ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance.

Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily.

Overexpression of caveolin-1 in inflammatory breast cancer cells enables IBC-specific gene delivery and prodrug conversion using histone-targeted polyplexes.

Gene therapy platforms offer a variety of potentially effective solutions for development of targeted agents that can be exploited for cancer treatment. The physicochemical properties of nanocarriers can be tuned to enhance their localization in tumors, and cell specificity can also be increased by appropriate selection of gene targets. A relatively underexploited approach to enhance therapeutic selectivity in cancer tissues is the use of nanocarriers whose nuclear targeting and uptake are triggered by the altered expression of specific endomembrane trafficking proteins in cancer cells.

Journey to the Center of the Cell: Current Nanocarrier Design Strategies Targeting Biopharmaceuticals to the Cytoplasm and Nucleus.

New biopharmaceutical molecules, potentially able to provide more personalized and effective treatments, are being identified through the advent of advanced synthetic biology strategies, sophisticated chemical synthesis approaches, and new analytical methods to assess biological potency. However, translation of many of these structures has been significantly limited due to the need for more efficient strategies to deliver macromolecular therapeutics to desirable intracellular sites of action. Engineered nanocarriers that encapsulate peptides, proteins, or nucleic acids are generally internalized into target cells via one of several endocytic pathways.

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

For successful gene delivery, plasmid DNA must be able to access the nucleus in order to be transcribed. Numerous studies have shown that gene delivery occurs more readily in dividing cells, which is attributed to increased nuclear access when the nuclear envelope disassembles during mitosis; however, nonviral carriers continue to have low transfection efficiencies and require large quantities of DNA per cell to achieve reasonable gene transfer, even in dividing cells. Therefore, we hypothesized that using histone-derived nuclear localization sequences (NLS)s to target polyplexes might enhance nuclear delivery by facilitating interactions with histone effectors that mediate nuclear partitioning and retention during mitosis.

Fluorescent dye incorporation causes weakened gene association and intracellular aggregate formation in nonviral carriers.

Background: The successful application of nonviral gene transfer technologies requires both improved understanding and control with respect to intracellular trafficking and release. However, the intracellular space is highly complex and hence well-defined, stable structures are necessary to probe the stages of the delivery pathway. Fluorescent labeling is a regularly used approach to monitor nonviral delivery and release, yet few studies investigate the effects of label incorporation on the structure and activity of gene-containing vehicles.

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

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.

Requirements for the nuclear entry of polyplexes and nanoparticles during mitosis.

Background: Nonviral gene delivery has a limited efficacy partly as a result of poor nuclear delivery, yet an understanding of the mechanisms of nuclear entry is limited. The present study aimed to test the common hypothesis that most nonviral vehicles enter the nucleus during cell division.

Methods: Polystyrene particles with diameters of 24-200 nm and carboxylate or amine surface groups, were either used as is or, alternatively, were functionalized with carboxyl-, hydroxyl- or amine- terminated poly(ethylene glycol) (PEG) and subsequently microinjected into the cytoplasm of NIH/3T3 mouse fibroblast cells.