Intratracheal Administration of siRNA Triggers mRNA Silencing in the Lung to Modulate T Cell Immune Response and Lung Inflammation.

Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c+ cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology.

Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit.

The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.

Quantification of Cy-5 siRNA signal in the intra-vital multi-photon microscopy images.

Transgenic mice with Tie2- green fluorescent protein (GFP) are used as a model to study the kinetic distribution of the Cy5-siRNA delivered by lipid nanoparticles (LNP) into the liver. After the mouse is injected with the LNP, it undergoes a procedure of intra-vital multi-photon microscopy imaging over a period of two hours, during which the process for the nanoparticle to diffuse into the hepatocytes from the vasculature system is monitored. Since the images are obtained in-vivo, the quantification of Cy5 kinetics suffers from the moving field of view (FOV). A method is proposed to register the sequence of images through template matching. Based on the semi-automatic segmentations of the vessels in the common FOV, the registered images are segmented into three regions of interest (ROI) in which the Cy5 signals are quantified. Computation of the percentage signal strength in the ROIs over time allows for the analysis of the diffusion of Cy5-siRNA into the hepatocytes, and helps demonstrate the effectiveness of the Cy5-siRNA delivery vehicle.

Mechanistically probing lipid-siRNA nanoparticle-associated toxicities identifies Jak inhibitors effective in mitigating multifaceted toxic responses.

A major hurdle for harnessing small interfering RNA (siRNA) for therapeutic application is an effective and safe delivery of siRNA to target tissues and cells via systemic administration. While lipid nanoparticles (LNPs) composed of a cationic lipid, poly-(ethylene glycol) lipid and cholesterol, are effective in delivering siRNA to hepatocytes via systemic administration, they may induce multi-faceted toxicities in a dose-dependent manner, independently of target silencing. To understand the underlying mechanism of toxicities, pharmacological probes including anti-inflammation drugs and specific inhibitors blocking different pathways of innate immunity were evaluated for their abilities to mitigate LNP-siRNA-induced toxicities in rodents. Three categories of rescue effects were observed: (i) pretreatment with a Janus kinase (Jak) inhibitor or dexamethasone abrogated LNP-siRNA-mediated lethality and toxicities including cytokine induction, organ impairments, thrombocytopenia and coagulopathy without affecting siRNA-mediated gene silencing; (ii) inhibitors of PI3K, mammalian target of rapamycin (mTOR), p38 and IκB kinase (IKK)1/2 exhibited a partial alleviative effect; (iii) FK506 and etoricoxib displayed no protection. Furthermore, knockout of Jak3, tumor necrosis factor receptors (Tnfr)p55/p75, interleukin 6 (IL-6) or interferon (IFN)-γ alone was insufficient to alleviate LNP-siRNA-associated toxicities in mice. These indicate that activation of innate immune response is a primary trigger of systemic toxicities and that multiple innate immune pathways and cytokines can mediate toxic responses. Jak inhibitors are effective in mitigating LNP-siRNA-induced toxicities.

Noninvasive imaging of lipid nanoparticle-mediated systemic delivery of small-interfering RNA to the liver.

Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.

Shedding of distinct cryptic collagen epitope (HU177) in sera of melanoma patients.

PURPOSE: Extracellular matrix remodeling during tumor growth plays an important role in angiogenesis. Our preclinical data suggest that a newly identified cryptic epitope (HU177) within collagen type IV regulates endothelial and melanoma cell adhesion in vitro and angiogenesis in vivo. In this study, we investigated the clinical relevance of HUI77 shedding in melanoma patient sera. EXPERIMENTAL DESIGN: Serum samples from 291 melanoma patients prospectively enrolled at the New York University Medical Center and 106 control subjects were analyzed for HU177 epitope concentration by a newly developed sandwich ELISA assay. HU177 serum levels were then correlated with clinical and pathologic parameters. RESULTS: Mean HU177 epitope concentration was 5.8 ng/mL (range, 0-139.8 ng/mL). A significant correlation was observed between HU177 concentration and nodular melanoma histologic subtype [nodular, 10.3 +/- 1.6 ng/mL (mean +/- SE); superficial spreading melanoma, 4.5 +/- 1.1 ng/mL; all others, 6.1 +/- 2.1 ng/mL; P = 0.01 by ANOVA test]. Increased HU177 shedding also correlated with tumor thickness (< or =1.00 mm, 3.8 +/- 1.1 ng/mL; 1.01-3.99 mm, 8.7 +/- 1.3 ng/mL; > or =4.00 mm, 10.3 +/- 2.4 ng/mL; P = 0.003 by ANOVA). After multivariate analysis controlling for thickness, the correlation between higher HU177 concentration and nodular subtype remained significant (P = 0.03). The mean HU177 epitope concentration in control subjects was 2.4 ng/mL. CONCLUSIONS: We report that primary melanoma can induce detectable changes in systemic levels of cryptic epitope shedding. Our data also support that nodular melanoma might be biologically distinct compared with superficial spreading type melanoma. As targeted interventions against cryptic collagen epitopes are currently undergoing phase I clinical trial testing, these findings indicate that patients with nodular melanoma may be more susceptible to such targeted therapies.

Intravital observation of Plasmodium berghei sporozoite infection of the liver.

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.

Recombinant alpha2(IV)NC1 domain inhibits tumor cell-extracellular matrix interactions, induces cellular senescence, and inhibits tumor growth in vivo.

