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1.  Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells 
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
PMCID: PMC3381593  PMID: 23344621
delivery; immune cell; siRNA
2.  A combinatorial library of lipid-like materials for delivery of RNAi therapeutics 
Nature biotechnology  2008;26(5):561-569.
The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2′-O-methyl (2′-O Me) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.
PMCID: PMC3014085  PMID: 18438401
3.  Hyperoxia causes angiopoietin 2–mediated acute lung injury and necrotic cell death 
Nature medicine  2006;12(11):1286-1293.
The angiogenic growth factor angiopoietin 2 (Ang2) destabilizes blood vessels, enhances vascular leak and induces vascular regression and endothelial cell apoptosis. We considered that Ang2 might be important in hyperoxic acute lung injury (ALI). Here we have characterized the responses in lungs induced by hyperoxia in wild-type and Ang2–/– mice or those given either recombinant Ang2 or short interfering RNA (siRNA) targeted to Ang2. During hyperoxia Ang2 expression is induced in lung epithelial cells, while hyperoxia-induced oxidant injury, cell death, inflammation, permeability alterations and mortality are ameliorated in Ang2–/– and siRNA-treated mice. Hyperoxia induces and activates the extrinsic and mitochondrial cell death pathways and activates initiator and effector caspases through Ang2-dependent pathways in vivo. Ang2 increases inflammation and cell death during hyperoxia in vivo and stimulates epithelial necrosis in hyperoxia in vitro. Ang2 in plasma and alveolar edema fluid is increased in adults with ALI and pulmonary edema. Tracheal Ang2 is also increased in neonates that develop bronchopulmonary dysplasia. Ang2 is thus a mediator of epithelial necrosis with an important role in hyperoxic ALI and pulmonary edema.
PMCID: PMC2768268  PMID: 17086189
4.  Very Late Antigen (VLA)-1 Blockade Markedly Promotes Survival of Corneal Allografts 
Archives of ophthalmology  2007;125(6):783-788.
To investigate the role of very late antigen 1 (VLA-1) (also known as integrin receptor α1β1) in corneal transplantation inflammation and allograft survival.
Cell infiltration and vasculogenesis (both angiogenesis and lymphangiogenesis) associated with allodisparate corneal transplantation were assessed in VLA-1—deficient conditions and controls by immunofluorescent microscopic studies. Corneal allograft survival was also assessed after anti—VLA-1 antibody treatment and in VLA-1 knockout recipient mice.
Anti—VLA-1 antibody treatment leads to a profound reduction in the granulocytic, monocytic, and T-cell infiltration after corneal transplantation. In addition, corneal angiogenesis and lymphangiogenesis were both significantly suppressed in VLA-1 knockout mice. Remarkably, universal graft survival was observed in both anti—VLA-1 antibody treatment and knockout mice.
Very late antigen 1 blockade markedly reduces inflammation and inflammation-induced tissue responses, including vasculogenic responses, associated with corneal transplantation and promotes allograft survival.
Clinical Relevance
These studies offer insights into important integrin-mediated mechanisms of corneal transplant—related inflammation and provide possible new integrin-based immunotherapies for transplant rejection.
PMCID: PMC2677688  PMID: 17562989
5.  Collagen-binding integrin α1β1 regulates intestinal inflammation in experimental colitis 
The Journal of Clinical Investigation  2002;110(12):1773-1782.
Central to inflammatory responses are the integrin-mediated adhesive interactions of cells with their ECM-rich environment. We investigated the role of the collagen-binding integrin α1β1 in intestinal inflammation using the mouse model of colitis induced by dextran sodium sulfate (DSS). mAb’s directed against murine α1 were found to significantly attenuate inflammation and injury in DSS-treated wild-type mice; similar protection was seen in mice deficient for α1β1 integrin. Blockade or loss of α1β1 was also associated with decreased mucosal inflammatory cell infiltrate and cytokine production. Importantly, we demonstrated that development and α1-mediated inhibition of DSS-induced colitis occurred independently of lymphocytes (Rag-2–/– mice), and identified the monocyte as a key α1β1-expressing cell type involved in the development of colitis in this model. In response to DSS, both α1 deficiency and anti-α1 mAb treatment significantly reduced monocyte accumulation and activation within the lamina propria. In summary, the data demonstrate that engagement of leukocyte-associated α1β1 receptors with ECM plays a pivotal role in mediating intestinal inflammation via promotion of monocyte movement and/or activation within the inflamed interstitium. Therapeutic strategies designed to disrupt such interactions may prove beneficial in treating intestinal inflammation.
PMCID: PMC151649  PMID: 12488427
6.  α5β1 Integrin Activates an NF-κB-Dependent Program of Gene Expression Important for Angiogenesis and Inflammation 
Molecular and Cellular Biology  2002;22(16):5912-5922.
GeneCalling, a genome-wide method of mRNA profiling, reveals that endothelial cells adhering to fibronectin through the α5β1 integrin, but not to laminin through the α2β1 integrin, undergo a complex program of gene expression. Several of the genes identified are regulated by the NF-κB transcription factor, and many are implicated in the regulation of inflammation and angiogenesis. Adhesion of endothelial cells to fibronectin activates NF-κB through a signaling pathway requiring Ras, phosphatidylinositol 3-kinase, and Rho family proteins, whereas adhesion to laminin has a limited effect. Retroviral transfer of the superrepressor of NF-κB, IκB-2A, blocks basic fibroblast growth factor-induced angiogenesis in vivo. These results suggest that engagement of the α5β1 integrin promotes an NF-κB-dependent program of gene expression that coordinately regulates angiogenesis and inflammation.
PMCID: PMC133962  PMID: 12138201
7.  Regulation of inflammation by collagen-binding integrins α1β1 and α2β1 in models of hypersensitivity and arthritis 
Journal of Clinical Investigation  2000;105(6):721-729.
Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix–rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, α1β1 and α2β1, in several in vivo models of inflammation. mAb’s against murine α1 and α2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were α1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of α1β1 and α2β1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.
PMCID: PMC377459  PMID: 10727440

Results 1-7 (7)