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Hołubowicz, R.* ; Gao, F.* ; Du, S.W.* ; Menezes, C.R.* ; Zhang, J.* ; Hołubowicz, M.W.* ; Chen, P.Z.* ; Armbrust, N. ; Geilenkeuser, J. ; Liu, D.R.* ; Truong, D.J.J. ; Westmeyer, G.G. ; Palczewska, G.* ; Palczewski, K.*

Scalable purification enables high-quality virus-like particles for therapeutic translation.

J. Biol. Chem. 301:110946 (2025)
Verlagsversion DOI PMC
Open Access Gold
Creative Commons Lizenzvertrag
Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus (MLV)-derived engineered VLPs (eVLPs) and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors (ENVLPEs+), we developed a workflow that involves single- and multi-modal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Crispr/cas ; Cas9 ; Base Editing ; Chromatography ; Nanotechnology ; Prime Editing ; Protein Delivery ; Ribonuclear Protein (rnp) ; Vesicles ; Virus-like Particles; Genomic Dna; Delivery; Reveals; Protein; Cells; Base
ISSN (print) / ISBN 0021-9258
e-ISSN 1083-351X
Zeitschrift Journal of Biological Chemistry, The
Quellenangaben Band: 301, Heft: 12, Seiten: , Artikelnummer: 110946 Supplement: ,
Verlag American Society for Biochemistry and Molecular Biology
Verlagsort Radarweg 29, 1043 Nx Amsterdam, Netherlands
Begutachtungsstatus Peer reviewed
Institut(e) Insitute of Synthetic Biomedicine (ISBM)
Förderungen University of California, Irvine School of Medicine Dean's Office grant
NIH
Howard Hughes Medical Institute
Foundation Fighting Blindness
Schmidt Science Fellows
Rhodes Trust
Natural Sciences and Engineering Research Council of Canada
Department of Ophthalmology
Research to Prevent Blindness
NIH core grant
Knights Templar Eye Foundation