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1.  Evaluation of Exogenous siRNA Addition as a Metabolic Engineering Tool for Modifying Biopharmaceuticals 
Biotechnology progress  2013;29(2):415-424.
Traditional metabolic engineering approaches, including homologous recombination, zinc finger nucleases, and short hairpin RNA (shRNA), have previously been employed to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors, This approach has the following potential advantages : no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 monoclonal antibody. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an ADCC assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.
doi:10.1002/btpr.1667
PMCID: PMC4226405  PMID: 23172735
2.  Therapeutic siRNA silencing in inflammatory monocytes 
Nature Biotechnology  2011;29(11):1005-1010.
Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.
doi:10.1038/nbt.1989
PMCID: PMC3212614  PMID: 21983520
3.  Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice 
The Journal of Clinical Investigation  2012;122(5):1677-1687.
Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease.
doi:10.1172/JCI61248
PMCID: PMC3336989  PMID: 22466652
4.  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.
doi:10.1038/mtna.2011.3
PMCID: PMC3381593  PMID: 23344621
delivery; immune cell; siRNA
5.  Empirically-controlled mapping of the Caenorhabditis elegans protein-protein interactome network 
Nature methods  2009;6(1):47-54.
To provide accurate biological hypotheses and inform upon global properties of cellular networks, systematic identification of protein–protein interactions has to meet high-quality standards. We present an expanded Caenorhabditis elegans protein-protein interaction network, or “interactome” map derived from testing a matrix of ~ 10,000 × ~ 10,000 proteins using a highly specific high-throughput yeast two-hybrid system. Through a new quality control empirical framework, We show that the resulting dataset (Worm Interactome 2007 or WI-2007) is similar in quality to low-throughput data curated from the literature. Previous interaction datasets have been filtered and integrated with WI-2007 to generate a high confidence consolidated map (Worm Interactome version 8 or WI8). This work allows us to estimate the size of the worm interactome at ~ 116,000 interactions. Comparison with other types of functional genomic data shows the complementarity of distinct experimental approaches in predicting different functional relationship features between genes or proteins.
PMCID: PMC3057923  PMID: 19123269
6.  Edgetic perturbation of a C. elegans BCL2 ortholog 
Nature methods  2009;6(11):843-849.
Genes and gene products do not function in isolation but within highly interconnected “interactome” networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or “edgetic” alleles. We establish a proof-of-concept with CED-9, a C. elegans BCL2 ortholog involved in apoptosis. Using ced-9 edgetic alleles, we uncover a new potential functional link between apoptosis and a centrosomal protein, demonstrating both the interest and efficiency of our strategy. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.
doi:10.1038/nmeth.1394
PMCID: PMC2865203  PMID: 19855391
7.  Edgetic perturbation models of human inherited disorders 
Cellular functions are mediated through complex systems of macromolecules and metabolites linked through biochemical and physical interactions, represented in interactome models as ‘nodes' and ‘edges', respectively. Better understanding of genotype-to-phenotype relationships in human disease will require modeling of how disease-causing mutations affect systems or interactome properties. Here we investigate how perturbations of interactome networks may differ between complete loss of gene products (‘node removal') and interaction-specific or edge-specific (‘edgetic') alterations. Global computational analyses of ∼50 000 known causative mutations in human Mendelian disorders revealed clear separations of mutations probably corresponding to those of node removal versus edgetic perturbations. Experimental characterization of mutant alleles in various disorders identified diverse edgetic interaction profiles of mutant proteins, which correlated with distinct structural properties of disease proteins and disease mechanisms. Edgetic perturbations seem to confer distinct functional consequences from node removal because a large fraction of cases in which a single gene is linked to multiple disorders can be modeled by distinguishing edgetic network perturbations. Edgetic network perturbation models might improve both the understanding of dissemination of disease alleles in human populations and the development of molecular therapeutic strategies.
doi:10.1038/msb.2009.80
PMCID: PMC2795474  PMID: 19888216
binary protein interaction; genotype-to-phenotype relationships; human Mendelian disorders; network perturbation
8.  A protein domain-based interactome network for C. elegans early embryogenesis 
Cell  2008;134(3):534-545.
Summary
Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or “interactome” networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed new insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.
doi:10.1016/j.cell.2008.07.009
PMCID: PMC2596478  PMID: 18692475
9.  A Map of the Interactome Network of the Metazoan C. elegans 
Science (New York, N.Y.)  2004;303(5657):540-543.
To initiate studies on how protein-protein interaction (or “interactome”) networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains ∼5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.
doi:10.1126/science.1091403
PMCID: PMC1698949  PMID: 14704431

Results 1-9 (9)