Vera M. Ruda

From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

Rab5 is necessary for the biogenesis of the endolysosomal system in vivo

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Influence of cationic lipid composition on gene silencing properties of lipid nanoparticle formulations of siRNA in antigen-presenting cells.

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.

IRE1α activation protects mice against acetaminophen-induced hepatotoxicity

Mice lacking the transcription factor XBP1 exhibit constitutive activation of the stress sensor IRE1α and are protected from acetaminophen overdose–induced acute liver failure.

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.

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Dutta et al. show that targeting VACM-1 expression in splenic macrophages impairs extramedullary hematopoiesis, thus reducing inflammation in mouse ischemic heart and atherosclerotic plaques.

Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination

Significance We explored the effect of nontraditional vaccine dosing profiles on antibody titers of vaccines and discovered that certain dosing profiles demonstrate >10-fold higher antibody production than the traditional single-dose prime–boost method. We also present a computational model that captures the experimental results and provides a mechanistic understanding of the biology behind the effectiveness of our strategy. This work has clinical significance in vaccine design because it is a simple method to increase the efficacy of subunit vaccines, which may lead to the development of efficacious vaccines for diseases such as HIV. Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1–2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.

The roles of individual mammalian argonautes in RNA interference in vivo.

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins.

RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver

BACKGROUND & AIMS The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver.