Intratumoral Administration of High-Concentration Nitric Oxide and Anti-mPD-1 Treatment Improves Tumor Regression Rates and Survival in CT26 Tumor-Bearing Mice.

BACKGROUND: Immune checkpoint inhibitors have transformed clinical oncology. However, their use is limited as response is observed in only ~20-50% of patients. Previously, we demonstrated that treating CT26 tumor-bearing mice with ultra-high-concentration gaseous nitric oxide (UNO) followed by tumor resection stimulated antitumor immune responses. Accordingly, UNO may improve tumor response to immune checkpoint inhibitors. Here, we investigated the ability of UNO to improve the efficacy of a programmed cell death protein-1 (PD-1) antibody in vitro and in treating CT26 tumor-bearing mice. METHODS: CT26 cells were injected into the flank of Balb/c mice (n = 15-16 per group). On day 6, CT26 cells were injected into the contralateral flank, and anti-mPD-1 injections commenced. Primary tumors were treated with intratumoral UNO on day 8. Tumor volume, response rates, toxicity, and survival were monitored. RESULTS: (1) Short exposure to 25,000-100,000 parts per million (ppm) UNO in vitro resulted in significant upregulation of PD-L1 expression on CT26 cells. (2) UNO treatment in vivo consistently reduced cell viability in CT26 tumors. (3) Treatment reduced regulatory T-cell (Treg) levels in the tumor and increased levels of systemic M1 macrophages. UNO responders had increased CD8+ T-cell tumor infiltration. (4) Nine days after treatment, primary tumor growth was significantly lower in the combination arm vs. anti-mPD-1 alone (p = 0.0005). (5) Complete tumor regression occurred in 8/15 (53%) of mice treated with a combination of 10 min UNO and anti-mPD-1, 100 days post-treatment, compared to 4/16 (25%) of controls treated with anti-mPD-1 alone (p = 0.1489). (6) There was no toxicity associated with UNO treatment. (7) Combination treatment showed a trend toward increased survival 100 days post-treatment compared to anti-mPD-1 alone (p = 0.0653). CONCLUSION: Combining high-concentration NO and immune checkpoint inhibitors warrants further assessment especially in tumors resistant to checkpoint inhibitor therapy.

Defining the timeline of periostin upregulation in cardiac fibrosis following acute myocardial infarction in mice.

After myocardial infarction (MI), the heart's reparative response to the ischemic insult and the related loss of cardiomyocytes involves cardiac fibrosis, in which the damaged tissue is replaced with a fibrous scar. Although the scar is essential to prevent ventricular wall rupture in the infarction zone, it expands over time to remote, non-infarct areas, significantly increasing the extent of fibrosis and markedly altering cardiac structure. Cardiac function in this scenario deteriorates, thereby increasing the probability of heart failure and the risk of death. Recent works have suggested that the matricellular protein periostin, known to be involved in fibrosis, is a candidate therapeutic target for the regulation of MI-induced fibrosis and remodeling. Different strategies for the genetic manipulation of periostin have been proposed previously, yet those works did not properly address the time dependency between periostin activity and cardiac fibrosis. Our study aimed to fill that gap in knowledge and fully elucidate the explicit timing of cellular periostin upregulation in the infarcted heart to enable the safer and more effective post-MI targeting of periostin-producing cells. Surgical MI was performed in C57BL/6J and BALB/c mice by ligation of the left anterior descending coronary artery. Flow cytometry analyses of cells derived from the infarcted hearts and quantitative real-time PCR of the total cellular RNA revealed that periostin expression increased during days 2-7 and peaked on day 7 post-infarct, regardless of mouse strain. The established timeline for cellular periostin expression in the post-MI heart is a significant milestone toward the development of optimal periostin-targeted gene therapy.

Gaseous nitric oxide tumor ablation induces an anti-tumor abscopal effect.