Cellular interaction with the extracellular matrix is thought to be a critical event in controlling angiogenesis and tumor growth. In our previous studies, genetically distinct noncollagenous (NC) domains of type-IV collagen were shown to interact with integrin receptors expressed on the surface of endothelial cells. Moreover, these NC1 domains were shown to inhibit angiogenesis in vivo. Here, we provide evidence that a recombinant form of the alpha2(IV)NC1 domain of type-IV collagen could bind integrins alpha1beta1 and alphavbeta3 expressed on melanoma cells and inhibit tumor cell adhesion in a ligand-specific manner. Systemic administration of recombinant alpha2(IV)NC1 domain potently inhibited M21 melanoma tumor growth within full thickness human skin and exhibited a dose-dependent inhibition of tumor growth in nude mice. Interestingly, alpha2(IV)NC1 domain enhanced cellular senescence in tumor cells in vitro and in vivo. Taken together, these results suggest that recombinant alpha2(IV)NC1 domain is not only a potent anti-angiogenic reagent, but it also directly impacts tumor cell behavior. Thus, alpha2(IV)NC1 domain represents a potent inhibitor of tumor growth by impacting both endothelial and tumor cell compartments.

Select forms of tumor cell apoptosis induce dendritic cell maturation.

Dendritic cells (DC) play a crucial role in initiating immune responses to tumors. DC can efficiently present antigens from apoptotic tumor cells, but apoptotic cells are thought to lack the inflammatory signals required to induce DC maturation. Here, we show that apoptosis of 67NR mouse carcinoma cells via the Fas (CD95) pathway or induced by the anticancer drug bortezomib (PS-341) but not by ultraviolet irradiation is associated with the production of maturation signals for DC. These data have important implications for the effects of chemotherapy on antitumor immunity in solid and hematologic malignancies.

Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated.

PURPOSE: Ionizing radiation can reduce tumor growth outside the field of radiation, known as the abscopal effect. Although it has been reported in multiple malignancies, the abscopal effect remains a rare and poorly understood event. Ionizing radiation generates inflammatory signals and, in principle, could provide both tumor-specific antigens from dying cells and maturation stimuli that are necessary for dendritic cells' activation of tumor-specific T cells. We therefore tested the hypothesis that the abscopal effect elicited by radiation is immune mediated. This was directly tested by enhancing the number of available dendritic cells using the growth factor Flt3-Ligand (Flt3-L). METHODS AND MATERIALS: Mice bearing a syngeneic mammary carcinoma, 67NR, in both flanks were treated with Flt3-L daily for 10 days after local radiation therapy (RT) to only 1 of the 2 tumors at a single dose of 2 or 6 Gy. The second nonirradiated tumor was used as indicator of the abscopal effect. Data were analyzed using repeated measures regression. RESULTS: RT alone led to growth delay exclusively of the irradiated 67NR tumor, as expected. Surprisingly, growth of the nonirradiated tumor was also impaired by the combination of RT and Flt3-L. As control, Flt3-L had no effect without RT. Importantly, the abscopal effect was shown to be tumor specific, because growth of a nonirradiated A20 lymphoma in the same mice containing a treated 67NR tumor was not affected. Moreover, no growth delay of nonirradiated 67NR tumors was observed when T cell deficient (nude) mice were treated with RT plus Flt3-L. CONCLUSIONS: These results demonstrate that the abscopal effect is in part immune mediated and that T cells are required to mediate distant tumor inhibition induced by radiation.

PS-341, a novel proteasome inhibitor, induces Bcl-2 phosphorylation and cleavage in association with G2-M phase arrest and apoptosis.

Treatment with the proteasome inhibitor, PS-341 resulted in concentration- and time-dependent effects on Bcl-2 phosphorylation and cleavage in H460 cells that coincided with the PS-341-induced G2-M phase arrest. The observed Bcl-2 cleavage paralleled the degree of PS-341-induced apoptosis but was detected to a similar extent with comparable concentrations of two other proteasome inhibitors (MG-132 and PSI). Calpain inhibitors, ALLM and ALLN, and the caspase inhibitors, Z-VAD and AC-YVAD did not induce BcI-2 phosphorylation and cleavage. Exposure to PS-341 resulted in an additional Mr 25,000 cleavage fragment of Bcl-2, whereas only a Mr 23,000 fragment was observed with other anticancer agents. The formation of the Mr 25,000 fragment was not prevented by caspase inhibitors unlike the Mr 23,000 fragment, which suggests mediation by a caspase-independent pathway. Cell fractionation studies revealed that the Bcl-2 cleaved fragments localize within membrane structures and was an early event (at approximately 12 h, posttreatment), and before the observed cleavage of poly(ADP-ribose) polymerase (PARP), beta-catenin, and DNA fragmentation (at approximately 36 h posttreatment). The Mr 23,000 Bcl-2 cleavage product was inhibited by the pan-caspase inhibitor and the inhibitors of capase-3, -8, -9; but the PARP cleavage was prevented only by the pan-caspase and caspase-3 inhibitors, which suggests that the Mr 23,000 Bcl-2 cleavage occurred at both the initiation and execution stages of apoptosis. The inhibition of the ubiquitin/proteasome pathway by PS-341 leads, at an early stage of apoptosis, to Bcl-2 phosphorylation and a unique proteolytic cleavage product, which are associated with G2-M phase arrest and the induction of apoptosis.