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bioRxiv · 10.1101/2022.01.19.476334

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Yadav, M. K.·Atala, A.·Lu, B.
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Abstract

Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Normal LVs contain the RNA genome, which is reverse transcribed into DNA and integrates into the host genome to mediate long-term gene expression. Recently, virus-like particles (VLPs) were developed for mRNA delivery. In these VLPs, packaging mRNA into the particles is achieved via interactions between the aptamer and aptamer-binding protein (ABP), and mRNA is not reverse transcribed or integrated. These VLPs are useful for delivering Cas9 mRNA for short-term endonuclease expression. Generating high-titer normal integrating LVs is challenging. Until recently, VLPs were not efficient for co-delivering Cas9 mRNA and single guide RNA (sgRNA). By fusing ABP to the N-terminus of HIV Gag protein, hybrid particles were developed to co-deliver Cas9 mRNA and sgRNA. But the method for modifying Gag impaired particle assembly. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on this observation, we developed hybrid particles to co-deliver Cas9 mRNA and sgRNA. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral transfer plasmids, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, we describe development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA and new LVs for enhanced vector production and gene expression.

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Yadav, M. K., Atala, A., Lu, B.. 2022-01-21. Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression. https://doi.org/10.1101/2022.01.19.476334

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