Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery

Abstract

Eukaryotic genomes contain domesticated genes from integrating viruses and mobile genetic elements. Among these are homologs of the capsid protein (known as Gag) of long terminal repeat (LTR) retrotransposons and retroviruses. We identified several mammalian Gag homologs that form virus-like particles and one LTR retrotransposon homolog, PEG10, that preferentially binds and facilitates vesicular secretion of its own messenger RNA (mRNA). We showed that the mRNA cargo of PEG10 can be reprogrammed by flanking genes of interest with Peg10's untranslated regions. Taking advantage of this reprogrammability, we developed selective endogenous encapsidation for cellular delivery (SEND) by engineering both mouse and human PEG10 to package, secrete, and deliver specific RNAs. Together, these results demonstrate that SEND is a modular platform suited for development as an efficient therapeutic delivery modality.

Fig. 1.. Identification of mammalian retroelement derived Gag homologs that form capsids and are secreted

A. Domain architectures of selected Capsid (CA)-containing mammalian Gag homologs compared to that of typical retrovirus and LTR retrotransposons. Each group of Gag homologs contains a distinct combination of predicted CA, Nucleocapsid (NC), Protease (PR), and Reverse Transcriptase (RT) domains. LTR, long terminal repeat; MA, matrix; IN, integrase. B. Fraction of the total bacterially-produced protein that forms oligomers (>600 kD), as determined by size exclusion chromatography. C. Representative negative stain transmission electron micrographs (TEM) of the Mus musculus (Mm) orthologues of the CA-domain containing proteins. Scale bar, 100 nm. D. Representative electron micrographs using cryogenic electron microscopy (cryoTEM) of a selected subset of the identified CA-domain containing proteins. Scale bar, 50 nm. E. Method for detecting extracellular forms of CA-domain containing homologs. F. Representative blots of CA-domain containing proteins in the cell-free fraction. CD81 was used as loading control for the ultracentrifuged cell-free fraction. Whole cell (W.C.) and VLP fraction blots for the endoplasmic reticulum marker CALNEXIN (CNX) ensure equal loading of whole cell protein and the purity of cell-free VLP fraction. G. Quantification of extracellular CA-domain containing proteins (as in F) based on n=3 replicates.

Fig 2.. MmPEG10 protein and mRNA is secreted in vesicles by cells in vitro.

A. Method for identifying nucleic acids that are secreted in the VLP fraction upon gene activation of CA-domain containing proteins. B. Differential RNA abundance and significance in the VLP fraction from N2a cells after CRISPR activation of endogenous MmPeg10. C. Alignment of sequencing reads showing sequencing coverage of the MmPeg10 mRNA from (B). D. Differential RNA abundance and significance in the VLP fraction from N2a cells after heterologous transfection of MmPeg10. n=3 replicates. E. The four domains of MmPEG10 are translated into two isoforms. These are self-processed by the PEG10 protease into separate domains, of which the NC and RT bind RNA. F. Fold enrichment of MmPeg10 mRNA compared to GFP in the VLP fraction from N2a cells transfected with wildtype MmPeg10 or deletions of the predicted nucleocapsid (ΔNC) and reverse transcriptase (ΔRT) domains. G. Log₂ fold change and significance of bound RNAs from eCLIP data comparing HA-GFP to WT MmPEG10-HA. H. Representative sequencing alignment histogram of the MmDdit4 locus generated from eCLIP of N2a cells transfected with wildtype or mutant MmPeg10. I. Representative sequencing alignment histogram of the MmPeg10 locus generated from eCLIP data of n = 3 HA-PEG10 and n = 3 untagged animals.

Fig 3.. Flanking mRNA with MmPeg10 5’ and 3’ UTRs enables functional intercellular transfer of mRNA into a target cell.

