Publications (12)166.83 Total impact
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Article: Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation.
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ABSTRACT: Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8(+) T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.Cancer Immunology and Immunotherapy 08/2012; · 3.70 Impact Factor -
Article: Origins of tumor-associated macrophages and neutrophils.
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ABSTRACT: Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b(+) Ly-6C(hi) monocytic and CD11b(+) Ly-6G(hi) granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.Proceedings of the National Academy of Sciences 02/2012; 109(7):2491-6. · 9.68 Impact Factor -
Article: Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells
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ABSTRACT: 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.Keywords: delivery; immune cell; siRNAMolecular Therapy — Nucleic Acids. 12/2011; 1(1):e4. -
Article: Therapeutic siRNA silencing in inflammatory monocytes in mice.
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ABSTRACT: Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.Nature Biotechnology 11/2011; 29(11):1005-10. · 29.50 Impact Factor -
Article: Allelic 'choice' governs somatic hypermutation in vivo at the immunoglobulin kappa-chain locus.
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ABSTRACT: Monoallelic demethylation and rearrangement control allelic exclusion of the immunoglobulin kappa-chain locus (Igk locus) in B cells. Here, through the introduction of pre-rearranged Igk genes into their physiological position, the critical rearrangement step was bypassed, thereby generating mice producing B cells simultaneously expressing two different immunoglobulin-kappa light chains. Such 'double-expressing' B cells still underwent monoallelic demethylation at the Igk locus, and the demethylated allele was the 'preferred' substrate for somatic hypermutation in each cell. However, methylation itself did not directly inhibit the activation-induced cytidine-deaminase reaction in vitro. Thus, it seems that the epigenetic mechanisms that initially bring about monoallelic variable-(diversity)-joining rearrangement continue to be involved in the control of antibody diversity at later stages of B cell development.Nature Immunology 08/2007; 8(7):715-22. · 26.01 Impact Factor -
Article: Antibody repertoires generated by VH replacement and direct VH to JH joining.
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ABSTRACT: The immunoglobulin heavy chain repertoire is generated by somatic rearrangement of variable (V(H)), diversity (D(H)), and joining (J(H)) elements. It can be further diversified by V(H) replacement, where nonrearranged V(H) genes invade preexisting V(H)D(H)J(H) joints. To study the impact and mechanism of V(H) replacement, we generated mice in which antibody production depends on the replacement of a nonproductive V(H)D(H)J(H) rearrangement inserted into its physiological position in the immunoglobulin heavy chain locus. In these mice a highly diverse heavy chain repertoire resulted from V(H) replacement and a second process of noncanonical V(D)J recombination, direct V(H) to J(H) joining. V(H) replacement rarely generated detectable sequence duplications but often proceeded through recombination between the conserved homologous sequences at the 3' end of V(H). Thus, V(H) replacement is an efficient mechanism of antibody diversification, and its impact on the overall antibody repertoire could be greater than anticipated because it frequently leaves no molecular footprint.Immunity 08/2006; 25(1):43-53. · 21.64 Impact Factor -
Article: Attenuated liver fibrosis in the absence of B cells.
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ABSTRACT: Analysis of mononuclear cells in the adult mouse liver revealed that B cells represent as much as half of the intrahepatic lymphocyte population. Intrahepatic B cells (IHB cells) are phenotypically similar to splenic B2 cells but express lower levels of CD23 and CD21 and higher levels of CD5. IHB cells proliferate as well as splenic B cells in response to anti-IgM and LPS stimulation in vitro. VDJ gene rearrangements in IHB cells contain insertions of N,P region nucleotides characteristic of B cells maturing in the adult bone marrow rather than in the fetal liver. To evaluate whether B cells can have an impact on liver pathology, we compared CCl4-induced fibrosis development in B cell-deficient and wild-type mice. CCl4 caused similar acute liver injury in mutant and wild-type mice. However, following 6 weeks of CCl4 treatment, histochemical analyses showed markedly reduced collagen deposition in B cell-deficient as compared with wild-type mice. By analyzing mice that have normal numbers of B cells but lack either T cells or immunoglobulin in the serum, we established that B cells have an impact on fibrosis in an antibody- and T cell-independent manner.Journal of Clinical Investigation 12/2005; 115(11):3072-82. · 15.39 Impact Factor -
Article: Direct in vivo VH to JH rearrangement violating the 12/23 rule.
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ABSTRACT: V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that VH to JH joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the DH-less allele are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular VH gene family or JH element in VHJH junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo.Journal of Experimental Medicine 03/2005; 201(3):341-8. · 13.85 Impact Factor -
Article: Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression.
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ABSTRACT: The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na+/Cl--coupled betaine/gamma-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function.Proceedings of the National Academy of Sciences 03/2004; 101(8):2392-7. · 9.68 Impact Factor -
Article: Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression
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ABSTRACT: The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na+/Cl–-coupled betaine/γ-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function.Proceedings of the National Academy of Sciences 02/2004; 101(8):2392-2397. · 9.68 Impact Factor -
Article: Expression of a targeted lambda 1 light chain gene is developmentally regulated and independent of Ig kappa rearrangements.
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ABSTRACT: Immunoglobulin light chain (IgL) rearrangements occur more frequently at Ig kappa than at Ig lambda. Previous results suggested that the unrearranged Ig kappa locus negatively regulates Ig lambda transcription and/or rearrangement. Here, we demonstrate that expression of a VJ lambda 1-joint inserted into its physiological position in the Ig lambda locus is independent of Ig kappa rearrangements. Expression of the inserted VJ lambda 1 gene segment is developmentally controlled like that of a VJ kappa-joint inserted into the Ig kappa locus and furthermore coincides developmentally with the occurrence of Ig kappa rearrangements in wild-type mice. We conclude that developmentally controlled transcription of a gene rearrangement in the Ig lambda locus occurs in the presence of an unrearranged Ig kappa locus and is therefore not negatively regulated by the latter. Our data also indicate light chain editing in approximately 30% of lambda 1 expressing B cell progenitors.Journal of Experimental Medicine 06/2003; 197(9):1165-72. · 13.85 Impact Factor -
Article: Expression of a Targeted λ1 Light Chain Gene Is Developmentally Regulated and Independent of Igκ Rearrangements
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ABSTRACT: Immunoglobulin light chain (IgL) rearrangements occur more frequently at Igκ than at Igλ. Previous results suggested that the unrearranged Igκ locus negatively regulates Igλ transcription and/or rearrangement. Here, we demonstrate that expression of a VJλ1-joint inserted into its physiological position in the Igλ locus is independent of Igκ rearrangements. Expression of the inserted VJλ1 gene segment is developmentally controlled like that of a VJκ-joint inserted into the Igκ locus and furthermore coincides developmentally with the occurrence of Igκ rearrangements in wild-type mice. We conclude that developmentally controlled transcription of a gene rearrangement in the Igλ locus occurs in the presence of an unrearranged Igκ locus and is therefore not negatively regulated by the latter. Our data also indicate light chain editing in ∼30% of λ1 expressing B cell progenitors.Journal of Experimental Medicine 05/2003; 197(9):1165-1172. · 13.85 Impact Factor
Top co-authors
Top Journals
Institutions
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2011–2012
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Alnylam Pharmaceuticals
Cambridge, MA, USA
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2003–2006
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Harvard University
- Center for Blood Research Institute
Boston, MA, USA
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2005
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Biogen idec
Weston, MA, USA
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