Gene-editing technologies, which include the CRISPR-Cas nucleases
and CRISPR base editors
, have the potential to permanently modify disease-causing genes in patients
. 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 worldwide
), 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.
Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms ...associated with this phenotypic distinction have remained a mystery for 50 y. Both Gram-negative and Gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the Gram-positive system responsible for D-alanylation of teichoic acids. Such machinery was not thought to be used by Gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between Gram-positive and Gram-negative organisms.
Ultraviolet photodissociation (UVPD) at 193 nm was implemented on a linear ion trap mass spectrometer for high-throughput proteomic workflows. Upon irradiation by a single 5 ns laser pulse, efficient ...photodissociation of tryptic peptides was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions. The factors that influence the UVPD mass spectra and subsequent in silico database searching via SEQUEST were evaluated. Peptide sequence aromaticity and the precursor charge state were found to influence photodissociation efficiency more so than the number of amide chromophores, and the ion trap q-value and number of laser pulses significantly affected the number and abundances of diagnostic product ions (e.g., sequence and immonium ions). Also, photoionization background subtraction was shown to dramatically improve SEQUEST results, especially when peptide signals were low. A liquid chromatography−mass spectrometry (LC−MS)/UVPD strategy was implemented and yielded comparable or better results relative to LC−MS/collision induced dissociation (CID) for analysis of proteolyzed bovine serum albumin and lysed human HT-1080 cytosolic fibrosarcoma cells.
O-Glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry ...(MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly and multiply deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. On the basis of the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and nonglycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the “superbug” A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis.
Host cell protein (HCP) impurities are generated by the host organism during the production of therapeutic recombinant proteins, and are difficult to remove completely. Though commonly present in ...small quantities, if levels are not controlled, HCPs can potentially reduce drug efficacy and cause adverse patient reactions. A high resolution approach for thorough HCP characterization of therapeutic monoclonal antibodies is presented herein. In this method, antibody samples are first depleted via affinity enrichment (e.g., Protein A, Protein L) using milligram quantities of material. The HCP-containing flow-through is then enzymatically digested, analyzed using nano-UPLC-MS/MS, and proteins are identified through database searching. Nearly 700 HCPs were identified from samples with very low total HCP levels (< 1 ppm to ∼ 10 ppm) using this method. Quantitation of individual HCPs was performed using normalized spectral counting as the number of peptide spectrum matches (PSMs) per protein is proportional to protein abundance. Multivariate analysis tools were utilized to assess similarities between HCP profiles by: 1) quantifying overlaps between HCP identities; and 2) comparing correlations between individual protein abundances as calculated by spectral counts. Clustering analysis using these measures of dissimilarity between HCP profiles enabled high resolution differentiation of commercial grade monoclonal antibody samples generated from different cell lines, cell culture, and purification processes.
The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan ...analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography-tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the "knowns" are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both "known" and "unknown" site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific "fingerprint" of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.
Peptides see the light: The photoactivation of peptide precursor cations during electron transfer dissociation (ETD) led to the generation of isotopic cluster peaks that more closely resembled ...theoretically predicted product‐ion distributions. This method should enable the application of ETD to low‐charge‐density peptide precursors, the gas‐phase secondary structure of which prevents the direct formation of c‐ and z.‐type fragment ions.
Summary
Similar to most Gram‐negative bacteria, the outer leaflet of the outer membrane of Vibrio cholerae is comprised of lipopolysaccharide. Previous reports have proposed that V. cholerae ...serogroups O1 and O139 synthesize structurally different lipid A domains, which anchor lipopolysaccharide within the outer membrane. In the current study, intact lipid A species of V. cholerae O1 and O139 were analysed by mass spectrometry. We demonstrate that V. cholerae serogroups associated with human disease synthesize a similar asymmetrical hexa‐acylated lipid A species, bearing a myristate (C14:0) and 3‐hydroxylaurate (3‐OH C12:0) at the 2′‐ and 3′‐positions respectively. A previous report from our laboratory characterized the V. cholerae LpxL homologue Vc0213, which transfers a C14:0 to the 2′‐position of the glucosamine disaccharide. Our current findings identify V. cholerae Vc0212 as a novel lipid A secondary hydroxy‐acyltransferase, termed LpxN, responsible for transferring the 3‐hydroxylaurate (3‐OH C12:0) to the V. cholerae lipid A domain. Importantly, the presence of a 3‐hydroxyl group on the 3′‐linked secondary acyl chain was found to promote antimicrobial peptide resistance in V. cholerae; however, this functional group was not required for activation of the innate immune response.
193‐nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a‐/x‐type, followed by d and w side‐chain loss ...ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1− through 3−) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge‐reduced radical products generated by UV photoexcitation were also subjected to collision‐induced dissociation (termed, activated‐electron photodetachment dissociation (a‐EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH∼11.5), piperidine‐modified mobile phase, LC‐MS/UVPD was implemented and resulted in the successful analysis of mitogen‐activated pathway kinases (MAPKs) using ultrafast activation times (5 ns).
Protein higher order structure (HOS) describes the three-dimensional folding arrangement of a given protein and plays critical roles in structure/function relationships. As such, it is a key product ...quality attribute that is monitored during biopharmaceutical development. Covalent labeling of surface residues, combined with mass spectrometry analysis, has increasingly played an important role in characterizing localized protein HOS. Since the label can potentially induce conformation changes, protocols generally use a small amount of label to ensure that the integrity of the protein HOS is not disturbed. The present study, however, describes a method that purposely uses high amounts of isobaric label (levels that induce denaturation) to enhance the sensitivity and resolution for detecting localized structural differences between two or more biological products. The method proved to be highly discriminative, detecting differences in HOS affecting as little as 2.5–5% of the molecular population, levels at which circular dichroism and nuclear magnetic resonance spectroscopy fingerprinting, both gold standard HOS techniques, were unable to adequately differentiate. The methodology was shown to have comparable sensitivity to differential scanning calorimetry for detecting HOS differences. In addition, the workflow presented herein can also quantify other product attributes such as post-translational modifications and site-specific glycosylation, using a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) run with automated data analysis. We applied this technique to characterize a large (>90 kDa), multiply glycosylated therapeutic protein under different heat stress conditions and aggregation states.
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