A. Schematic showing reprogramming MmPEG10 for functional delivery of a cargo RNA flanked with the MmPeg10 5′ and 3′ UTRs (hereafter, “cargo(RNA)”) B epresentative TEM micrographs of VLP fraction immunogold labeled for MmPEG10. Text labels indicate transfection of cells with MmPeg10 or mock (negative). Arrowheads indicate gold labeling. Scale bar, 50 nm. C. Representative images of loxP-GFP N2a cells treated with VSVg pseudotyped MmPEG10 VLPs produced by transfecting Mm.cargo(Cre) or Cre mRNA, and a lentivirus encoding Cre. Scale bar, 100 um. D. Functional transfer of RNA into loxP-GFP N2a cells mediated by VSVg pseudotyped MmPEG10 VLPs. Data quantified by flow cytometry 72 hours after VLP addition, n = 3 replicates. E. Functional transfer of RNA into loxP-GFP N2a cells mediated by VSVg pseudotyped VLPs produced with MmPeg10 or mCherry and Mm.cargo(Cre) constructs encoding tiles of the MmPeg10 3’UTR. Data quantified by flow cytometry 72 hours after VLP addition, n = 3 replicates. F. Functional transfer of RNA into loxP-GFP N2a cells mediated by VSVg pseudotyped VLPs produced with HsPEG1010 or mCherry and Hs.cargo(Cre) constructs encoding tiles of the HsPeg10 3’UTR. Data quantified by flow cytometry 72 hours after VLP addition, n = 3 replicates. G. Functional transfer of RNA into loxP-GFP N2a cells mediated by VSVg pseudotyped VLPs produced with rMmPeg10 and Mm.cargo(Cre) or Cre mRNA. Data quantified by flow cytometry 72 hours after VLP addition, n = 3 replicates. H. Functional transfer of RNA into loxP-GFP N2a cells mediated by VSVg pseudotyped VLPs produced with rHsPeg10 and Hs.cargo(Cre) or Cre mRNA. Data quantified by flow cytometry 72 hours after VLP addition, n = 3 replicates. For all panels *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, One-Way ANOVA.

Fig. 4.. SEND is a modular system capable of delivering gene editing tools into human and mouse cells.

A. Representative images demonstrating functional transfer of Mm.cargo(Cre) or Cre mRNA in rMmPEG10 VLPs pseudotyped with VSVg (V), MmSYNA (A), or MmSYNB (B) in Ai9 (loxP-tdTomato) tail tip fibroblasts. Scale bar, 200 μm. B ercent of tdTomato positive cells out of the total number of H2A stained nuclei from high content imaging of n=3 replicates of (A). C. Schematic representing the retooling of SEND for genome engineering. D. Indels at the MmKras locus in MmKras1-sgRNA-N2a cells treated with SEND (VSVg pseudotyped rMmPEG10 VLPs) containing either SpCas9 mRNA, Mm.UTR(SpCas9), or Mm.cargo(SpCas9) and a lentivirus encoding SpCas9. Indels quantified by NGS 72 hours after VLP or lentivirus addition, n=3 replicates. E. Indels at the mouse MmKras locus in a constitutively expressing SpCas9 N2a cell line either transfected with a plasmid carrying the MmKras sgRNA or treated with SEND (rMmPEG10, VSVg, MmKras sgRNA). Indels quantified by NGS after 72 hours, n=3 replicates. F. Indels at the MmKras locus in N2a cells treated with SEND (VSVg pseudotyped rMmPeg10 SEND VLPs) containing either SpCas9 mRNA or Mm.cargo(SpCas9) and sgRNA. Indels quantified by NGS 72 hours after VLP addition, n=3 replicates. G. Indels at the HsVEGFA locus in HEK293FT cells treated with SEND (VSVg pseudotyped rHsPEG10 VLPs) containing either SpCas9 mRNA or Hs.cargo(SpCas9) and an unmodified sgRNA. Indels determined by NGS 72 hours after VLP addition, n=3 replicates. H. SEND is a modular delivery platform combining an endogenous Gag homolog, cargo mRNA, and fusogen, which can be tailored for specific contexts.