Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression

Significance The importance of viral icosahedral capsid-based nanoparticles (NPs) as cell-delivery vehicles is now being recognized. Virtually any specific protein and nucleic acid can be encapsidated together into a phage T4 capsid that can display surface-binding ligands for tissue targeting. In this study, T4 NPs packed in vivo with active cyclic recombination (Cre) recombinase and in vitro with fluorescent mCherry expression plasmid DNA were delivered into cancer cells. When released into cells together, the packaged active Cre recombinase within the capsid circularizes the packaged DNA of the linear expression plasmid, enhancing the expression of the mCherry gene. The efficient and specific packaging and unpackaging of DNA and active protein together into targeted cells has potential applications in targeted gene therapy and cancer therapy. Packaging specific exogenous active proteins and DNAs together within a single viral-nanocontainer is challenging. The bacteriophage T4 capsid (100 × 70 nm) is well suited for this purpose, because it can hold a single long DNA or multiple short pieces of DNA up to 170 kb packed together with more than 1,000 protein molecules. Any linear DNA can be packaged in vitro into purified procapsids. The capsid-targeting sequence (CTS) directs virtually any protein into the procapsid. Procapsids are assembled with specific CTS-directed exogenous proteins that are encapsidated before the DNA. The capsid also can display on its surface high-affinity eukaryotic cell-binding peptides or proteins that are in fusion with small outer capsid and head outer capsid surface-decoration proteins that can be added in vivo or in vitro. In this study, we demonstrate that the site-specific recombinase cyclic recombination (Cre) targeted into the procapsid is enzymatically active within the procapsid and recircularizes linear plasmid DNA containing two terminal loxP recognition sites when packaged in vitro. mCherry expression driven by a cytomegalovirus promoter in the capsid containing Cre-circularized DNA is enhanced over linear DNA, as shown in recipient eukaryotic cells. The efficient and specific packaging into capsids and the unpackaging of both DNA and protein with release of the enzymatically altered protein–DNA complexes from the nanoparticles into cells have potential in numerous downstream drug and gene therapeutic applications.

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