Explore the vast range of titles available.

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. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs.Su Chul Jang et al. develop exoSTING, consisting of an engineered extracellular vesicle loaded with a potent cyclic dinucleotide (CDN) agonist of the STING pathway. They find that exoSTING shows more than 100-fold increased potency in in vivo tumor models and has increased tumor retention and lower levels of systemic inflammatory cytokine production as compared to free CDN.

AAV gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. Here, we characterize the innate immune response to AAV in human whole blood. We identified neutrophils, monocyte-related dendritic cells, and monocytes as the most prevalent cell subsets able to internalize AAV particles, while conventional dendritic cells were the most activated in terms of the CD86 co-stimulatory molecule upregulation. Although low titers (≤1:10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (≥1:100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. Interestingly, both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, we demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.

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.

BackgroundExosomes are natural, abundant extracellular vesicles capable of transferring complex molecules between neighboring and distant cell types. Translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. Important strategies to maximize the therapeutic potential of exosomes therefore include payload loading, functionalization of the exosome surface with pharmacologically active proteins, and delivery to target cells of interest.MethodsThrough comparative proteomic analysis of purified exosomes, we identified several highly enriched and exosome-specific proteins, including a transmembrane glycoprotein (PTGFRN) belonging to the immunoglobulin superfamily. Leveraging PTGFRN as a scaffold for exosome surface display, we developed our engExTM platform to generate engineered exosomes functionalized with a variety of structurally and biologically diverse proteins.Systemically administered exosomes are primarily taken up by macrophages in the liver and spleen. To redirect exosome uptake to other cell types, we employed our engineering platform to display functional targeting ligands, including single domain antibodies, single chain variable fragments, single chain Fabs (scFabs), and receptor ligands, on the exosome surface at high density. To demonstrate that exosome surface modifications can alter cellular tropism, we generated exosomes displaying anti-Clec9A scFabs to target conventional type 1 dendritic cells (cDC1s), anti-CD3 scFabs to target T cells, and CD40 ligand to target B cells. The engineered exosomes exhibited functional antigen binding that led to greater association with the cell types expressing the cognate receptor both in vitro and in vivo.ResultsIn mice, systemic administration of exosomes engineered to display scFabs targeting Clec9A resulted in a 4-fold increase in the percentage of cDC1 cells in the blood that had taken up exosomes over controls, and a 6-fold increase in the number of exosomes taken up per cell. We further showed that compared to untargeted exosomes, those with altered tropism achieved increased functional payload delivery to the target cell of interest. In primary mouse dendritic cells, anti-Clec9A exosomes loaded with a cyclic dinucleotide STING agonist achieved greater pathway induction, 2.3-fold greater as measured by IFNβ production, 2-fold by IFNα, and 15-fold by IL-12, when compared to an untargeted control. Preliminary in vivo data show that intra-tumorally administered anti-Clec9A exosomes reduce the required STING agonist dose 10-fold to achieve single-agent tumor control and induce immune responses against tumor-associated antigen, compared to controls.ConclusionsThese results demonstrate the potential of our engExTM platform to generate targeted exosome therapeutics capable of immune cell stimulation and tumor growth inhibition in vivo.Ethics ApprovalAll experimental animal studies were performed according to Codiak BioSciences IACUC approved AUP CB2020-001.

It is well documented that cancer is disproportionately distributed in racial/ethnic minority groups and medically underserved communities. In addition, cancer prevention and early detection represent the key defenses to combat cancer. The purpose of this article is to showcase the comprehensive health education and community outreach activities at the H. Lee Moffitt Cancer Center and Research Institute (Moffitt) designed to promote and increase access to and utilization of prevention and early detection services among underserved populations. One of Moffitt’s most important conduits for cancer prevention and early detection among underserved populations is through its community education and outreach initiatives, in particular, the Moffitt Program for Outreach Wellness Education and Resources (M-POWER). M-POWER works to empower underserved populations to make positive health choices and increase screening behaviors through strengthening collaboration and partnerships, providing community-based health education/promotion, and increasing access to care. Effective, empowering, and culturally and linguistically competent health education and community outreach, is key to opening the often impenetrable doors of cancer prevention and early detection to this society’s most vulnerable populations.

Delivery of nucleic acids for therapeutic applications is an exciting field, promising innovative approaches for regulating protein expression towards the treatment of diseases including multiple varieties of cancer. The focus of this field is to develop efficacious delivery vehicles which can shuttle therapeutic nucleic acids into cells in a nontoxic manner, leading to treatment of diseases on a molecular level. However, the efficiency of non-viral delivery vehicles has been limited by an incomplete understanding of their cellular uptake, subcellular trafficking, and intracellular delivery to the nucleus. This dissertation focuses on experiments designed to elucidate the biological properties of the histone H3 tail peptide utilized for DNA delivery vehicles, and how they enhance gene delivery. We have found that these H3 tail peptides, in combination with the cationic polymer poly(ethylenimine) (PEI), can effectively bind and protect plasmid DNA (pDNA), for efficient delivery to the nucleus. The studies described in this dissertation focus on elucidating the endocytic pathway and nuclear delivery mechanisms of H3-targeted polyplexes in mammalian cell lines. These studies demonstrate that (1) the caveolar endocytic pathway plays a vital role in the fate of polyplexes, (2) H3-targeted polyplexes interact with histone effectors to effectively enter the nucleus during mitosis, and (3) H3-targeted polyplexes selectively target cancer cells that overexpress caveolin. In total, this dissertation provides new evidence for the potential role for histone-based materials as effective gene transfer vehicles, and supports the importance of subcellular trafficking for non-viral gene delivery. Ultimately, gene therapies will rely on the development of methods to control gene targeting and trafficking along the delivery pathway, requiring properties that enable them to appropriately navigate the intracellular space. This dissertation contributes to the growing body of literature to improve our fundamental understanding of the processing of non-viral carriers.