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Combining steric exclusion with anion exchange - development of a universal and scalable adeno-associated virus downstream process
No full textAdeno-associated viruses (AAV) are among the leading vectors for in vivo gene therapy. The purification of AAV remains a bottleneck as it typically requires multiple individual process steps, often resulting in product loss and high costs. Current downstream processes are usually serotype-specific and rely primarily on expensive affinity resins. To address these limitations, we developed a serotype-independent purification method using steric exclusion chromatography (SXC) that can be combined with a subsequent anion exchange full/empty separation step. This alternative approach eliminates the need for intermediate concentration and buffer exchange, thereby reducing the number of process steps required while achieving high-purity full AAV particles. SXC conditions were optimized using a design of experiments approach. Isocratic separation of full and empty AAV resulted in further purification of the sample. The overall process achieved a viral genome recovery of 51.7 %, along with impurity depletions of 99.9 % for DNA and 99.8 % for protein. The process was successfully adapted to different AAV serotypes and genes of interest, demonstrating its robustness and versatility. In addition, the scalability of SXC was demonstrated, highlighting its potential for large-scale manufacturing. This streamlined, universal, and scalable process provides a robust and efficient alternative to traditional AAV purification processes, addressing critical challenges in gene therapy production and paving the way for broader implementation in research and manufacturing
Affinity maturation endows potent activity onto class 6 SARS-CoV-2 broadly neutralizing antibodies
No full textThe emergence of SARS-CoV-2 variants of concern (VOCs) has greatly diminished the neutralizing activity of previously FDA-approved monoclonal antibodies (mAbs), including that of antibody cocktails and of first-generation broadly neutralizing antibodies such as S309 (Sotrovimab). In contrast, antibodies targeting cryptic conformational epitopes of the receptor binding domain (RBD) have demonstrated broad activity against emerging variants, but exert only moderate neutralizing activity, which has so far hindered clinical development. Here, we utilize in vitro display technology to identify and affinity-mature antibodies targeting the cryptic class 6 epitope, accessible only in the "up" conformation of the SARS-CoV-2 spike trimer. Increasing antibody affinity into the low picomolar range endowed potent neutralization of VOCs and protection of hACE2 mice from viral challenge. Cryoelectron microscopy and crystal structures of two affinity-matured antibodies (4C12-B12 and 4G1-C2) in complex with RBD highlighted binding modes and epitopes distal from mutational hotspots commonly overserved in VOCs, providing direct structural insights into the observed mutational resistance. Moreover, we further demonstrate that antibodies targeting the class 6 epitope, rather than being an artifact of in vitro selection, are common in the IgG1+ memory B cell repertoire of convalescent patients and can be induced in human antibody V-gene transgenic mice through immunization. Our results highlight the importance of very high (picomolar) affinity in the development of neutralizing antibodies and vaccines and suggest an affinity threshold in the provision of broad and long-lasting immunity against SARS-CoV-2
Contrasting effects of arsenic on mycorrhizal-mediated silicon and phosphorus uptake by rice
No full textBlood proteomics and multimodal risk profiling of human volunteers after incision injury: A translational study for advancing personalized pain management after surgery
No full textCombination of searches for singly and doubly charged Higgs bosons produced via vector-boson fusion in proton–proton collisions at s = 13 TeV with the ATLAS detector
No full textNutritional and Sensory Properties of Meat Analogues: A Current Overview and Future Considerations
No full textOH-Detected Aromatic Microsolvation of an Organic NO Radical: Halogenation Controls the Solvation Side
No full textTransient Triplet Metallopnictinidenes M–Pn (M = Pd II , Pt II ; Pn = P, As, Sb): Characterization and Dimerization
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Molecular basis of human nuclear and mitochondrial tRNA 3′ processing
No full textAbstract Eukaryotic transfer RNA (tRNA) precursors undergo sequential processing steps to become mature tRNAs. In humans, ELAC2 carries out 3′ end processing of both nucleus-encoded (nu-tRNAs) and mitochondria-encoded (mt-tRNAs) tRNAs. ELAC2 is self-sufficient for processing of nu-tRNAs but requires TRMT10C and SDR5C1 to process most mt-tRNAs. Here we show that TRMT10C and SDR5C1 specifically facilitate processing of structurally degenerate mt-tRNAs lacking the canonical elbow. Structures of ELAC2 in complex with TRMT10C, SDR5C1 and two divergent mt-tRNA substrates reveal two distinct mechanisms of pre-tRNA recognition. While canonical nu-tRNAs and mt-tRNAs are recognized by direct ELAC2–RNA interactions, processing of noncanonical mt-tRNAs depends on protein–protein interactions between ELAC2 and TRMT10C. These results provide the molecular basis for tRNA 3′ processing in both the nucleus and the mitochondria and explain the organelle-specific requirement for additional factors. Moreover, they suggest that TRMT10C–SDR5C1 evolved as a mitochondrial tRNA maturation platform to compensate for the structural erosion of mt-tRNAs in bilaterian animals