Poly(l-glutamic acid) augments the transfection performance of lipophilic polycations by overcoming tradeoffs among cytotoxicity, pDNA delivery efficiency, and serum stability.

Polycations are scalable and affordable nanocarriers for delivering therapeutic nucleic acids. Yet, cationicity-dependent tradeoffs between nucleic acid delivery efficiency, cytotoxicity, and serum stability hinder clinical translation. Typically, the most efficient polycationic vehicles also tend to be the most toxic. For lipophilic polycations-which recruit hydrophobic interactions in addition to electrostatic interactions to bind and deliver nucleic acids-extensive chemical or architectural modifications sometimes fail to resolve intractable toxicity-efficiency tradeoffs. Here, we employ a facile post-synthetic polyplex surface modification strategy wherein poly(l-glutamic acid) (PGA) rescues toxicity, inhibits hemolysis, and prevents serum inhibition of lipophilic polycation-mediated plasmid (pDNA) delivery. Importantly, the sequence in which polycations, pDNA, and PGA are combined dictates pDNA conformations and spatial distribution. Circular dichroism spectroscopy reveals that PGA must be added last to polyplexes assembled from lipophilic polycations and pDNA; else, PGA will disrupt polycation-mediated pDNA condensation. Although PGA did not mitigate toxicity caused by hydrophilic PEI-based polycations, PGA tripled the population of transfected viable cells for lipophilic polycations. Non-specific adsorption of serum proteins abrogated pDNA delivery mediated by lipophilic polycations; however, PGA-coated polyplexes proved more serum-tolerant than uncoated polyplexes. Despite lower cellular uptake than uncoated polyplexes, PGA-coated polyplexes were imported into nuclei at higher rates. PGA also silenced the hemolytic activity of lipophilic polycations. Our work provides fundamental insights into how polyanionic coatings such as PGA transform intermolecular interactions between lipophilic polycations, nucleic acids, and serum proteins, and facilitate gentle yet efficient transgene delivery.

Mutations in yeast Pcf11, a conserved protein essential for mRNA 3' end processing and transcription termination, elicit the Environmental Stress Response.

The Pcf11 protein is an essential subunit of the large complex that cleaves and polyadenylates eukaryotic mRNA precursor. It has also been functionally linked to gene-looping, termination of RNA Polymerase II (Pol II) transcripts, and mRNA export. We have examined a poorly characterized but conserved domain (amino acids 142-225) of the Saccharomyces cerevisiae  Pcf11 and found that while it is not needed for mRNA 3' end processing or termination downstream of the poly(A) sites of protein-coding genes, its presence improves the interaction with Pol II and the use of transcription terminators near gene promoters. Analysis of genome-wide Pol II occupancy in cells with Pcf11 missing this region, as well as Pcf11 mutated in the Pol II CTD Interacting Domain, indicates that systematic changes in mRNA expression are mediated primarily at the level of transcription. Global expression analysis also shows that a general stress response, involving both activation and suppression of specific gene sets known to be regulated in response to a wide variety of stresses, is induced in the two pcf11 mutants, even though cells are grown in optimal conditions. The mutants also cause an unbalanced expression of cell wall-related genes that does not activate the Cell Wall Integrity pathway but is associated with strong caffeine sensitivity. Based on these findings, we propose that Pcf11 can modulate the expression level of specific functional groups of genes in ways that do not involve its well-characterized role in mRNA 3' end processing.