Chemically Tuned Intracellular Gene Delivery by Core-Shell Nanoparticles: Effects of Proton Buffering, Acid Degradability, and Membrane Disruption.

Nanoparticles consisting of a condensed nucleic acid core surrounded by protective layers which aid to overcome extracellular and intracellular hurdles to gene delivery (i. e., core-shell nanoparticles, CSNPs) synthetically mimic viruses. The outer shells shield the core and are particularly designed to enable facilitated release of the gene payload into the cytoplasm, the major limiting step in intracellular gene delivery. The hypothetical proton sponge effect and degradability in response to a stimulus (i. e., mildly acidic pH in the endosome) are two prevailing, although contested, principles in designing effective carriers for intracellular gene delivery via endosomal escape. Utilizing the highly flexible chemical-tuning of the polymeric shell via surface-initiated photo-polymerization of the various monomers at different molecular ratios, the effects of proton buffering capacity, acid-degradability, and endosomal membrane-lysis property on intracellular delivery of plasmid DNA by CSNPs were investigated. This study demonstrated the equivalently critical roles of proton buffering and acid-degradability in achieving efficient intracellular gene delivery, independent of cellular uptake. Extended proton buffering resulted in further improved transfection as long as the core structure was not compromised. The results of the study present a promising synthetic strategy to the development of an efficient, chemically-tunable gene delivery carrier.

Synthetically Engineered Adeno-Associated Virus for Efficient, Safe, and Versatile Gene Therapy Applications.

Gene therapy directly targets mutations causing disease, allowing for a specific treatment at a molecular level. Adeno-associated virus (AAV) has been of increasing interest as a gene delivery vehicle, as AAV vectors are safe, effective, and capable of eliciting a relatively contained immune response. With the recent FDA approval of two AAV drugs for treating rare genetic diseases, AAV vectors are now on the market and are being further explored for other therapies. While showing promise in immune privileged tissue, the use of AAV for systemic delivery is still limited due to the high prevalence of neutralizing antibodies (nAbs). To avoid nAb-mediated inactivation, engineered AAV vectors with modified protein capsids, materials tethered to the capsid surface, or fully encapsulated in a second, larger carrier have been explored. Many of these engineered AAVs have added benefits, including avoided immune response, overcoming the genome size limit, targeted and stimuli-responsive delivery, and multimodal therapy of two or more therapeutic modalities in one platform. Native and engineered AAV vectors have been tested to treat a broad range of diseases, including spinal muscular atrophy, retinal diseases, cancers, and tissue damage. This review will cover the benefits of AAV as a promising gene vector by itself, the progress and advantages of engineered AAV vectors, particularly synthetically engineered ones, and the current state of their clinical translation in therapy.

Computational Models for Activated Human MEK1: Identification of Key Active Site Residues and Interactions.

MEK1 is a protein kinase in the MAPK cellular signaling pathway that is notable for its dual specificity and its potential as a drug target for a variety of cancer therapies. While much is known about the key role of MEK1 in signaling events, understanding of the structural features that sustain MEK1 function remains limited because of the absence of crystal or NMR structural insights into the phosphorylated and activated form of MEK1. In this work, homology modeling was used to overcome this limitation and generate computational models of the doubly phosphorylated active MEK1 conformation. A variety of models were generated using crystal structures of active protein kinases as homology model templates. These models were equilibrated using molecular dynamics simulations, and each model was validated against several known structural characteristics of activated kinases. The best model structures were used in docking studies with ATP and a small peptide sequence that represents the activation loop of ERK2 to identify the most important residues in stabilizing protein docking and phosphorylation. These results provide insights for the pursuit of structure-guided mutagenesis and drug design.