Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification—currently reliant on filtration and anion-exchange or size-exclusion chromatography—suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%–60% of viral genomes; 40%–50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·10⁹ TU per mL of resin, or 5·10¹¹ vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality

Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival

Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse–chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells

De novo and inherited variants in ZNF292 underlie a neurodevelopmental disorder with features of autism spectrum disorder

International audiencePURPOSE:Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous neurodevelopmental disorders. We sought to delineate the clinical, molecular, and neuroimaging spectrum of a novel neurodevelopmental disorder caused by variants in the zinc finger protein 292 gene (ZNF292).METHODS:We ascertained a cohort of 28 families with ID due to putatively pathogenic ZNF292 variants that were identified via targeted and exome sequencing. Available data were analyzed to characterize the canonical phenotype and examine genotype-phenotype relationships.RESULTS:Probands presented with ID as well as a spectrum of neurodevelopmental features including ASD, among others. All ZNF292 variants were de novo, except in one family with dominant inheritance. ZNF292 encodes a highly conserved zinc finger protein that acts as a transcription factor and is highly expressed in the developing human brain supporting its critical role in neurodevelopment.CONCLUSION:De novo and dominantly inherited variants in ZNF292 are associated with a range of neurodevelopmental features including ID and ASD. The clinical spectrum is broad, and most individuals present with mild to moderate ID with or without other syndromic features. Our results suggest that variants in ZNF292 are likely a recurrent cause of a neurodevelopmental disorder manifesting as ID with or without ASD