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. 2024 Mar 12;121(11):e2307812120.
doi: 10.1073/pnas.2307812120. Epub 2024 Mar 4.

Human paraneoplastic antigen Ma2 (PNMA2) forms icosahedral capsids that can be engineered for mRNA delivery

Victoria Madigan #  1   2   3   4   5 , Yugang Zhang #  1   2   3   4   5 , Rumya Raghavan #  1   2   3   4   5 , Max E Wilkinson  1   2   3   4   5 , Guilhem Faure  1   2   3   4   5 , Elena Puccio  1   2   3   4   5 , Michael Segel  1   2   3   4   5 , Blake Lash  1   2   3   4   5 , Rhiannon K Macrae  1   2   3   4   5 , Feng Zhang  1   2   3   4   5
Affiliations

Human paraneoplastic antigen Ma2 (PNMA2) forms icosahedral capsids that can be engineered for mRNA delivery

Victoria Madigan et al. Proc Natl Acad Sci U S A. .

Abstract

A number of endogenous genes in the human genome encode retroviral gag-like proteins, which were domesticated from ancient retroelements. The paraneoplastic Ma antigen (PNMA) family members encode a gag-like capsid domain, but their ability to assemble as capsids and traffic between cells remains mostly uncharacterized. Here, we systematically investigate human PNMA proteins and find that a number of PNMAs are secreted by human cells. We determine that PNMA2 forms icosahedral capsids efficiently but does not naturally encapsidate nucleic acids. We resolve the cryoelectron microscopy (cryo-EM) structure of PNMA2 and leverage the structure to design engineered PNMA2 (ePNMA2) particles with RNA packaging abilities. Recombinantly purified ePNMA2 proteins package mRNA molecules into icosahedral capsids and can function as delivery vehicles in mammalian cell lines, demonstrating the potential for engineered endogenous capsids as a nucleic acid therapy delivery modality.

Keywords: PNMA; VLP; capsid; gene therapy; mRNA delivery.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Genomic location and domain architecture of human PNMA genes. (A) Genomic location of human PNMA genes. (B) Phylogenetic tree of PNMA family including all human PNMAs (PNMA1-8 and CCDC8), the marsupial PNMA (MePNMA), and a turtle Ty3/mdg4 (Materials and Methods) from which the tree is rooted. Domain architecture of each protein is deduced from the structural models (Materials and Methods). Domain architecture encompasses an RRM-like fold domain (pink), dimerization domain that forms only upon interaction (in orange), and capsid domain in light blue for N-terminal capsid domain and dark blue for C-terminal capsid domain. Additional domains predicted to fold are shown in gray. Zinc fingers are shown in yellow, and regions with a high concentration of K-R are shown in green. RRM, RNA recognition motif.
Fig. 2.
Fig. 2.
PNMA2 capsids are secreted from human cells without encapsidated RNA. (A) Schematic of isolation of cell lysate and viral-like particle (VLP) fraction. (B) Western blot showing PNMA protein expression in HEK293FT cells in either cell lysate (Top) or the VLP fraction (Bottom). (C) Example TEM micrographs of a single HA-immunoprecipitated VLP fraction from negative control cells (Top panels) or cells overexpressing HA-tagged PNMA2 (Bottom panels) (Scale bar, 100 nm). (D) Expression of PNMA2 or a mutant of PNMA2 lacking the start codon in whole-cell lysate (cells) or the VLP fraction (Top). Quantification of PNMA2 mRNA (cDNA) in either the whole-cell lysate or VLP fraction in cells expressing either wild-type PNMA2 (gray) or a mutant lacking the start codon (pink) (Bottom). Samples were compared via the unpaired t test where ns represents a P > 0.05, * represents P ≤ 0.05, ** represents P ≤ 0.01, and *** represents P ≤ 0.001. (E) (Top) Schematic of the experimental procedure to identify mRNA packaged in PNMA2 capsids. (Bottom) Volcano plots showing differential mRNA expression of PNMA2 CRISPRa samples versus a non-targeting guide control in either the cell lysate (Left) or VLP fraction (Right). Three transcripts (ZNF142, GOLGA2P9, and TTC25) were above the significance threshold but were found to be insignificant after adjusting for multiple hypothesis testing (Materials and Methods).
Fig. 3.
Fig. 3.
In vitro assembly and RNA packaging of PNMA2 capsids. (A) TEM micrograph of PNMA2 purified from E. coli (Scale bar, 100 nm). (B) Size-exclusion chromatography (SEC) trace of PNMA2 particles purified from E. coli, where Vo indicates void volume. (C) Schematic of workflow for in vitro production of PNMA2 capsids with representative TEM images below.
Fig. 4.
Fig. 4.
Cryo-EM structure of human PNMA2 capsids. (A) Cryo-EM density of PNMA2 with I4 symmetry imposed, colored by radial distance from the center of the capsid, with the exterior of the PNMA2 capsid pictured on the Left and a cross-section of the capsid shown on the Right. (B) Model of the PNMA2 capsid with a protein monomer outlined. (C) Details of the interactions of a PNMA2 monomer (pink highlight) with adjacent monomers, with symmetry axes indicated. (D) Electrostatic potential of the inside of the PNMA2 capsid. Red indicates negative charge. (E) Central slice of the PNMA2 cryo-EM density. The projected positions of the modeled N and C termini are shown as blue and red circles.
Fig. 5.
Fig. 5.
Engineering of ePNMA2 for RNA encapsidation and delivery. (A) Schematic of ePNMA2 protein and TEM micrograph of ePNMA2 capsids (Scale bar, 100 nm). (B) Quantification of RNAs per capsid packaged by ePNMA2 during reassembly across a range of salt conditions following RNaseA treatment. (C) Immunofluorescence showing ePNMA2 entry into Neuro2A cells with either no co-treatment or LAH4 (Scale bar, 40 µm). (D) Schematic of workflow for in vitro production of ePNMA2 capsids. (E) Quantification of live (DAPI negative) GFP positive cells by flow cytometry following delivery by ePNMA2 of Cre mRNA to Neuro2A-loxP-GFP recipient cells. Data shown are three technical triplicates that are representative from three biological replicates. Samples were compared via the unpaired t test where ns represents a P > 0.05, *represents P ≤ 0.05, **represents P ≤ 0.01, and ***represents P ≤ 0.001. (F) White light (WL) and GFP fluorescence images of Neuro2A-loxP-GFP recipient cells 96 h after treatment with RNA, RNase treated RNA, or RNase treated ePNMA2, -Cre all with LAH4 cell penetrating peptide (Scale bar, 100 µm).
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