BACKGROUND: In-situ tumor ablation provides the immune system with the appropriate antigens to induce anti-tumor immunity. Here, we present an innovative technique for generating anti-tumor immunity by delivering exogenous ultra-high concentration (> 10,000 ppm) gaseous nitric oxide (UHCgNO) intratumorally. METHODS: The capability of UHCgNO to induce apoptosis was tested in vitro in mouse colon (CT26), breast (4T1) and Lewis lung carcinoma (LLC-1) cancer cell lines. In vivo, UHCgNO was studied by treating CT26 tumor-bearing mice in-situ and assessing the immune response using a Challenge assay. RESULTS: Exposing CT26, 4T1 and LLC-1 cell lines to UHCgNO for 10 s-2.5 min induced cellular apoptosis 24 h after exposure. Treating CT26 tumors in-situ with UHCgNO followed by surgical resection 14 days later resulted in a significant secondary anti-tumor effect in vivo. 100% of tumor-bearing mice treated with 50,000 ppm UHCgNO and 64% of mice treated with 20,000 ppm UHCgNO rejected a second tumor inoculation, compared to 0% in the naive control for 70 days. Additionally, more dendrocytes infiltrated the tumor 14 days post UHCgNO treatment versus the nitrogen control. Moreover, T-cell penetration into the primary tumor was observed in a dose-dependent manner. Systemic increases in T- and B-cells were seen in UHCgNO-treated mice compared to nitrogen control. Furthermore, polymorphonuclear-myeloid-derived suppressor cells were downregulated in the spleen in the UHCgNO-treated groups. CONCLUSIONS: Taken together, our data demonstrate that UHCgNO followed by the surgical removal of the primary tumor 14 days later induces a strong and potent anti-tumor response.

Transcription Factor Binding in Embryonic Stem Cells Is Constrained by DNA Sequence Repeat Symmetry.

Transcription factor (TF) recognition is dictated by the underlying DNA motif sequence specific for each TF. Here, we reveal that DNA sequence repeat symmetry plays a central role in defining TF-DNA-binding preferences. In particular, we find that different TFs bind similar symmetry patterns in the context of different developmental layers. Most TFs possess dominant preferences for similar DNA repeat symmetry types. However, in some cases, preferences of specific TFs are changed during differentiation, suggesting the importance of information encoded outside of known motif regions. Histone modifications also exhibit strong preferences for similar DNA repeat symmetry patterns unique to each type of modification. Next, using an in vivo reporter assay, we show that gene expression in embryonic stem cells can be positively modulated by the presence of genomic and computationally designed DNA oligonucleotides containing identified nonconsensus-repetitive sequence elements. This supports the hypothesis that certain nonconsensus-repetitive patterns possess a functional ability to regulate gene expression. We also performed a solution NMR experiment to probe the stability of double-stranded DNA via imino proton resonances for several double-stranded DNA sequences characterized by different repetitive patterns. We suggest that such local stability might play a key role in determining TF-DNA binding preferences. Overall, our findings show that despite the enormous sequence complexity of the TF-DNA binding landscape in differentiating embryonic stem cells, this landscape can be quantitatively characterized in simple terms using the notion of DNA sequence repeat symmetry.

Co-assembled Ca2+ Alginate-Sulfate Nanoparticles for Intracellular Plasmid DNA Delivery.

Successful gene therapy requires the development of suitable carriers for the selective and efficient delivery of genes to specific target cells, with minimal toxicity. In this work, we present a non-viral vector for gene delivery composed of biocompatible materials, CaCl2, plasmid DNA and the semi-synthetic anionic biopolymer alginate sulfate (AlgS), which spontaneously co-assembled to form nanoparticles (NPs). The NPs were characterized with a slightly anionic surface charge (Zeta potential [ζ] = -14 mV), an average size of 270 nm, and their suspension was stable for several days with no aggregation. X-ray photoelectron spectroscopy (XPS) validated their ternary composition, and it elucidated the molecular interactions among Ca2+, the plasmid DNA, and the AlgS. Efficient cellular uptake (>80%), associated with potent GFP gene expression (22%-35%), was observed across multiple cell types: primary rat neonatal cardiac fibroblasts, human breast cancer cell line, and human hepatocellular carcinoma cells. The uptake mechanism of the NPs was studied using imaging flow cytometry and shown to be via active, clathrin-mediated endocytosis, as chemical inhibition of this pathway significantly reduced EGFP expression. The NPs were cytocompatible and did not activate the T lymphocytes in human peripheral blood mononuclear cells. Proof of concept for the efficacy of these NPs as a carrier in cancer gene therapy was demonstrated for Diphtheria Toxin Fragment A (DT-A), resulting in abrogation of protein synthesis and cell death in the human breast cancer cell line. Collectively, our results show that the developed AlgS-Ca2+-plasmid DNA (pDNA) NPs may be used as an effective non-viral carrier for pDNA.

