The intracellular transport of therapeutic gene carriers is poorly understood, limiting the rational design of efficient new vectors. We used live-cell real-time multiple particle tracking to quantify the intracellular transport of hundreds of individual nonviral DNA nanocarriers with 5-nm and 33-ms resolution. Unexpected parallels between several of nature's most efficient DNA viruses and nonviral polyethylenimine͞DNA nanocomplexes were revealed to include motor protein-driven transport through the cytoplasm toward the nucleus on microtubules. Active gene carrier transport led to efficient perinuclear accumulation within minutes. The results provide direct evidence to dispute the common belief that the efficiency of nonviral gene carriers is dramatically reduced because of the need for their relatively slow random diffusion through the cell cytoplasm to the nucleus and, instead, focuses the attention of rational carrier design on overcoming barriers downstream of perinuclear accumulation. G ene delivery to the cell nucleus has been implicated as the Achilles' heel of gene therapy (1). Synthetic, nonviral DNA delivery systems have been used to improve the transfer of foreign genetic material into cells, both in vitro (2) and in vivo (3). However, without evolution working to carefully hone and optimize the delivery process, manmade delivery vectors suffer from lower efficiencies compared with nature's DNA viruses. Despite this drawback, reduced immunogenicity, improved safety, and the ability to carry larger DNA loads make nonviral carriers attractive for gene therapy (4, 5). For scientists and engineers to ''evolve'' synthetic vectors into more efficient gene delivery vehicles, the key steps in the transfection process where viral systems show superior efficiency must first be identified.Investigation of the intracellular trafficking of DNA carriers promises to improve the efficiency of nonviral delivery vectors by determining the rate-limiting steps of gene transfection, thereby allowing for the development of strategies to overcome these barriers (6-8). Currently, the transport of nonviral DNA carriers through the cytoplasm is poorly characterized, but is thought to be inefficient and potentially rate limiting because of their need to ''randomly migrate'' to the nucleus (9). Confocal microscopy has been used to study intracellular trafficking of nonviral systems (6, 10, 11), allowing the locations of complexes at discrete times to be determined, yielding an insightful but qualitative description of the transport process. Fluorescence recovery after photobleaching has recently been used to quantify overall ''effective diffusion'' rates of DNA molecules in the cytoplasm (12). With this ensemble-averaged technique, however, information associated with individual DNA carriers [the rates of individual particle movements, the mode of transport (e.g., random versus directed or active), and the trajectory and directionality of the transport] remains a black box. To achieve single-particle resolution at the nanometer ...
The ICP0 protein of herpes simplex virus type 1 is an E3 ubiquitin ligase and transactivator required for the efficient switch between latent and lytic infection. As DNA damaging treatments are known to reactivate latent virus, we wished to explore whether ICP0 modulates the cellular response to DNA damage. We report that ICP0 prevents accumulation of repair factors at cellular damage sites, acting between recruitment of the mediator proteins Mdc1 and 53BP1. We identify RNF8 and RNF168, cellular histone ubiquitin ligases responsible for anchoring repair factors at sites of damage, as new targets for ICP0-mediated degradation. By targeting these ligases, ICP0 expression results in loss of ubiquitinated forms of H2A, mobilization of DNA repair proteins and enhanced viral fitness. Our study raises the possibility that the ICP0-mediated control of histone ubiquitination may link DNA repair, relief of transcriptional repression, and activation of latent viral genomes.
In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.
A number of neurodegenerative disorders may potentially be treated by the delivery of therapeutic genes to neurons. Nonviral gene delivery systems, however, typically provide low transfection efficiency in post-mitotic differentiated neurons. To uncover mechanistic reasons for this observation, we compared gene transfer to undifferentiated and differentiated SH-SY5Y cells using polyethylenimine (PEI)/DNA nanocomplexes. Differentiated cells exhibited substantially lower uptake of gene vectors. To overcome this bottleneck, RGD or HIV-1 Tat peptides were attached to PEI/DNA nanocomplexes via poly(ethylene glycol) (PEG) spacer molecules. Both RGD and Tat improved the cellular uptake of gene vectors and enhanced gene transfection efficiency of primary neurons up to 14-fold. RGD functionalization resulted in a statistically significant increase in vector escape from endosomes, suggesting it may improve gene delivery by more than one mechanism.
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