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

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.