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Gene-editing technologies, which include the CRISPR–Cas nucleases1–3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a ‘once-and-done’ approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs. In a cynomolgus macaque model, CRISPR base editors delivered in lipid nanoparticles are shown to efficiently and stably knock down PCSK9 in the liver to reduce levels of PCSK9 and low-density lipoprotein cholesterol in the blood.

Reducing the production of larger aggregation-prone amyloid β-peptides (Aβ) remains an untested therapeutic approach for reducing the appearance and growth of Aβ plaques in the brain, which are a hallmark pathological feature of Alzheimer's disease. γ-Secretase modulators (GSMs) are therapeutics that impact γ-secretase-dependent cleavage of amyloid precursor protein to promote the production of shorter Aβ peptides that are less prone to aggregation and plaque deposition. This is accomplished without inhibiting overall γ-secretase function and cleavage of other substrates, which is believed to be a source of deleterious side effects. Here, we report the pharmacokinetic and pharmacodynamic properties of BIIB042, a novel bioavailable and brain-penetrant GSM. In cell-based assays, BIIB042 reduced the levels of Aβ42, increased the levels of Aβ38 and had little effect on the levels of Aβ40, the most abundant Aβ species. Similar pharmacodynamic properties were confirmed in the central nervous system and in plasma of mice and rats, and also in plasma of cynomolgus monkeys after a single oral dose of BIIB042. BIIB042 reduced Aβ42 levels and Aβ plaque burden in Tg2576 mice, which overexpress human amyloid precursor protein and serve as a model system for Alzheimer's disease. BIIB042 did not inhibit cleavage of other γ-secretase substrates in cell-based and in vivo signaling and cleavage assays. The pharmacodynamic effects of lowering Aβ42 in the central nervous system coupled with demonstrated efficacy in reducing plaque pathology suggests modulation of γ-secretase, with molecules like BIIB042, is a compelling therapeutic approach for the treatment of Alzheimer's disease.

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aβ42 levels in mice and rats.

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (−)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl) propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

Information on the identity of inks adds to the circumstantial evidence in legal cases involving fraudulent documents. In combination with optical methods, multiple thin-layer chromatography (TLC) is currently the analytical tool used by forensic chemists to separate, compare and distinguish inks based on their dye composition. In our studies, capillary electrophoresis (CE) was used for the analysis of water-soluble fountain pen inks. Inks are complex mixtures of synthetic organic and inorganic dyes, surfactants, resins and other components. The investigations included the development of an electrophoretic separation method, the optimization of an extraction procedure for inks from paper as well as the evaluation of ultraviolet/visible (UV/VIS) absorbance and laser-induced fluorescence (LIF) detection for the analysis of inks by CE. Good results for the separation of 17 blue and black inks of different manufacturers and countries of origin were obtained with 100 mM borate buffer, pH 8.0, containing 20% methanol. The electropherograms of the inks and their extracts from paper showed patterns that were in most cases distinctly different from each other. Ultraviolet/visible scans can be used to compare spectra of the separated main and trace components of inks. Fluorescence detection at different excitation and emission wavelengths was more sensitive, but added to the complexity of the electropherograms due to the excitation of coextracted fluorescing paper components. The resolution power of CE combined with the information content provided by the detection modes investigated prove CE to be a powerful tool for the identification of water-soluble inks used on paper documents.

Background: In recent years, there has been a growing interest in automating small-molecule bioanalytical sample preparations specifically using the Hamilton MicroLab® STAR liquid-handling platform. In the most extensive work reported thus far, multiple small-molecule sample preparation assay types (protein precipitation extraction, SPE and liquid–liquid extraction) have been integrated into a suite that is composed of graphical user interfaces and Hamilton scripts. Using that suite, bioanalytical scientists have been able to automate various sample preparation methods to a great extent. However, there are still areas that could benefit from further automation, specifically, the full integration of analytical standard and QC sample preparation with study sample extraction in one continuous run, real-time 2D barcode scanning on the Hamilton deck and direct Laboratory Information Management System database connectivity. Results: We developed a new small-molecule sample-preparation automation system that improves in all of the aforementioned areas. Conclusion: The improved system presented herein further streamlines the bioanalytical workflow, simplifies batch run design, reduces analyst intervention and eliminates sample-handling error.

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.