Specificity-Determining DNA Triplet Code for Positioning of Human Preinitiation Complex.

The notion that transcription factors bind DNA only through specific, consensus binding sites has been recently questioned. No specific consensus motif for the positioning of the human preinitiation complex (PIC) has been identified. Here, we reveal that nonconsensus, statistical, DNA triplet code provides specificity for the positioning of the human PIC. In particular, we reveal a highly nonrandom, statistical pattern of repetitive nucleotide triplets that correlates with the genomewide binding preferences of PIC measured by Chip-exo. We analyze the triplet enrichment and depletion near the transcription start site and identify triplets that have the strongest effect on PIC-DNA nonconsensus binding. Using statistical mechanics, a random-binder model without fitting parameters, with genomic DNA sequence being the only input, we further validate that the nonconsensus nucleotide triplet code constitutes a key signature providing PIC binding specificity in the human genome. Our results constitute a proof-of-concept for, to our knowledge, a new design principle for protein-DNA recognition in the human genome, which can lead to a better mechanistic understanding of transcriptional regulation.

Mechanisms of cellular uptake and endosomal escape of calcium-siRNA nanocomplexes.

Ca2+-siRNA nanocomplexes represent a simple yet an effective platform for siRNA delivery into the cell cytoplasm, with subsequent successful siRNA-induced target gene silencing. Herein, we aimed to elucidate the roles played by calcium ions in siRNA nanocomplex formation, cell uptake, and endosomal escape. We investigated whether the replacement of Ca2+in the nanocomplex by other bivalent cations would affect their cell entry and subsequent gene silencing. Our results indicate that Mg2+ and Ba2+ lead to the formation of nanocomplexes of similar physical features (size=100nm, surface charge ζ=-8mV) as the Ca2+-siRNA nanocomplexes. Yet, these nanocomplexes were not uptaken by the cells to the same extent as those prepared with Ca2+, and siRNA-induced target gene silencing was not obtained. Cell internalization of Ca2+--siRNA nanocomplexes, examined by employing chemical inhibitors to clathrin-, caveolin- and dynamin-mediated endocytosis pathways, indicated the involvement of all mechanisms in the process. Inhibition of endosome acidification by bafilomycin completely abolished the siRNA-mediated silencing by Ca2+-siRNA nanocomplexes. Collectively, our results indicate that Ca2+ promotes cell internalization and rapid endosomal escape, thus leading to the efficient siRNA-induced target gene silencing elicited by the Ca2+-siRNA nanocomplexes.

A bridge to silencing: Co-assembling anionic nanoparticles of siRNA and hyaluronan sulfate via calcium ion bridges.

Therapeutic implementation of RNA interference (RNAi) through delivery of short interfering RNA (siRNA) is still facing several critical hurdles, which mostly can be solved through the use of an efficient delivery system. We hereby introduce anionic siRNA nanoparticles (NPs) co-assembled by the electrostatic interactions of the semi-synthetic polysaccharide hyaluronan-sulfate (HAS), with siRNA, mediated by calcium ion bridges. The NPs have an average size of 130nm and a mild (-10mV) negative surface charge. Transmission electron microscopy (TEM) using gold-labeled components and X-ray photoelectron spectroscopy (XPS) demonstrated the spatial organization of siRNA molecules in the particle core, surrounded by a layer of HAS. The anionic NPs efficiently encapsulated siRNA, were stable in physiological-relevant environments and were cytocompatible, not affecting cell viability or homeostasis. Efficient cellular uptake of the anionic siRNA NPs, associated with potent gene silencing (>80%), was observed across multiple cell types, including murine primary peritoneal macrophages and human hepatocellular carcinoma cells. In a clinically-relevant model of acute inflammatory response in IL-6-stimulated human hepatocytes, STAT3 silencing induced by HAS-Ca(2+)-siRNA NPs resulted in marked decrease in the total and activated STAT3 protein levels, as well as in the expression levels of downstream acute phase response genes. Collectively, anionic NPs prove to be an efficient and cytocompatible delivery system for siRNA.