Histone-targeted Polyplexes Avoid Endosomal Escape and Enter the Nucleus During Postmitotic Redistribution of ER Membranes

Abstract

Nonviral gene delivery is a promising therapeutic approach because of its safety and controllability, yet limited gene transfer efficacy is a common issue. Most nonviral strategies rely upon endosomal escape designs; however, endosomal escape is often uncorrelated with improved gene transfer and membranolytic structures are typically cytotoxic. Previously, we showed that histone-targeted polyplexes trafficked to the nucleus through an alternative route involving caveolae and the Golgi and endoplasmic reticulum (ER), using pathways similar to several pathogens. We hypothesized that the efficacy of these polyplexes was due to an increased utilization of native vesicular trafficking as well as regulation by histone effectors. Accordingly, using confocal microscopy and cellular fractionation, we determined that a key effect of histone-targeting was to route polyplexes away from clathrin-mediated recycling pathways by harnessing endomembrane transfer routes regulated by histone methyltransferases. An unprecedented finding was that polyplexes accumulated in Rab6-labeled Golgi/ER vesicles and ultimately shuttled directly into the nucleus during ER-mediated nuclear envelope reassembly. Specifically, super resolution microscopy and fluorescence correlation spectroscopy unequivocally indicated that the polyplexes remained associated with ER vesicles/membranes until mitosis, when they were redistributed into the nucleus. These novel findings highlight alternative mechanisms to subvert endolysosomal trafficking and harness the ER to enhance gene transfer.

Importin-4 Regulates Gene Delivery by Enhancing Nuclear Retention and Chromatin Deposition by Polyplexes

“…These results demonstrate a delayed tri colocalization with importin-4 as compared with Rab6, which correlates with the trafficking pattern we identified with our H3-targeted polyplexes and Rab6. 19 We note that in dual colocalization analyses of importin-4 and Sec61 alone, we observed a maximum of 12% colocalization, and in fact, there was less colocalization during mitosis (at the 12 h time point) as compared with the triple colocalization of these two proteins with the H3-targeted polyplexes. The higher coincidence of importin-4 and Sec-61 in the presence of the polyplexes suggested that binding between the H3-targeted polyplexes and importin-4 may drive interactions between importin-4 and Sec61.…”

“…19 In these prior studies, we specifically showed that the transport behavior of the H3-targeted polyplexes was conferred in part by interactions with histone H3 effectors, such as H3K4 methyltransferase subunits involved in regulating vesicular transport between late endosomes and the Golgi. 19 These findings motivated the analyses herein to determine whether other H3 effectors, such as the H3 importins, might be involved in shuttling H3-targeted polyplexes into the nucleus during mitosis.…”

“…Importin-4 is a known cytoplasmic protein, and in our previous studies, we showed that the H3-targeted polyplexes trafficked to the nucleus using an endomembrane transport pathway that resulted in polyplex accumulation in Rab6-linked ER vesicles prior to mitosis, followed by redistribution into the nucleus during mitosis. 19 The Sec61 translocon functions to insert secretory and transmembrane proteins into the ER during protein synthesis, or to retrotranslocate misfolded proteins in the ER to the cytosol for degradation. 26 It is also known to retrotranslocate certain toxins, such as cholera toxin and ricin, which are trafficked from the cell surface to the ER to the cytosol.…”

“…In particular, we demonstrated that incorporation of the H3 peptides improved gene transfection by engaging the histone effector H3K4 methyltransferase to enhance endocytic trafficking via caveolae-mediated endocytosis and retrograde routing through the Golgi and endoplasmic reticulum (ER). 19 The H3-targeted polyplexes accumulated in the ER prior to mitosis, and the ER-localized population of polyplexes redistributed into the nucleus following cell division. In nature, histones are also transferred through the ER and cytoplasm during synthesis and post-translational processing as well as during cellular division.…”

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

“…14 Non-endolysosomal trafficking that does not require endosomal escape, but enables nuclear entry through the endoplasmic reticulum, has been shown to occur for certain nanoparticulates, such as specific histone-targeted nanoparticles, but it has not been demonstrated for the majority of nanoparticle formulations. 15,16 The presence of intracellularly delivered nucleic acid at an acidic pH indicates that the nucleic acid is not in the cytoplasm and is not in the environment required for successful gene expression. Failure to escape the endosome to the cytoplasm can result in nucleic acid degradation when the early endosome transitions to a late endosome/lysosome, typically within an hour following uptake.…”