Drew Weissman

Small Interfering RNAs Mediate Sequence-Independent Gene Suppression and Induce Immune Activation by Signaling through Toll-Like Receptor 3

Small interfering (si) and short hairpin (sh) RNAs induce robust degradation of homologous mRNAs, making them a potent tool to achieve gene silencing in mammalian cells. Silencing by siRNAs is used widely because it is considered highly specific for the targeted gene, although a recent report suggests that siRNA also induce signaling through the type I IFN system. When human embryonic kidney 293 (HEK293) or keratinocyte (HaCaT) cell lines or human primary dendritic cells or macrophages were transfected with siRNA or shRNAs, suppression of nontargeted mRNA expression was detected. Additionally, siRNA and shRNA, independent of their sequences, initiated immune activation, including IFN-α and TNF-α production and increased HLA-DR expression, in transfected macrophages and dendritic cells. The siRNAs induced low, but significant, levels of IFN-β in HEK293 and HaCaT cells. Secretion of these cytokines increased tremendously when HEK293 cells overexpressed Toll-like receptor 3 (TLR3), and the increased secretion of IFN-β was inhibited by coexpression of an inhibitor of TIR domain-containing adapter-inducing IFN-β, the TLR3 adaptor protein linked to IFN regulatory factor 3 signaling. Although siRNA and shRNA knockdown of genes represents a new and powerful tool, it is not without nonspecific effects, which we demonstrate are mediated in part by signaling through TLR3.

Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro

DC‐SIGN is a C‐type lectin, highly expressed on the surface ofimmature dendritic cells (DCs), that mediates efficient infection of Tcells in trans by its ability to bind HIV‐1, HIV‐2, and SIV. Inaddition, the ability of DC‐SIGN to bind adhesion molecules on surfacesof naïve T cells and endothelium also suggests its involvementin T‐cell activation and DC trafficking. To gain further insights intothe range of expression and potential functions of DC‐SIGN, weperformed a detailed analysis of DC‐SIGN expression in adult and fetaltissues and also analyzed its regulated expression on cultured DCs andmacrophages. First, we show that DC‐SIGN expression is restricted tosubsets of immature DCs in tissues and on specialized macrophages inthe placenta and lung. There were no overt differences between DC‐SIGNexpression in adult and fetal tissues except that DC‐SIGN expression inalveolar macrophages was only present after birth. Similarly, intissues, DC‐SIGN was observed primarily on immature (CD83‐negative)DCs. Secondly, in the peripheral blood, we found expression of DC‐SIGNon a small subset of BDCA‐2+ plasmacytoid DC precursors (pDC2),concordant with our finding of large numbers of DC‐SIGN‐positive cellsin allergic nasal polyps (previously shown to be infiltrated by DC2).Triple‐label confocal microscopy indicated that DC‐SIGN was colocalizedwith BDCA‐2 and CD123 on DCs in nasal polyp tissue. Consistent withthis finding is our observation that DC‐SIGN can be up‐regulated onmonocyte‐derived macrophages upon exposure to the Th2 cytokine, IL‐13. In summary, our data demonstrate the relevant populations of DC andmacrophages that express DC‐SIGN in vivo where it may impact theefficiency of virus infection and indicate that DC‐SIGN expression maybe involved in the Th2 axis of immunity.

Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) enter target cells by forming a complex between the viral envelope protein and two cell-surface membrane receptors: CD4 and a 7-span transmembrane chemokine receptor (reviewed in refs 1,2,3). Isolates of HIV that differ in cellular tropism use different subsets of chemokine receptors as entry cofactors: macrophage-tropic HIVs primarily use CCR5, whereas T-cell-tropic and dual-tropic isolates use CXCR4 (refs 1,2,3) receptors. HIV-mediated signal transduction through CCR5 is not required for efficient fusion and entry of HIV in vitro. Here we show that recombinant envelope proteins from macrophage-tropic HIV and SIV induce a signal through CCR5 on CD4+ T cells and that envelope-mediated signal transduction through CCR5 induces chemotaxis of T cells. This chemotactic response may contribute to the pathogenesis of HIV in vivo by chemo-attracting activated CD4+ cells to sites of viral replication. HIV-mediated signalling through CCR5 may also enhance viral replication invivo by increasing the activation state of target cells. Alternatively, envelope-mediated CCR5 signal transduction may influence viral-associated cytopathicity or apoptosis.

Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference

RNA interference (RNAi) is an ancient antiviral response that processes dsRNA and associates it into a nuclease complex that identifies RNA with sequence homology and specifically cleaves it. We demonstrate that RNAi mediated by 21-bp dsRNA specifically inhibits HIV-1 infection of permanent cell lines and primary CD4+ T cells. Inhibition of HIV replication was measured by p24 Gag protein content in supernatant, Northern blot analysis, and DNA PCR for products of reverse transcription. The inhibition occurred at two points in the viral life cycle, after fusion and before reverse transcription and during transcription of viral RNA from integrated provirus. Treatment of HIV-infected activated CD4+ T cells with a fluorine-derivatized siRNA that is resistant to RNase A yielded similar inhibition of HIV infection. In addition, the derivatized siRNA could be delivered without lipofectin complexing and in the presence of serum. The identification of RNAi activity against HIV-1 presents a new approach to study viral infections and a proof of concept of RNAi antiviral activity in mammalian cells.

Differential N-Linked Glycosylation of Human Immunodeficiency Virus and Ebola Virus Envelope Glycoproteins Modulates Interactions with DC-SIGN and DC-SIGNR

ABSTRACT The C-type lectins DC-SIGN and DC-SIGNR [collectively referred to as DC-SIGN(R)] bind and transmit human immunodeficiency virus (HIV) and simian immunodeficiency virus to T cells via the viral envelope glycoprotein (Env). Other viruses containing heavily glycosylated glycoproteins (GPs) fail to interact with DC-SIGN(R), suggesting some degree of specificity in this interaction. We show here that DC-SIGN(R) selectively interact with HIV Env and Ebola virus GPs containing more high-mannose than complex carbohydrate structures. Modulation of N-glycans on Env or GP through production of viruses in different primary cells or in the presence of the mannosidase I inhibitor deoxymannojirimycin dramatically affected DC-SIGN(R) infectivity enhancement. Further, murine leukemia virus, which typically does not interact efficiently with DC-SIGN(R), could do so when produced in the presence of deoxymannojirimycin. We predict that other viruses containing GPs with a large proportion of high-mannose N-glycans will efficiently interact with DC-SIGN(R), whereas those with solely complex N-glycans will not. Thus, the virus-producing cell type is an important factor in dictating both N-glycan status and virus interactions with DC-SIGN(R), which may impact virus tropism and transmissibility in vivo.

HIV Gag mRNA Transfection of Dendritic Cells (DC) Delivers Encoded Antigen to MHC Class I and II Molecules, Causes DC Maturation, and Induces a Potent Human In Vitro Primary Immune Response

Dendritic cells (DC) are the major APCs involved in naive T cell activation making them prime targets of vaccine research. We observed that mRNA was efficiently transfected, resulting in superior translation in DC compared with other professional APCs. A single stimulation of T cells by HIV gag-encoded mRNA-transfected DC in vitro resulted in primary CD4+ and CD8+ T cell immune responses at frequencies of Ag-specific cells (5–12.5%) similar to primary immune responses observed in vivo in murine models. Additionally, mRNA transfection also delivered a maturation signal to DC. Our results demonstrated that mRNA-mediated delivery of encoded Ag to DC induced potent primary T cell responses in vitro. mRNA transfection of DC, which mediated efficient delivery of antigenic peptides to MHC class I and II molecules, as well as delivering a maturation signal to DC, has the potential to be a potent and effective anti-HIV T cell-activating vaccine.

Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA

In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.

Increased Erythropoiesis in Mice Injected With Submicrogram Quantities of Pseudouridine-containing mRNA Encoding Erythropoietin

Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10–100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA–LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA–LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA–LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Cutting Edge: Innate Immune System Discriminates between RNA Containing Bacterial versus Eukaryotic Structural Features That Prime for High-Level IL-12 Secretion by Dendritic Cells

RNA derived from bacterial but not eukaryotic sources, when transfected into human monocyte-derived dendritic cell precursors, induces high-level IL-12 secretion in conjunction with dendritic cell maturation stimuli. In vitro-transcribed mRNA that mimics the structure of bacterial mRNA in the lack of a long 3′-poly(A) tail likewise induces IL-12 secretion, but this property is lost upon efficient enzymatic 3′-polyadenylation. Among other tested RNAs, only polyuridylic acid induced IL-12 p70. This RNA response phenomenon appears biologically distinct from the classically defined response to dsRNA. RNA-transfected APC also polarize T cells in an IL-12-dependent manner toward the IFN-γhighIL-5 low Th1 phenotype, suggesting a link between the detection of appropriately structured RNA and the skewing of immune responses toward those best suited for controlling intracellular microbes. RNA structured to emulate bacterial patterns constitutes a novel vaccine strategy to engender polarized Th1-type immune responses.