A substantial proportion of the human genome is made up of retroelements — ancient transposable DNA tracts including retroviruses that have integrated into our genome during evolution. A recent study in Science reports the development of a modular system based on some of these endogenous retroelements that can be used for delivery of therapeutic mRNA cargo. This platform might support the burgeoning field of mRNA-based therapies, such as mRNA vaccines for SARS-CoV-2.
Many endogenous retroelements have lost their original function, but some have been co-opted for physiological processes, sometimes involving transfer of mRNA between cells. This led Segel et al. to wonder whether retroelements in the human genome might be programmed to deliver specific nucleic acids, which would then be translated into protein therapies inside the cell.
The researchers began by searching for homologues of the retroelement structural gene gag, and in particular for gag homologues containing a capsid domain, in the human genome. Capsid proteins form virus-like particles (VLPs) around secreted retroelement RNA, which could be a useful component for an RNA delivery platform.
By computational survey, Segel et al. looked for capsid-containing gag homologues that were common to the human and mouse genome, reasoning that such hits would be most likely to serve physiological roles in mammalian cells. The researchers used Escherichia coli to express mouse versions of the identified gene candidates, and observed by electron microscopy that several of the resultant protein products formed VLPs and are secreted. Of these proteins, PEG10 showed the greatest propensity for VLP secretion.
PEG10 is derived from a homologue of a common type of retroelement called a long-terminal repeat retrotransposon. In vitro studies including CRISPR-mediated activation of endogenous Peg10 in mouse cells showed PEG10 binds to and facilitates secretion of Peg10 transcripts. Studies in transgenic mice suggested a function of PEG10 in mammals could be to stabilize mRNAs involved in neurodevelopment.
The authors sought to reprogramme PEG10 to bind and package a different RNA cargo. They chose the gene encoding Cre recombinase (Cre) as a test cargo, which they aimed to transfer to mouse N2a cells expressing a loxP–GFP reporter.
They found that flanking Cre with the untranslated regions of PEG10 provided a ‘packaging signal’ for Cre mRNA secretion in VLPs. Addition of the fusogenic envelope protein from vesicular stomatitis virus (VSV) — a mix-and-match process known as virus pseudotyping — enabled entry of the Cre mRNA cargo into target cells.
To make the system fully endogenous, the team looked for alternative fusogens to the VSV envelope protein for pseudotyping. They focused on the syncytins, which are fusogenic proteins in mammalian cells that evolved from retroviral envelope proteins. In the mouse RNA delivery system, replacement of the VSV fusogen with the mouse syncytin A gene enabled transfer of Cre mRNA cargo to target cells in vitro.
The authors named their tripart system — the recoded PEG10 sequence, the target gene and the fusogen — selective endogenous encapsidation for cellular delivery (SEND).
To demonstrate the modular nature of SEND for delivery of a cargo mRNA of choice, the researchers used the system to deliver Cas9 mRNA to mouse N2a cells that constitutively express a single guide RNA against Kras. The SEND system achieved functional delivery of Cas9 mRNA to recipient cells, 60% of which contained insertions or deletions (indels) in Kras after delivery.
In addition, by co-packaging Cas9 mRNA and vascular endothelial growth factor A (VEGFA) guide RNA within the SEND system, the researchers created an all-in-one vector that produced indels at the VEGFA locus in 40% of HEK293 cells.
The ability to swap in different mRNA cargoes makes SEND a potentially broadly applicable platform for delivery of nucleic acids. Moreover, the authors note that the system could be less immunogenic than other methods of mRNA delivery, such as viral vectors, as SEND uses endogenous proteins. Indeed, PEG10 is highly expressed in the developing human thymus, which is a key site for the induction of T cell tolerance.
Future studies might characterize and develop other capsid proteins and fusogens encoded in the human genome to provide additional components to optimize the SEND platform.
Segel, M. et al. Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. Science 373, 882–889 (2021)
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