Ferns are the second largest clade of vascular plants with over 10,000 species, yet the generation of genomic resources for the group has lagged behind other major clades of plants. Transcriptomic data have proven to be a powerful tool to assess phylogenetic relationships, using thousands of markers that are largely conserved across the genome, and without the need to sequence entire genomes. We assembled the largest nuclear phylogenetic dataset for ferns to date, including 2884 single-copy nuclear loci from 247 transcriptomes (242 ferns, five outgroups), and investigated phylogenetic relationships across the fern tree, the placement of whole genome duplications (WGDs), and gene retention patterns following WGDs. We generated a well-supported phylogeny of ferns and identified several regions of the fern phylogeny that demonstrate high levels of gene tree–species tree conflict, which largely correspond to areas of the phylogeny that have been difficult to resolve. Using a combination of approaches, we identified 27 WGDs across the phylogeny, including 18 large-scale events (involving more than one sampled taxon) and nine small-scale events (involving only one sampled taxon). Most inferred WGDs occur within single lineages (e.g., orders, families) rather than on the backbone of the phylogeny, although two inferred events are shared by leptosporangiate ferns (excluding Osmundales) and Polypodiales (excluding Lindsaeineae and Saccolomatineae), clades which correspond to the majority of fern diversity. We further examined how retained duplicates following WGDs compared across independent events and found that functions of retained genes were largely convergent, with processes involved in binding, responses to stimuli, and certain organelles over-represented in paralogs while processes involved in transport, organelles derived from endosymbiotic events, and signaling were under-represented. To date, our study is the most comprehensive investigation of the nuclear fern phylogeny, though several avenues for future research remain unexplored.

Mutated RAS proteins are potent oncogenic drivers and have long been considered "undruggable". While RAStargeting therapies have recently shown promise, there remains a clinical need for RAS inhibitors with more diverse targets. Small proteins represent a potential new therapeutic option, including K27, a designed ankyrin repeat protein (DARPin) engineered to inhibit RAS. However, K27 functions intracellularly and is incapable of entering the cytosol on its own, currently limiting its utility. To overcome this barrier, we have engineered a lipid nanoparticle (LNP) platform for potent delivery of functional K27-D30�a charge-modified version of the protein�intracellularly in vitro and in vivo. This system efficiently encapsulates chargemodified proteins, facilitates delivery in up to 90% of cells in vitro, and maintains potency after at least 45 days of storage. In vivo, these LNPs deliver K27-D30 to the cytosol of cancerous cells in the liver, inhibiting RAS-driven growth and ultimately reducing tumor load in an HTVI-induced mouse model of hepatocellular carcinoma. This work shows that K27 holds promise as a new cancer therapeutic when delivered using this LNP platform. Furthermore, this technology has the potential to broaden the use of LNPs to include new cargo types�beyond RNA�for diverse therapeutic applications.

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