to target drugs to specific cells and tissues. Structurally, liposomes are sphere-like vesicles of phospholipid molecules that are surrounded by equal number of aqueous compartments. The spherical shell encapsulates an aqueous interior which contains substances such as peptides and proteins, hormones, enzymes, antibiotics, antifungal and anticancer agents. This structural property of liposomes makes it an important nano-carrier for drug delivery. Extrusion is one of the most frequently used technique for preparing monodisperse uni-lamellar liposomes as the technique is used to control vesicle size. The process involves passage of lipid suspension through polycarbonate membrane with a fixed pore size to produce vesicles with a diameter near the pore size of the membrane used in preparing them. An advantage of this technique is that there is no need to remove the organic solvent or detergent from the final preparation. This review focuses on composition of liposome formulation with special emphasis on factors affecting drug release and drug-loading.

Abstract Background: Tau is a neuronal scaffolding protein which aggregates intracellularly upon hyperphosphorylation to form neurofibrillary tangles (NFT). NFTs are a hallmark of various microtubule associated protein tau (MAPT)‐associated neurodegenerative diseases, including Alzheimer’s disease, frontotemporal dementia, and progressive supranuclear palsy. Currently, there is no approved treatment targeting Tau pathology, nor any other disease‐modifying therapy for these diseases. Wave Life Sciences has developed a stereopure antisense oligonucleotide to reduce MAPT expression in patients with MAPT‐associated neurodegenerative diseases. Method: Using Wave’s proprietary PRISM platform, we designed and screened stereopure oligonucleotides targeting MAPT in vitro and identified a lead sequence. PN chemistry was applied to the lead sequence in subsequent studies to further improve pharmacological properties. In vitro target engagement was measured in human iCell neurons after treatment with Wave’s oligonucleotide, WVE‐005, or a published reference stereorandom oligonucleotide under gymnotic conditions. For in vivo analysis, transgenic mice received a single dose of 12.5, 25, 50, or 100µg oligonucleotide, and target engagement was measured after 4 weeks. To measure duration, transgenic mice received a single 100µg ICV dose and target engagement was tracked over 6 months. Finally, oligonucleotides were tested in non‐human primates (NHP) in a 12mg single‐dose intrathecal (IT) study. MAPT mRNA levels were quantified by qPCR, and intracellular distribution was evaluated by ViewRNA. Result: WVE‐005 showed dose‐dependent silencing of MAPT mRNA in iPSC‐derived neurons with an IC50 of 84nM. In a dose‐response study, 12.5µg and 25µg WVE‐005 led to 50% knockdown after 4 weeks in hippocampus and cortex, respectively, in transgenic mice. WVE‐005 led to >77% MAPT mRNA knockdown at 12 weeks post 100µg dosing, with knockdown of approximately 50% persisting 6 months post‐injection. In NHPs, WVE‐005 showed improved distribution and potency compared to non‐PN oligonucleotide with the same sequence. WVE‐005 decreased MAPT expression across NHP brain regions 28 days post‐single dose much greater than the non‐PN oligonucleotide and was detected in neuronal and glial cells. Conclusion: WVE‐005 potently and durably decreased MAPT mRNA expression in vitro and in multiple animal models, including throughout CNS upon IT administration in NHPs. These data support continued evaluation of WVE‐005 as a potential therapeutic for MAPT‐associated neurodegenerative diseases.

Alternative splicing is frequently involved in the diversification of protein function and can also be modulated for therapeutic purposes. Here we develop a predictive model, called Exon ByPASS (predicting Exon skipping Based on Protein amino acid SequenceS), to assess the criticality of exon inclusion based solely on information contained in the amino acid sequence upstream and downstream of the exon junctions. By focusing on protein sequence, Exon ByPASS predicts exon skipping independent of tissue and species in the absence of any intronic information. We validate model predictions using transcriptomic and proteomic data and show that the model can capture exon skipping in different tissues and species. Additionally, we reveal potential therapeutic opportunities by predicting synthetically skippable exons and neo-junctions arising in cancer cells.

Abstract A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. We utilised the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the repeat expansion in CSF of people with C9orf72-associated FTD/ALS. We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intra- and inter-plate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) DPRs in the CSF of samples collected through the Genetic FTD Initiative. We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had 8-fold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.

Volumetric magnetic resonance imaging (vMRI) has been widely studied in Huntington's disease (HD) and is commonly used to assess treatment effects on brain atrophy in interventional trials. Global and regional trajectories of brain atrophy in HD, with early involvement of striatal regions, are becoming increasingly understood. However, there remains heterogeneity in the methods used and a lack of widely-accessible multisite, longitudinal, normative datasets in HD. Consensus for standardized practices for data acquisition, analysis, sharing, and reporting will strengthen the interpretation of vMRI results and facilitate their adoption as part of a pathobiological disease staging system. The Huntington's Disease Regulatory Science Consortium (HD-RSC) currently comprises 37 member organizations and is dedicated to building a regulatory science strategy to expedite the approval of HD therapeutics. Here, we propose four recommendations to address vMRI standardization in HD research: (1) a checklist of standardized practices for the use of vMRI in clinical research and for reporting results; (2) targeted research projects to evaluate advanced vMRI methodologies in HD; (3) the definition of standard MRI-based anatomical boundaries for key brain structures in HD, plus the creation of a standard reference dataset to benchmark vMRI data analysis methods; and (4) broad access to raw images and derived data from both observational studies and interventional trials, coded to protect participant identity. In concert, these recommendations will enable a better understanding of disease progression and increase confidence in the use of vMRI for drug development.

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.

PurposeAntisense oligonucleotides have been under investigation as potential therapeutics for many diseases, including inherited retinal diseases. Chemical modifications, such as chiral phosphorothioate (PS) backbone modification, are often used to improve stability and pharmacokinetic properties of these molecules. We aimed to generate a stereopure MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) antisense oligonucleotide as a tool to assess the impact stereochemistry has on potency, efficacy, and durability of oligonucleotide activity when delivered by intravitreal injection to eye.MethodsWe generated a stereopure oligonucleotide (MALAT1-200) and assessed the potency, efficacy, and durability of its MALAT1 RNA-depleting activity compared with a stereorandom mixture, MALAT1-181, and other controls in in vitro assays, in vivo mouse and nonhuman primate (NHP) eyes, and ex vivo human retina cultures.ResultsThe activity of the stereopure oligonucleotide is superior to its stereorandom mixture counterpart with the same sequence and chemical modification pattern in in vitro assays, in vivo mouse and NHP eyes, and ex vivo human retina cultures. Findings in NHPs showed durable activity of the stereopure oligonucleotide in the retina, with nearly 95% reduction of MALAT1 RNA maintained for 4 months postinjection.ConclusionsAn optimized, stereopure antisense oligonucleotide shows enhanced potency, efficacy, and durability of MALAT1 RNA depletion in the eye compared with its stereorandom counterpart in multiple preclinical models.Translational RelevanceAs novel therapeutics, stereopure oligonucleotides have the potential to enable infrequent administration and low-dose regimens for patients with genetic diseases of the eye.